Wah Chiu, PhD - US grants

Our long term objective is to understand the molecular interaction of DNA with proteins which would influence the function and metabolism of nucleic acids. As first step towards achieving this goal we plan to determine the 3 dimensional structures of the individual proteins and the macromolecular complexes which are involved in DNA replication, recombination and repair. Subsequently we will relate the structural data to genetic, biochemical and biophysical information in order to advance our understanding of the molecular mechanism of these biolobical events. This type of detailed knowledge will provide profound insights into a variety of important research areas such as cancer, aging and genetic engineering. In this proposal, we plan to use electron microscopy to determine the 3 dimensional structures of gp32*I, a T4 DNA helix destabilizing protein at 7Angstrom resolution, of RecA protein, an E. coli recombination enzyme and of RecA and DNA complex at low resolution (greater than 25 Angstrom). We will preserve these specimens in a frozen, hydrated state, image them under low dose, low temperature conditions to reduce radiation damage and use computer processing methods to reconstruct the 3 dimensional density maps. We will develop a number of image processing procedures needed to achieve the specified resolution in the reconstructions. The work on gp32*I protein will represent the first example of a high resolution structural study on the crystal of a soluble protein by electron microscopy. The methodology resulting from this project will have broad applicability to other thin protein crystals. At 7 Angstrom we expect to identify the secondary structure of the protein. This will provide answers to some of the questions about structure-function relationships in the protein. Moreover this structural data will form the basis of future high resolution study of the protein by electron or x-ray diffraction analysis. We will attempt to grow large crystals of gp32 and of gp32*I for atomic structure determination by X-ray diffraction method. The low resolution study of RecA and its complex with DNA in a filamentous form will provide information about the size and shape of the protein and its complex with DNA. These will enable us to evaluate current models of RecA-DNA interaction. Because this proposal involves a considerable effort to develop novel methodology in electron microscopy, a 5 year support is requested to complete the proposed tasks.

Our objectives are to determine the three-dimensional (3D) crystal structures of macromolecules at near atomic resolution and the 3-D structures of non-crystalline macromolecular assemblies at intermediate resolution, by a combination of electron microscopy and computer processing methods. The present proposal is a resource related project. Requests include the acquisition of a medium high voltage (300 kV) electron microscope with appropriate accessories and an operational fund to develop the technology for the above aims. Technical development will be focused on specimen preparation, 3-D data collection at low dose and low temperature conditions and implementation of computer programs for 3-D reconstruction. The developed technology will be immediately applied to the high resolution crystal structure determination of crotoxin complex, T4 DNA helix destabilizing protein, fungus lysozyme and Fc fragments; and to the intermediate resolution structure determination of muscle Z disc and flagella axoneme. Collaborative projects will be established with investigators outside our research groups after the midpoint of the grant period. We plan to complete a thorough analysis of the potential as well as limitations of this technology in its general applicability to a variety of biological systems. We expect to present a final recommendation on the feasibility and desirability of establishing a national shared facility of this technology at the end of this grant period.

The proposed conference will be the first Gordon Conference on Three-dimensional Electron Microscopy of Macromolecules. Electron microscopy has developed extremely rapidly over the last few years as a tool for structure research at the molecular level. Important technical breakthroughs have recently been made which enable three-dimensional structure to be determined more accurately than before, and which open the way to a study of structure essentially under controlled physiological conditions. As a result there are now exciting new opportunities for understanding biological mechanisms and principles of molecular design. The focus of this meeting will be on the novel electron microscopic methods being developed for preserving structure in the native hydrated state, for imaging molecules in frozen solution, for reducing radiation damage by low dose and low temperature and for extracting high resolution three-dimensional information from images of molecules in isolation, in complex assemblies and in crystalline arrays. Important recent structural findings, relating especially to membrane and contractile proteins, will also be emphasized. This conference will help catalyze the further development of this field by bringing together many of the key investigators and allowing them to discuss their latest and perhaps even incomplete studies in a unique and informal setting. Speakers are being chosen on the basis of the importance of their recent research contributions and the potential of their particular approach in future structure research. Topics to be covered include: electron microscopy of specimens preserved in frozen solution, high resolution structure determination from two-dimensional crystals, combined electron microscopy and X-ray diffraction, correlation averaging of isolated molecules, crystallization methods, related approaches in structural cell biology, and recent structural findings dealing with membrane proteins, filamentous proteins and other complex molecular assemblies.

There are three objectives in the present proposal: (1) to determine the structure of the crotoxin complex from Crotalus d. terrificus by low dose electron diffraction methods, (2) to determine the molecular interaction between the crotoxin and the purified acetylcholine receptor membrane from T. Californica by binding kinetic analysis together with electron microscopy, and (3) to search for experimental conditions in reconstituting the receptor proteins that would form a highly ordered two dimensional array and would also maintain its specific activity in binding with the neurotoxin. The three dimensional structure of the crotoxin complex will be reconstructed to 5 angstrom units resolution by computer processing techniques from the low dose electron diffraction and image intensities collected at different tilting angles in a 100 keV transmission electron microscope. The glucose embeddment technique will be used to maintain the hydration of the crotoxin complex thin crystal inside the microscope vaccum. Nuclear track emulsion (NTB3), which has been found to be a more effective recording medium for low dose microscopy, will be used for the data recording. Acetylcholine receptor membrane will be isolated from the T. Californica according to the published procedures. Kinetic studies will be conducted to demonstrate the presence of specific interaction between the crotoxin and the purified receptor membrane, and to determine the dissociation constant and the possible binding domain in the acetylcholine receptor membrane. Frozen hydrated, unstained specimen preparation methods will be utilized in obtaining electron images of the acetylcholine receptor membrane and of the crotoxin-acetylcholine receptor membrane complex in order to visualize the quarternary structure of the receptor proteins and the molecular arrangement of the toxin-receptor complex. If the purified receptor membrane is found non-periodic, we will attempt to reconstitute the receptor membrane protein in a highly crystalline array by using different lipid compositions and temperatures. The objective of the last part of the proposal is to prepare the membrane in a crystalline form so that the structure of the membrane receptor-neurotoxin complex can be studied at high resolution.

Rotavirus; virus morphology; virus protein; protein structure; virus envelope; antiviral antibody; genetic transcription; monoclonal antibody; virus RNA; peptidases; image processing; electron microscopy; X ray crystallography; laboratory mouse;

Electron cryo-microscopy and image analysis have become useful tools for determining 3-dimensional structures of macromolecular assemblies at near atomic resolution. Our laboratory is uniquely equipped with a 400 kV electron microscope funded by the National Center for Research Resources of NIH as a national facility. To complement our NIH support, this proposal is aimed at improving the specimen preparation procedure,, cryo-specimen holder performance and data collection on our 400 kV electron cryo- microscope. The first half of this proposed technology development will be carried out in collaboration with Gatan Inc. to build a carbon cryo-coater for vacuum deposit of a thin carbon film onto frozen, hydrated specimens, and high resolution cryo-specimen holder for the JEOL 400 kV electron microscope. The performance of this equipment will be evaluated with a variety of biological specimens under investigation in our Resources Center. The final version of this equipment will become standard products of Gatan. The second half of this proposal is to develop software for capture and analysis of electron diffraction patterns and images of biological specimens with our newly acquired 1024x1024 slow scan charge-coupled device (CCD) camera. This software will be made available to the research community. These enchanced technologies will be used for several collaborative and user projects in our NIH supported Resource Center.

This award to a group of 27 Faculty will provide positions for 5 graduate students in a new computational biology training program at the Center for Computational Biology established jointly by three institutions (Rice University, Baylor College of Medicine and the University of Houston). The faculty come from Departments of Cell Biology, Biochemistry, Chemistry, Computer Science, Statistics and Electrical Engineering. A few also participate in the STC for Research on Parallel Computation at Rice University. The training program will emphasize visualization, algorithm development and advanced computation in biology, biochemistry and biophysics. Also included are courses offered by the individual institutions, a course taught jointly by faculty from all three, and a Center-sponsored seminar series. The award includes funds to be used to aid in recruiting and to promote student participation in meetings & workshops.

9413229 Phillips This award to large group of outstanding faculty from Rice University, Baylor College of Medicine and the University of Houston provides funds for support of a training program on computational aspects of modern biology, including experimental and theoretical analyses of macromolecular structure using crystallographic, microscopic and other experimental techniques, and computational analysis of genomic structure. The faculty groups from each institution provide complementary skills in biochemistry, biophysics and computer science, resulting in a program broader and stronger than could be provided by the individual institutions. Important aspects of the training include dual mentorship for postdoctoral trainees, a large summer undergraduate program and a biannual national symposium on selected topics in computational biology. This award is also supported by the New Technology Program of the Division of Advanced Scientific Computing and by the Computational Biology Activity of the Division of Biological Instrumentation and Resources. ***

[unreadable] DESCRIPTION (provided by applicant): The National Center for Macromolecular Imaging (NCMI) has been supported by NCRR since 1985. We focus on advancing electron cryomicroscopy for structural biology towards atomic resolution. This period, we determined the structures of 7 viruses and molecular machines at 6.5-9.5 A. Most secondary structure elements of the proteins can be seen. This resulted from innovation in instrument set-up, data collection, refinement, feature extraction and visualization. We published 72 manuscripts related to core, collaboration, service, training and dissemination, and sponsored 11 workshops and symposia. In the coming period, we aim 1) to extend the resolution of single particle reconstructions towards 3-4 A; and 2) to transform our experimental and computational steps so that biologists can quickly obtain structures at sub-nanometer resolution. We will install a new cryomicroscope, upgrade instruments, and improve the quality and efficiency of specimen preparation, data collection, pre-processing, refinement, structure analysis, data mining, deposition, visualization, animation and data management. Our staff will partner in some cases with instrument and software developers at other centers and industry. This core development is motivated by 13 collaborative and 16 service projects. These span the spectrum of specimens, including subcellular ordered assemblies, viruses, ion channels, receptors, sensors, molecular machines, multi-subunit enzymes and nano-particles with sizes from ~300 kDa to 200 MDa. We will disseminate our technology via publications, workshops, symposia, a book on cryo-EM and video conferencing. We will adopt an open-source code policy for our software. We anticipate a continuous growth in demand for our facility from global users and will emulate the management model of a synchrotron beamline. A committee of prominent scientists will annually advise us. NCMI will lead in integrating all aspects of cryo-EM structure determination to [unreadable] [unreadable]

A number of human herpes viruses are significant pathogens which cause a variety of well known syndromes. They form a particular hazard to immunologically compromised patients such as those undergoing transplant surgery or suffering from AIDS. The Herpes simplex viruses, HSV-1 and -2, are the best known herpes viruses and cause recurrent facial and genital cold sores, respectively. In more severe cases, HSV-1 may cause herpes keratitis (leading to sight impairment) or a potentially fatal encephalitis. The HSV-1 genome has been sequenced and contains over 70 genes, approximately half of which have a role in virion structure and assembly. The HSV-1 virion (2,400 Angstrom units) consists of a large (1,250 Angstrom unit) icosahedral capsid embedded in an amorphous protein layer, called the tegument, which is in turn enveloped by a glycoprotein containing lipid membrane. Following infection of cells, herpes virions are uncoated at the cell membrane and the capsids are transported across the cytoplasm to the nuclear pore complex at which point, the viral DNA is released into the nucleus. New capsids (containing 7 proteins) are assembled inside the nucleus and, following packaging of viral DNA, fill capsids (containing 5 proteins) exit the nucleus by budding through the nuclear membrane. The long term objective of our research is to understand the structural basis of assembly mechanism for herpesvirus capsids and its interactions with other non-capsid proteins and cellular carriers/receptors. Such an understanding should prove valuable in developing interventionist strategies directed against herpesvirus infection. This proposals aimed at improving our knowledge of the herpesvirus capsid structure and molecular interactions using high resolution electron cryomicroscopy and computer reconstruction together with molecule biology techniques. The first phase of the proposed research is to demonstrate the feasibility of attaining a 10 Angstrom Unit structure of the herpesvirus capsid by employing our 400 kV electron cryomicroscopy coupled with improved image processing procedures. The reconstruction at 10 Angstrom resolution can be expected to define protein subdomains and reveal characteristic features, including the alpha helices under favorable circumstance, of individual capsid shell protein subunits. The second phase of the work envisaged in this proposal is to employ the developed procedures to study the molecular structures of naturally occurring, this proposal and genetically engineered herpesvirus capsids. The recent development of a baculovirus based system for HSV-1 capsid assembly allows extensive manipulation of the constituent capsid proteins and creates a great need of structural information on the newly generated capsids. Our structural studies will be aimed at answering biological questions relating to the structural transformation of the capsid proteins at various morphogenetic stages involving scaffold assembly, proteolysis, scaffold exit and DNA packaging; the molecular boundaries and interactions among the major capsid shell proteins and their respective contribution to the capsid stability; the mode of interactions of the capsid shell proteins with the tegument proteins; and the mapping of regions of interest of various proteins by inserting or deleting short fragments of amino acid sequences. We anticipate that this research will not only advance a useful tool or virus structure research but also shed critical insights into structure-based mechanism of biological activities of herpesvirus capsids.

Recent advances in computing and networking technology have opened a new frontier in biological research. Baylor College of Medicine (BCM) has recognized this opportunity and its potential impact on understanding fundamental biological processes and developing biotechnological applications. The field of computational and structural biology was designated as one of the highest priority areas for development and expansion at BCM in the 1994 report of the College's Strategic Planning Committee. This proposal will create a new core laboratory for high performance computation, communication and visualization applied specifically to biological research. The proposed facility will include a parallel array of 12 processors with advanced visualization capability, and a high bandwidth network to four research and teaching clusters throughout the College. The core laboratory will be administered by an executive director and an executive committee of seven faculty and a student representative. It will be staffed with a computing application specialist and a system administrator. Twenty-five faculty members at BCM are key participants in this proposal, which covers four research areas. These include electron image reconstruction of macromolecular assemblies, x-ray crystallography, molecular biology informatics and computational neurobiology. All of the investigators are leaders in their respective scientific disciplines and use computationally intensive methods in their research efforts. Establishment of this proposed core laboratory will provide highspeed network access to a high performance computing and visualization resource that cannot be reasonably acquired by any individual investigator. The impact of this proposed bio-computing core laboratory at BCM will be enormous. This facility will: x improve our ability to tackle problems of great importance in cell biology, biochemistry, virology, molecular genetics and neurobiology which rely heavily on computa tionally intensive methods; x facilitate the development of novel methods of analysis and simulation using parallel and distributed computation; enhance our capability for visualization of large and complex biological data; x enable direct high-speed network linkage between the proposed high performance computers and the groups engaged in computationally intensive research at different locations throughout the College; x enrich student training in the use of computers for understanding complex biophysical and structural concepts; x attract new faculty members and trainees at postdoctoral, predoctoral and undergraduate levels to join our recently established Ph.D. Program of Structural and Computational Biology and Molecular Biophysics (SCBMB). The total cost of establishing the proposed core laboratory is $2.3 million, including equipment purchase, high-speed network connection, room renovation, software installation, equipment maintenance and personnel salary support for a period of three years. BCM will provide 34 % matching funds totaling $0.78 million. The total project cost that NSF is asked to fund is $1.5 million.

With the support of NSF Computational Biology Training Group, we have an active program throughout the year to give experience in structural and computational biology research to both undergraduate and high school students. We have a summer seminar series for a group of 10 undergraduate and high school students working in various computational and structural biology labs. This program has been shown to be useful in recruiting future graduate students. One of the current graduate students in Dr. Chiu s lab was a previous summer trainee at Baylor. Chit-Kwan Lin is an exceptional student from Harvard who has made an impressive contribution to our computer data analysis on the studies of actin bundle. He plans to continue his senior thesis research with our collaborator, Professor Paul Matsudaira at MIT when he returns to Cambridge this fall.

Abstract The National Center for Macromolecular Imaging (NCMI) URL:HTTP://ncmi.bioch.bcm.tmc.edu at Baylor College of Medicine (BCM) requests partial funding to acquire a 300 keV electron microscope equipped with a field emission gun and a liquid helium specimen stage. NCMI is a national resource center supported by the National Center for Research Resources of NIH. Its mission is to develop electron imaging of macromolecular assemblies near atomic resolution. We also conduct methodology development, collaboration, service, technology dissemination and training. The improved imaging capability will be used in all these components of the NCMI research mission.

For single particles, it is impractical to record electron diffraction intensities. We are developing a strategy to make corrections of the amplitudes and phases of the structure factors computed from electron images of single particles. Our computational procedure of summing the Fourier transforms of the single particle images has proven effective in determining the zeros of the contrast transfer function and thus the defocus value of the image. This defocus determination is used to correct the phase reversal of the structure factors. For the amplitudes, we attempted to use x ray solution scattering to determine the scattering profile of the particles with which we would correct for the amplitudes of the computed structure factors. The x-ray data has been collected at the Stanford Linear Accelerator Laboratory with the assistance of Hiro Tsuruta. Initially, we collected low angle solution scattering data in the range of 300 to 50 [unreadable]using a multi-layer crystal and then high angle data in the range of 100 to 5 [unreadable]. Subsequently, we used a better positionally calibrated Si(111) monochromator crystal for data collection at resolution ranges from 400 to 13 [unreadable] and from 50 to 4 [unreadable]. Using these data, we are able to determine quantitatively the amount of amplitude contrast and the extent of damping in the computed transforms of our 400 keV images P22 phages. These parameters will be corrected in our final reconstruction in order to obtain a more accurate structure.

Our long term goal is to determine the molecular structures of DNA-containing viruses such as P22 bacteriophage and herpes simplex virus type 1 capsid which have diameters of 600 E and 1,250 E, respectively. Our primary structural tool is electron cryomicroscopy and computer reconstruction and the resolution of the reconstruction is targeted to beyond 10 E. The electron imaging technique has an intrinsic inaccuracy of measuring the amplitudes of the structure factors due to the microscope instrumental factors. This proposal is aimed at using X-ray solution scattering to provide the scattering amplitudes of these viral capsids at a broad range of frequencies from 1/1000 to 1/10 E-1. These measurements will be used to correct those computed from the electron images of the corresponding capsids.

structural biology; technology /technique development; virus; communicable diseases; spectrometry; biomedical resource;

This project is aimed at evaluating the potential of a 300 keV electron cryomicroscope with a field emission gun and a liquid helium cryostage. Such an instrument is only available in Japan. We froze samples of P22 phage on EM grids in Houston and shipped them under liquid nitrogen to Dr. Y. Fujiyoshi s laboratory in Japan. Joanita Jakana and Wah Chiu performed the imaging in Japan. A few sets of quality images were obtained in a week of intensive work. Our subsequent computer analysis indicated that the damping of the computed transform of the images taken in Japan was not as steep as that of the 400 keV images taken with equivalent defocus in Houston. This preliminary data has provided the results needed to develop a grant proposal submitted to NSF for acquisition of such an instrument, which costs ~US$2 million, in the US. We have obtained a strong matching fund commitment from Baylor s administration to support this grant application. Notice of funding of $ 1 million from NSF has just been received today.

Bluetongue virus (BTV) (Reoviridae family, Orbivirus genus) is a non-enveloped, icosahedral double-stranded RNA virus. Several protein layers enclose the genome. Upon cell entry the outer layer is stripped away leaving a core whose surface is composed of VP7. The structure of the trimeric VP7 molecule has been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one largely alpha-helical and the other a smaller domain with the familiar 'jelly-roll' topology. The relative orientations of these two domains vary in different crystal forms. A 23 resolution map of the core determined, using electron cryo microscopy (cryoEM) data, reveals 260 trimers of VP7 organized on a rather precise T=13 laevo icosahedral lattice. The VP7 layer occupies a shell between 260[unreadable] and 345[unreadable] from the center of the core. Below this radius (230-260[unreadable]) lies the T=1 layer of 120 molecules of VP3. By fitting the X-ray structure of the individual VP7 trimer to the cryoEM structure, we have generated an atomic model of the VP7 layer of BTV and demonstrated that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the conformation of the molecule in the core. The beta barrel domains of VP7 are external to the core and interact with the protein in the outer layer of the mature virion. The lower alpha-helical domains of VP7 interact with the inner layer of VP3. Adjacent VP7 trimers in the T=13 layer interact principally through well defined regions in the broader lower domains and conform well to the structure expected from the theory of quasi-equivalence, with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth substrate upon which the two dimensional lattice of VP7 trimers is laid down.

There are 6-8 graduate students at any one time using the NCMI s facilities to pursue their Ph.D. thesis work. We have been quite generous with our resource to let these students work freely, and with our own time for informal teaching interactions. The senior scientists in the Center have held special tutorials to help train the junior scientists to understand not only the operations of the instruments but also the physical principles. A number of publications reported here involve the graduate students' efforts, justifying this expenditure of time and effort.

Bacteriophages have long been used as model systems for the study of gene expression, macromolecular assembly, and for cloning vectors in genetic engineering. Bacteriophage P22 is among the most comprehensively studied bacteriophages using biochemical and genetic tools. The availability of numerous P22 mutant capsids and assembly intermediates makes it possible to identify key proteins within the capsids and to study assembly pathways and mechanisms. Based on our previous success on low resolution structural studies of P22 capsids in different functional and chemical states in collaboration with Jonathan King and Peter Prevelige, we propose to embark on a more systematic and co-ordinated effort to further characterize the structural basis of the P22 morphogenesis. In this proposal, we specifically aim at understanding the structural mechanisms for scaffolding protein assisted procapsid assembly, capsid shell expansion upon maturation, and the modes of internal DNA organization and encapsidation. To achieve this, we will employ electron cryomicroscopy and computer reconstruction as the structural tools to study a variety of P22 assembly intermediates and chemically or genetically modified particles. We will target the structural determinations to approximately 7 Angstrom units resolution using Fourier Bessel reconstruction algorithm for particles with icosahedral symmetry. We also plan to develop algorithms to determine the structure of the portal protein complex within the capsid through a hybridized image processing tools aimed at understanding the mismatched symmetry between the non-icosahedrally organized DNA entry port and the icosahedrally packed coat proteins.

This award provides partial support for a Symposium on Electron Crystallography of Macromolecules at Granlibakken Conference Center in Lake Tahoe, California, between December 9-12, 1998. The motivation of this symposium is to be based on the recent advances in this field of structural biology capable of resolving biological structure details at 3-10 angstrom resolution range. The Symposium will focus on recent dramatic advances in four branches of electron crystallography, emphasizing both methodology development and the elucidation of inter-molecular interactions in large macromolecular assemblies. Particular attention will be paid to proteins - both integral membrane components and non-membrane proteins - in crystalline monolayers; probing inter-molecular interactions by fitting high resolution subunit structures into lower resolution maps of intact complexes; analysis of filaments and free-standing particles at subnanometer resolution; and a novel method of phasing native X-ray data based on exploiting EM-derived molecular envelopes and non-crystallographic symmetry. The goal of the Symposium is to summarize these and related lines of investigation, assess the prospects for further methodological improvement (higher resolution, accelerated analysis), and stimulate their application to a more extensive set of biological problems. Another important purpose of this Symposium is to disseminate this emerging structural biology tool to the cell and molecular biology community. The organizers will make an attempt to attract participants from non structural biology discipline attending this meeting via a variety of means of advertising the Symposium and scheduling it right before the 1998 Cell Biology Annual Conference to be held in San Francisco. For a broader dissemination, it is planned to publish a special issue of the envisaged topics in the Journal of Structural Biology contributed from the invited participants.

We propose to organize a Workshop on Electron Crystallography of Macromolecules at Granlibakken Conference Center in Lake Tahoe, California, between December 9-12, 1998. The motivation of this workshop is to based on the recent advances in this field of structural biology capable of resolving biological structure details at 3-10 Angstrom units resolution range. The Workshop will focus on recent dramatic advances in four branches of electron crystallography, emphasizing both methodology development and the elucidation of intermolecular interactions in large macromolecular assemblies. Particular attention will be paid to proteins - both integral membrane components and non-membrane proteins - in crystalline monolayers; probing intermolecular interactions by fitting high resolution subunit structures into lower resolution maps of intact complexes; analysis of filaments and free-standing particles at subnanometer resolution; and a novel method of phasing native X-ray data based on exploiting EM- derived molecular envelopes and non-crystallographic symmetry. The goal of the Workshop is to summarize these and related lines of investigation, assess the prospects for further methodological improvement (higher resolution, accelerated analysis), and stimulate their application to a more extensive set of biological problems. Another important purpose of this Workshop is to disseminate this emerging structural biology tool to the cell and molecular biology community. We will make an attempt to attract participants from non structural biology discipline attending this meeting via a variety of means of advertising the Workshop and scheduling it right before the 1998 Cell Biology Annual Conference to be held in San Francisco. For a broader dissemination, we plan to publish a special issue of the envisaged topics in the Journal of Structural Biology contributed from the invited participants.

bioimaging /biomedical imaging; electron microscopy; biomedical equipment resource; biomedical resource;

DESCRIPTION (provided by applicant): Our laboratory has demonstrated the capability of solving medium resolution (7-9 Angstroms) structures of several icosahedral particles by combining several thousand-particle images recorded in an electron cryomicroscope. At these resolutions, we have been able to identify most of the alpha helices and beta sheets of the proteins. Presently, the process of data collection and computer image reconstruction is very labor intensive, even for experienced users, and has a very steep learning curve for new users. We propose to develop a high throughput procedure so that the process of structure determination of icosahedral particles at medium resolution - from data collection to structure determination and interpretation - can be completed within 3-5 weeks. This represents a factor of 10-20x reduction in time for completion of each project. This technological advance will allow molecular virologists to use such structures as a standard approach to understand structure and function relationship in the context of virus assembly and interactions with cellular factors such as antibodies and receptors. This proposal consists of two developmental components. The first is to develop semi-automatic on-line digital image acquisition with a 4k x 4k CCD camera in our newly acquired 200 kV electron cryomicroscope with a field emission gun. The second is to improve the computational efficiency of existing software and to integrate much of the stand-alone software, which includes particle picking, microscope parameter determination, particle orientation and center refinement, 3D reconstruction and structural interpretation. We will use the Python scripting language to coordinate these programs and use an object-oriented database to manage the metadata throughout the project data acquisition and analysis. This software will be made freely available to the research community. We will deploy these technology enhancements to our already funded projects on P22 bacteriophage and herpes virus in addition to a new project with the Venezuelan Equine Encephalitis virus, a select agent with potential bioterrorism threat.

EIA-0325004 -Baylor College of Medicine Wah Chi COLLABORATIVES: ITR: Subnanometer Structure Based Fold Determination of Biological Complexes .We propose to form a team of three independent investigators with different expertises to co-develop the necessary computation and visualization methodology for biologists to quickly, easily and accurately deduce the folds of the domain components of large macromolecular complexes. While structures of macromolecular complexes are becoming more prevalent, and the size of some density maps would increase up to 1GB, the visualization and analysis requirements to extract important biological information are far beyond the currently available software. Our research task will explore different algorithms to enhance the signal through denoising of the 3-dimensional structure, visualize these large and filtered maps effectively, extract the individual protein components and their salient structural features such as a helices and b sheets accurately, and ultimately construct pseudo atomic model with novel structure prediction methods using protein sequence and structure considerations as constraints. We will set up a test data set, comprised of both simulated and experimental structures, to validate each of our development steps. The software developments in each of the research sites will be glued together using a scripting language (Python) and made easy to use through a graphical interface. Additionally, the software will be extensively documented and distributed via an open source policy, encouraging continued collaboration and development.

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computational biology; molecular biology information system; structural biology;

computational biology; molecular biology information system;

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DESCRIPTION (application abstract): The chaperonin TRiC is a protein folding nanomachine necessary for the growth of all eukaryotic cells. The protein substrates of this barrel-shaped 16-subunit nanomachine include actins, tubulins, and tumor suppressor proteins. Similarly to its archaeal homolog Mm-cpn, it couples ATP hydrolysis to internalization, folding, and release of newly synthesized polypeptide chains. The folding cycle includes opening and closing of a built-in lid of the chaperonin critical for binding and release. Our Nanomedicine Development Center (NDC) will extend and integrate the current techniques in electron cryomicroscopy, single-molecule imaging, computational biology, and X-ray crystallography to quantify the chaperonin subunit conformations and dynamics as well as the protein folding intermediates bound within the chaperonin cavity. The marriage of these advanced technologies will allow us to visualize chaperonin machinery functioning not only in vitro but also within cells. Building on a more comprehensive and quantitative description of these protein folding nanomachines, we will engineer modified chaperonins to provide a novel therapeutic tool for inhibiting and promoting the folding of selected proteins whose misfolding or aggregation are associated with human diseases. These proteins include actin, tumor suppressor proteins p53 and Von Hippel Lindau, the aggregating A-beta peptide and the cataract related lens protein, gamma-crystallin. Through visualizing conformations and sites in which these chains are folded by the chaperonin, together with their experimentally observed and computed dynamics, we will also design novel substrates that will be folded efficiently in the naturally occurring or newly engineered chaperonin, opening new avenues in protein design. The corresponding approaches will include design of adaptor peptides for modifying the substrates or chaperonins to enhance or inhibit substrate-chaperone interactions. We have assembled a team of 15 investigators from 6 institutions with expertise in chaperones, protein folding, electron cryomicroscopy, computer simulation and modeling, X-ray crystallography, singlemolecule imaging and trapping, and clinical research. We will work together in developing a strategic set of experimental and computational tools that will enable characterization of biological nanomachines, both in vitro and in vivo. We will interact with complementary expertise of other NDC in synthetic chemistry and fluorescence technologies. Our Center can be a critical resource to other NDC who need assistance in solving protein folding and aggregation problems. Our clinical investigators will contribute to the design of pilot studies for therapeutic applications in cell culture models of disease states. We also plan new educational tools via virtual courses in design and application of biological nanomachines, aimed at bridging the gap between biology and mechanical engineering for students in our 6 participating institutions. Finally, the organization, collaborations and communications in our NDC exemplify the 21st century goal of conducting interdisciplinary research via new mechanisms of data sharing and analysis.

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The National Center for Macromolecular Imaging (NCMI) seeks to collaborate with NBCR by migrating NCMI's Scientific Animation &Integration Library (SAIL) to NBCR's visual programming environment Vision. These tools developed by NCMI have produced numerous visualizations used in various critical applications such as to evaluate the validity of electron cryomicroscopy 3-D reconstructions at different refinement stages;segmenting protein components in a macromolecular context;discovering structural features such as alpha helices and beta sheets in the extracted molecular component maps;production of animations to model complex biological phenomena;production of animations for presentations of complex structures;generation of graphics for publications and presentations;assisting collaborators in understanding NCMI's products;and providing comparable visualization environments to peer level laboratories.

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This project, impacting genome analysis, structural proteomics, and macromolecular imaging by linking massive and exponentially growing data in each of these fields to the molecular basis of biological functions, aims at acquiring a 220-CPU Opteron distributed memory computing cluster. The Structural and Computational Biology and Molecular Biophysics (SCBMB) Program using the cluster encompasses six institutions: Baylor College of Medicine, Rice University, the University of Houston, the University of Texas MD Anderson, the University of Texas Health Sciences Center-Houston, and the University of Texas Medical Branch-Galveston. The infrastructure will service the following research activities:
Electron Cryo-Microscopic Reconstruction of Biological Assemblies at the National Center for Macromolecular Imaging (NCMI) (mainly Electronic Cryomicroscopy and Image Reconstruction), Characterization of Functional Surfaces for Structural Proteomics (involving the Evolutionary Trace Method (ET) and utilizing High-Throughput Identification and Geometric Matching of Functional Surfaces), Comparative Genomics (including Parallel Comparison of DNA sequences via Positional Hashing (Pash) and Comparative Sequence Assembly and Mapping)
Micro-RNA (miRNA) (binding target complementary mRNS by regulating gene expressions)
Each research project has high computational demands that will be met with the proposed parallel environments. The derivation of meaningful inferences from raw biological data requires intensive computation involving data sets. For example, to reconstruct 3-D images of macromolecular machines, the labs need to transform and auto-correlate gigabytes of voxels from electron cryomicroscope data; to identify functional sites in protein structures, the lab requires all-against-all comparisons of thousands of protein structures; and to identify mammalian genes and detect novel micro-RNAs, the labs require cross-comparisons of billions of basepairs of DNA sequence. These applications all share the common feature of repeated but relatively independent computations that can be split among many CPUs with modest need of communication through a common file server. These shared characteristics can be serviced well by the requested cluster.

Broader Impact: The infrastructure enhances the educational experience at participating institutions. Students in the SCBMB graduate program (encompassing 6 institutions) will use the system, significantly enhancing their research opportunities. Baylor has outreach programs for undergraduates and for high school. Workshops, software tool development, and technology transfer will serve to disseminate the results.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The success in genome sequencing has led to new initiatives in structural genomics and proteomics. We anticipate a growth of research in the structural biology of large biomolecular complexes (>0.5 M Da). These types of specimens present new challenges in each step of the structure determination pipeline. Once the structures are determined there are significant challenges in archiving and visualizing the results. Every step requires significant investment in the development of appropriate computational algorithms, implementation of efficient codes and the creation of tools for use by biologists. In this P20 pre-center proposal, we have assembled a group of four investigators who have complementary expertise in electron cryo-microscopy, crystallography, data representation and archiving, visualization and computer science. We will develop a virtual Computational Center for Biomolecular Complexes (C2BC), which will provide an enabling computational environment as well as a strong interdisciplinary cadre of researchers beyond the current investigators for the structural biology of biomolecular complexes. In this P20 proposal, we will engage in two sets of activities: workshops and pilot studies. These activities are organized under three leadership teams on computational interface to experiments, data representation, and structural analysis and visualization. Each team leader will solicit and encourage community input through workshops and video-conferences that will come up with recommendations on the computational demands of the experimental data and user interface requirements of the scientific undertaking. Each team will conduct pilot studies on selected systems to demonstrate the mechanism of collaboration across disciplines and institutions among the four PI and Co-PIs. We will have a project manager to organize the workshops and communications among different investigators and institutions. There will be an external advisory committee to guide the formation of the proposed center. [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are collaborating with industrial developers to improve the design of the cryo-specimen plungers.

DESCRIPTION (provided by applicant): Many spherical viruses and some enzyme complexes form particles having icosahedral symmetry. The use of electron cryomicroscopy and single particle reconstruction has allowed us to visualize the 3-dimensional structures of this type of particle to 7-9 A resolution. This resolution is sufficient to resolve long alpha helices and recognize large beta sheets. In the past three years, we have determined 6 structures of icosahedral particles with sizes ranging from 600 -1250 A in this resolution range. They include herpesvirus capsid, rice dwarf virus, procapsid and mature forms of P22 bacteriophage, rotavirus and cytoplasmic polyhedrosis virus. With this experience, we have identified a number of possible improvements in the suite of software specific to the icosahedral particle reconstruction in order to improve the ease of its future maintenance and upgrade, the accuracy of determined structure, the ease of usage by biology end-users and the computational efficiency. In this proposal, we will have four specific goals. The first aim is to rewrite the major codes in C++, which includes the icosahedral particle center and orientation refinement and the Fourier Bessel reconstruction. They will be optimized for both shared and distributed computer platforms and will be written in modular form to make them easy to maintain and modify. The second aim is to improve the algorithms for particle orientation parameter refinement, contrast transfer function and B factor corrections and Fourier Bessel reconstruction respectively. We expect that these improved codes will lead to a more accurate structure determination of icosahedral particles. The third aim is to integrate the improved codes with a Python language binder together with a user-friendly graphical interface and to output relevant intermediate data processing results that would allow the users to decide if the refinement is heading to the correct structure convergence. The fourth aim is to document the codes adequately with a help menu and to disseminate them broadly and freely through the internet access. Before the software release, we will confirm their reliability and accuracy by testing with simulated and experimental data. The experimental data consist of structures that we have previously determined to subnanometer resolution and for which the crystal structures of some of their components are known.

cryoelectron microscopy; biomedical resource; bioimaging /biomedical imaging;

bioimaging /biomedical imaging; three dimensional imaging /topography

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The success in genome sequencing has led to new initiatives in structural genomics and proteomics. We anticipate a growth of research in the structural biology of large biomolecular complexes (>0.5 M Da). These types of specimens present new challenges in each step of the structure determination pipeline. Once the structures are determined there are significant challenges in archiving and visualizing the results. Every step requires significant investment in the development of appropriate computational algorithms, implementation of efficient codes and the creation of tools for use by biologists. In this P20 pre-center proposal, we have assembled a group of four investigators who have complementary expertise in electron cryo-microscopy, crystallography, data representation and archiving, visualization and computer science. We will develop a virtual Computational Center for Biomolecular Complexes (C2BC), which will provide an enabling computational environment as well as a strong interdisciplinary cadre of researchers beyond the current investigators for the structural biology of biomolecular complexes. In this P20 proposal, we will engage in two sets of activities: workshops and pilot studies. These activities are organized under three leadership teams on computational interface to experiments, data representation, and structural analysis and visualization. Each team leader will solicit and encourage community input through workshops and video-conferences that will come up with recommendations on the computational demands of the experimental data and user interface requirements of the scientific undertaking. Each team will conduct pilot studies on selected systems to demonstrate the mechanism of collaboration across disciplines and institutions among the four PI and Co-PIs. We will have a project manager to organize the workshops and communications among different investigators and institutions. There will be an external advisory committee to guide the formation of the proposed center. [unreadable] [unreadable]

bioimaging /biomedical imaging; three dimensional imaging /topography

bioimaging /biomedical imaging; three dimensional imaging /topography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Both of our JEM3000 and JEM3200 electron cryomicroscopes are equipped with a Gatan 4kx4k CCD camera. Quantitative tests for its performance in terms of spectral signal to noise ratio at different frequencies are conducted with a variety of specimens including carbon film, protein crystals and single particles.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our expansion of the collaborative and service research requires an efficient and cost effective means of communication with the remote collaborators. We are implementing different capabilities and options with improving successes.

DESCRIPTION (provided by applicant): Cryo-electron microscopy (cryoEM) is a maturing methodology in structural biology that enables the determination of 3D structures of macromolecular complexes and cells at a broad range of resolution from 2 to 100 A with information that bridges the gap between cell biology and crystallography/NMR. It is now well accepted by structural, molecular and cell biologists that a cryoEM map and its associated model can form the basis for subsequent hypothesis-driven research and knowledge discovery. In the past four years, several independent workshops organized by the European Bioinformatics Institute (EBI) in Hinxton, UK and the Research Collaboratory for Structural Bioinformatics (RCSB) at Rutgers, the State University of New Jersey, Piscataway, NJ, USA, have reached a community consensus that it is time to establish publicly supported, one-stop deposition and retrieval facilities for cryoEM density maps, atomic models and associated metadata. This proposal is a joint effort among investigators of the EBI, RCSB and the National Center for Macromolecular Imaging (NCMI) at Baylor College of Medicine, Houston. These investigators have complementary expertise in archiving density maps (Kim Henrick), archiving coordinates of atomic models (Kim Henrick, Helen Berman), and all aspects of cryoEM technology development, including data collection, map generation, cryoEM map restrained modeling, visualization, segmentation and annotation (Wah Chiu). We propose to integrate the expertise and infrastructure of the three well-established research centers to create a global deposition and retrieval network for cryoEM maps, models and their associated metadata, as well as a web service for a number of software tools for standardized map format conversion, map segmentation and assessment, model assessment, visualization, and data integration. These tools are essential for verification of the deposited data and would also facilitate efficient use of the archived data. We will develop these tools in consultation with the major developers of cryoEM software. Before inclusion in the set of web services, we will evaluate the suitability of these tools for the intended purposes with the map and coordinate data available in the current data resources. We will fully document the usage and limitations of these tools. Moreover, the new, comprehensive cryoEM data resource will be built in a manner that will be straightforward to maintain and extend as the field grows and matures. Finally, an important component activity of this proposed research is to gather community input throughout the entire grant period so that the final system will be designed and implemented at the highest standards to serve the needs of both cryoEM experts and biological end-users.

Biological nanomachines are the assemblies that carry out all the basic biological processes in a living organism. Electron cryo-microscopy (cryoEM) is the most appropriate structural tool to determine molecular structures of biological nanomachines that generally consist of multiple protein subunits and/or nucleic acids with a total mass greater than 0.5 million Daltons. The goal is to develop information discovery and integration methodologies for deriving atomic models of nanomachines. Such models will be derived from 3-dimensional (3-D) cryoEM mass density function (i.e. a volumetric density map) in conjunction with physics of protein folding and informatics data. This project is made possible by an integration of the expertise of five investigators in computer graphics, computational biophysics, structural informatics and cryoEM. The intellectual merit of this research is highlighted by the computational approaches of extracting structural information from low-resolution, complex cryoEM volume densities and integrating this information into classical protein structure modeling paradigms, such as comparative modeling and ab initio modeling, for understanding biological nanomachines. The three research goals involve information discovery, information integration and validation of the proposed algorithms. The proposed research will have significant impacts in three disparate disciplines: computer science, molecular modeling, and cryoEM. Furthermore, the team will disseminate their resulting tools freely to the academic community and will host a workshop towards the end of the project. To enhance the impact of their research, the investigators will integrate research with education at each member institution with an eye towards diversity. In particular, these investigators will develop a virtual didactic course in modeling of biological nanomachines for graduate and senior undergraduate students at the five participating institutions.

CCD camera; CRISP; Computer Retrieval of Information on Scientific Projects Database; Condition; Data; Electrons; Funding; Grant; He element; Helium; Image; Institution; Investigators; Liquid substance; NIH; National Institutes of Health; National Institutes of Health (U.S.); Negative Beta Particle; Negatrons; Performance; Research; Research Personnel; Research Resources; Research Specimen; Researchers; Resolution; Resources; Source; Specimen; United States National Institutes of Health; charge coupled device camera; design; designing; electron optics; fluid; imaging; instrument; liquid; particle

AIDS; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; CRISP; Computer Retrieval of Information on Scientific Projects Database; Development and Research; Funding; Grant; Immunologic Deficiency Syndrome, Acquired; Institution; Investigators; NIH; National Institutes of Health; National Institutes of Health (U.S.); R &D; R&D; Research; Research Personnel; Research Resources; Researchers; Resources; Source; United States National Institutes of Health; research and development

Algorithms; CRISP; Computer Programs; Computer Retrieval of Information on Scientific Projects Database; Computer software; Funding; Grant; Image; Institution; Investigators; Modeling; NIH; National Institutes of Health; National Institutes of Health (U.S.); Research; Research Personnel; Research Resources; Researchers; Residual; Residual state; Resolution; Resources; Simulate; Software; Source; Structure; United States National Institutes of Health; Virion; Virus Particle; computer program/software; imaging; nano meter; nanometer; novel; particle; reconstruction

Program Director/Principal Investigator (Last, First, Middle): Chiu, Wah 1R01 AI075208-01A1 ABSTRACT Herpesviruses are major pathogens in human populations, causing disease in normal and immuno-compromised individuals. Double-stranded DNA (dsDNA) tailed bacterial viruses and herpesviruses share surprising similarities in capsid assembly and organization. In both viral classes, a precursor (procapsid) shell is formed with the help of scaffolding proteins. During viral/phage morphogenesis, newly replicated dsDNA is inserted into the procapsid by a terminase protein complex through a unique portal vertex, which contains multiple gene products. In addition, both viruses deliver their nucleic acid into a membrane bound compartment while the emptied capsids remain outside this compartment. These similarities in assembly, structure, and infection suggest that herpesviruses and dsDNA phages arose from a common ancestor. The portal and associated proteins are critical in the transport of the DNA genome from the virion across the bacterial envelope for phage, and across the nuclear envelope for Herpesvirus. Until very recently the structures of such complexes - lacking icosahedral symmetry - could not be resolved by current structure analysis methods. Recent advances in asymmetric single particle reconstructions from electron cryo-microscopy images of the infectious virions of epsilon15 and P22 phages have revealed details of components of the portal vertex. However, the structural organization of the portal vertex before DNA entry and later when the DNA exits, remains to be determined. Our recent results with the herpesvirus capsid and virion suggest that its portal organization is also similar to that of the phage portal. The herpesvirus portal vertex may act like the phage portal vertex, and rearrange to release or deliver DNA into the cell nucleus. Such an apparatus presents a potential target for anti-viral therapies. We propose to use electron cryo-microscopy and electron cryo-tomography to visualize higher resolution features in the portal vertex in the herpesvirus virion and to determine the structural rearrangements of the phage portal vertex during the process of DNA ejection into host cells. The structures to be determined include not only the isolated virus particles but also the forms generated upon interaction with the host cell and release of DNA. The imaging and computational tools developed for these investigations should be generally applicable to the infection processes of many other viruses. The proposed studies, when combined with other biological and modeling information, will yield valuable structural information for developing anti-viral and gene delivery strategies targeted at these processes.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. So far, cryo-EM reconstruction of single particles determined at subnanometer resolution have been readily, convincingly and successfully applied to relatively large nanomachines. It is known that many biological processes are carried out by macromolecular complexes which have a molecular mass between 50-300 kDa. Such relatively small sizes pose a challenge for single particle reconstruction because these complexes are often difficult to localize under typical cryo-EM conditions, thus making it difficult or impossible to determine the particles'orientations. We are exploring the use of a heated phase plate to record images. Nagayama and colleagues in Japan have demonstrated images with strikingly high contrast for a number of biological specimens ranging from single particles to whole cells. Their most recent work using a heated phase plate positioned in the focal plane of an objective lens having a longer focal length allowed them to image GroEL and reconstruct it to 12 [unreadable] resolution (Danev and Nagayama, 2008). Our goal in this project is to explore the use of such imaging technology.

DESCRIPTION (provided by applicant): The overall goal of this initiative is to investigate the cellular uptake, trafficking, and sub-cellular localization of different classes and subtypes of nanoparticles (NPs) with well-defined physiochemical properties for the creation of a reference table that relates the sub-cellular distribution of NPs to their intrinsic physiochemical properties across a range of cell lines. The subcellular fate of NPs is relevant both in terms of the therapeutic efficacy and biosafety of the NPs. The effective impact of size, shape, charge, and chemical composition of nanomaterials, in the presence of serum opsonins, on both cellular entry and subsequent subcellular localization will be investigated. The expected outcome of this project is to create a reference table that accelerates the transition of nanomaterials from the bench to the clinic by rapidly expanding our knowledge of the effect of a material's intrinsic characteristics on its intracellular destination. The final product, a comprehensive table of NPs and their subcellular locations, will guide the future development of NP drug delivery systems for rapid expansion of biomedical applications, including cancer therapy, cardiovascular imaging, and gene therapy. PUBLIC HEALTH RELEVANCE: What this project seeks to deliver is a multi-dimensional reference table that relates the subcellular distribution and toxicity of NPs to their intrinsic physiochemical properties across a range of diverse cells and cell lines. It is our hope that the data generated from this project will serve as a resource for future research and encourage model development and new insights into nanotechnologies for imaging and drug delivery.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Though EMAN has been used successfully for icosahedral and asymmetric reconstruction of virus particles, the EMAN method of particle orientation refinement is a very CPU intensive process for large virus particles. One million CPU hours were consumed to determine the 4.5 [unreadable] map of the epsilon15 phage (Jiang et al., 2008) using the NSF supported TeraGrid computing facility . A similar structure determination using the clusters available at the NCMI combined with outside supercomputer allocations has taken over 12 months to refine a single structure of P22 phage from ~23,000 particle images. We, therefore, look for alternative methods for such large objects, with the same accuracy but a higher CPU efficiency.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our Center has two 300 kV JEOL electron cryomicroscopes. They have different cryo-specimen holder design and electron optics. Both of them are equipped with Gatan 4kx4k CCD camera. They can be operated at different temperatures and are evaluated their damage effects for molecular structure determinations.

B. Abstract and Specific Aims The goal of our Nanomedicine Development Center has been to use outside-the-box, high-risk, high-payoff and highly interdisciplinary approaches and methodologies to obtain a quantitative understanding of cellular protein folding nanomachines such as mammalian TRiC and the archaeal Mm-cpn chaperonins. Our objective is to re-engineer them for biomedical applications. Central to our approach is the iterative application of scientific, engineering and clinical principles. Our ultimate goal is to harness our knowledge of protein folding machines to create novel therapeutic agents for inhibiting and/or promoting the folding bf selected proteins whose misfolding or aggregation are associated with human diseases. At the inception of our Center, we assembled a team of 15 investigators from 6 institutions with expertise in chaperone biochemistry, protein folding, electron cryomicrosopy, computer simulation and design. X-ray crystallography, single-molecule imaging and trapping, and clinical research. Through the translational supplement available in the mid-course of the current grant cycle, we recruited 8 medical investigators who are specialists in different disease targets, all of which are tied to protein misfolding or aggregation. In the past 5 years, we have made substantial advances in the development of technologies for characterizing chaperonins and other cellular nanomachines in unprecedented detail and in identifying protein folding, misfolding and aggregation disease targets to pave the way for the development of novel nanomedicine therapeutics. In this renewal, we seek to continue the interdisciplinary collaborations among 17 basic and physician scientists from 8 institutions to implement the second phase of our NDC We aim to bring our basic research closer to translational reality in alleviating the deleterious effects of misfolded or aggregated proteins in human diseases including Von Hippel Landau syndrome, stroke and ischemic injury, cystic fibrosis, Huntington?s disease, cataracts, and Alzheimer's disease. Our goals are (i) to utilize the deep insights we have gained into TRiC and Mm-cpn structure and function in the past few years to engineer novel modified forms of chaperonins, chaperonin subunits or peptides for diagnostic and therapeufic purposes; (ii) to design and screen novel adaptor molecules that expand or modify chaperonin-substrate recognition, alter protein folding and prevent protein aggregation; and (iii) to use the chaperonin nanomachine as a novel target for the identification of small molecules from existing NIH libraries that can be developed for therapeufic use. The main philosophy of the Center is to continue our highly interdisciplinary strategy and collaboration to develop cutting-edge methodologies and therapeufic solutions. Our approach consists of 4 major aims aligned with stages in the therapeutics discovery process: ? Aim 1: Identify and validate therapeutic strategies for six selected disease targets ? Aim 2: Design and screen novel therapeutic agents ? Aim 3: Assess molecular mechanism of therapeufic action ? Aim 4: Perform pre-clinical model testing. Although our team's nucleus was a group of basic scientists, we recognize the need to scale down the level of basic science research in order to accomplish the NDC goal, which is to transform our research into medical applications as quickly as possible. Nevertheless, we plan to maintain sufficient basic science investigation to validate and to measure the functional properties of the newly discovered therapeutics in the context of the cell or cell-like environment Such validation is critical to guide and adjust the design process, thus improving the quality of the engineered products finally administered in vivo. After an initial evaluation period, we will select among the initial disease targets and concentrate on a reduced number of targets in the third year of this grant cycle. This will enable us to better develop and optimize therapeutics that will lead to clinical trials. In preparation for the later phases of the translational process, we will engage in a dialogue and collaborations with necessary specialists both in academia and in industry; including bioengineers, pharmacologists and toxicologists so that our intellectual concepts and discoveries can be brought to human trials promptly.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. CCD camera is routinely used for cryo-EM data collection. However, Quantitative tests for its performance in terms of spectral signal to noise ratio at different frequencies are needed to assess their practical limits.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. AgsA(aggregation-suppressing protein) is a novel small heat shock protein (sHsp) discovered in the thermally aggregated fraction from a Salmonella enterica serovar Typhimurium dnaK-null strain. It is strongly induced by high temperatures. The similarity between AgsA and the other two sHsps of Salmonella serovar Typhimurium, IbpA and IbpB, is rather low (around 30% amino acid sequence identity). Phylogenetic analysis suggested that AgsA arose from an ancient gene duplication or amplification at an early evolutionary stage of gram-negative bacteria. Biochemistry data suggest that AgsA is an effective chaperone capable of preventing aggregation of nonnative proteins and maintaining them in a state competent for refolding in Salmonella serovar Typhimurium at high temperatures. Sample of AgsA was multiplied in colibacillus by Xiaodong Shi, one coworker worked in ZengYi Chang's lab of Peking University. Preliminary data (mostly negative stain results) suggest that the particle of AgsA has a olive-like feature with a symmetry of D3. Comparing with its preventing aggregation stage(treat with insuline in room temperature), we can find remarkable distortion in the middle of it. One problem was found in the early research, AgsA seems likely to aggregate itself.This may effect the step of boxing the particles. Another problem is that AgsA are able to construct a fiber-like structure. Due to the similarity in width, it is really difficult to distinguish between particles and the small parts of the fiber. In the following experiment, we'd like to get a sub-nanometer model of two stages, one is the original stage and another is the preventing aggregation stage. We wish to find out what is the different between them. This may lead to finding a clue to how AgsA combines and prevents protein aggregation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. So far, cryo-EM reconstruction of single particles determined at subnanometer resolution have been readily, convincingly and successfully applied to relatively large nanomachines. It is known that many biological processes are carried out by macromolecular complexes which have a molecular mass between 50-300 kDa. Such relatively small sizes pose a challenge for single particle reconstruction because these complexes are often difficult to localize under typical cryo-EM conditions, thus making it difficult or impossible to determine the particles? orientations. We are exploring the use of a heated phase plate to record images. Nagayama and colleagues in Japan have demonstrated images with strikingly high contrast for a number of biological specimens ranging from single particles to whole cells. Their most recent work using a heated phase plate positioned in the focal plane of an objective lens having a longer focal length allowed them to image GroEL and reconstruct it to 12 [unreadable] resolution (Danev and Nagayama, 2008). Our goal in this project is to explore the use of such imaging technology.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Zernike phase plate technology is being implemented in our JEM2200FSC instrument which has been equipped with a FEG, an in column energy filter, a turbo molecular pump and a Gatan 4k CCD camera. The phase plate holder is installed in the instrument. Our goal is to test the resolution of this technology and apply it to biological specimens of various sorts.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Overall goal of the project is to characterize size and shapes of nano particles using cryo- TEM. This is part of a cross institutional studies on morphology, toxicity and functionalization of various nano particles intended for therapeutic purposes.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Herpes Simplex Virus (HSV) infects almost 2 billion people worldwide, due in part to its low mortality rate and its ability to cause latent infections. HSV is an enveloped, dsDNA virus. Infection of host cells begins with binding of HSV to the cell membrane, after which the HSV capsid and some bound tegument proteins are released into the cytoplasm using either a direct membrane fusion or a pH-dependent endocytosis pathway. Once in the cytoplasm, HSV travels along microtubules towards the nucleus. Upon reaching the nucleus, the capsid interacts with Nuclear Pore Complex (NPC) filaments (Nup214-Nup88 and/or Nup358) emanating from the NPC. Subsequently HSV releases its dsDNA genome at which point the dsDNA is translocated through the NPC. We hypothesize that structural studies will elucidate the method by which binding to the NPC causes the subsequent release and translocation of the viral genome. Using cryo-electron tomography, we will examine how HSV interacts with the NPC in Xenopus Laevis oocytes. Ultimately, the information from this study may aid in the development of better anti-viral drugs against HSV while helping researchers gain insight into the mechanisms other viruses employ for NPC attachment and genome translocation into the nucleus.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Direct electron detection (DDD) sensor images high-energy electrons directly. By avoiding the electron-to-light conversion process of a scintillator, the DDD achieves unprecedented sensitivity and resolution. The DE-12 Camera System developed by Direct Electron in San Diego is based on a 12 Mpixel DDD sensor featuring a 3K by 4K array of 6 micron pixels. It offers very high signal-to-noise ratio (SNR) for primary electrons (>30:1) enables single electron counting.It also has a fast readout rate with 25 msec per 12 Megapixel frame. It can be placed in the camera chamber in any of our JEOL electron microscopes (JEM3200FSC and JEM2200FSC). We perform a resolution test with a demo unit from the company.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Though EMAN has been used successfully for icosahedral and asymmetric reconstruction of virus particles, the EMAN method of particle orientation refinement is a very CPU intensive process for large virus particles. One million CPU hours were consumed to determine the 4.5 [unreadable] map of the epsilon15 phage using the NSF supported TeraGrid computing facility . A similar structure determination using the clusters available at the NCMI combined with outside supercomputer allocations has taken over 12 months to refine a single structure of P22 phage from ~23,000 particle images. We, therefore, look for alternative methods for such large objects, with the same accuracy but a higher CPU efficiency.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We collaborate with Dr. M. O'Malley at Rice University to develop an automated freezing apparatus. Such automation will eliminate the human training and will enhance the reproducibility of this critical step in cryo-specimen preparation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. CCD camera is routinely used for cryo-EM data collection. However, Quantitative tests for its performance in terms of spectral signal to noise ratio at different frequencies are needed to assess their practical limits.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. JEM3200 is our primary instrument for high resolution data collection. It is necessary to have periodic performance test by imaging c film at both different magnifications.

HIV; RNA;

DESCRIPTION (provided by applicant): The technology development cores in our parent grant include the near atomic resolution structural determination of large molecular machines, single particle averaging of subtomograms in cryo-ET of complex assemblies and cells, data integration from the wet lab through 3-D reconstruction and deposition, and exploring the technique of Zenike phase contrast optics. This supplement focuses primarily on Zernike phase contrast imaging, which among all of our cores, shows the greatest potential for revolutionary change in the field. With other new resource, we are also installing a direct electron detection device which has the potential, for the first time, to surpass the quantum efficiency of photographic film. For traditional single particle imaging, per-image efficiency does not fundamentally limit what can be achieved. However, for the applications where Zernike phase contrast is the most effective, per-image efficiency is a major factor. Combining these two technologies, with the addition of an in-column energy filter, offers the potential for tackling previously impossible structural projects at higher resolutions. However, these technologies are far from maturity, and in this supplement, we work to resolve some of the current limiting factors in these technologies. Our aims are: 1) Assessing the limiting factors contributing to the short lifetime of the current Zernike phase plate. 2) Fabricating and testing Zernike phase plates with improved lifetime and high resolution imaging capabilities using the clean room nanofabrication facility at Caltech. 3) Testing the performance specifications of the new direct electron detection device for high quality image collection. 4)Developing new image processing protocols for optimally working with Zernike images recorded on the direct detection device. This technology development is driven by recently established collaborative structural projects including (i) biochemically purified RNA molecules as small as 50 kDa, which are generally considered to be difficult or impossible due to their small size; and (ii) phages at different stages of infection and assembly. These specimens are highly relevant to cancer therapeutics and diagnostics and to viral infection.

Abstract Three-dimensional transmission electron microscopy (3DEM) has become a cutting-edge method for determining structures of large biological assemblies due to recent technical advances. Hundreds of 3DEM experiments are reported in the literature each year and more than 4400 structures are now available in public archives through our EMDataBank.org website. Since 2014, following the cryo-electron microscopy resolution revolution, more than 500 maps with resolutions better than 5.0 Å have been released, most with associated coordinate models. It is critically important that 3DEM maps and the models derived from them are of the highest possible quality, and to achieve this aim it is necessary to have community-accepted validation measures. In our current funding period we developed and promoted new methods and infrastructure to determine map accuracy and resolution, as well as novel methods to build and validate map-derived models. In addition, we hosted challenges and meetings to involve the 3DEM and modeling communities in developing new map and model validation standards. We also significantly expanded the underlying representation for 3DEM experimental methods, and collaborated to fully integrate 3DEM into the wwPDB Deposition and Annotation system. We propose here to continue to develop innovative solutions for validating 3DEM-derived structures of macromolecular assemblies, focusing on the moderate to high resolution range, 5-2 Å. To achieve this we will host new challenges formulated to engage the community using increasingly complex data derived by 3DEM methods and provide statistical analysis of their outcomes. In addition, we will develop and evaluate computational protocols that yield quantifiable parameters for validating 3DEM-derived structures, in terms of density map resolvability, map and model correlation and atom position uncertainty of the associated model. We will also improve the validation criteria for nucleic acids, which occur in 30% of all 3DEM derived models. We will use the EMDataBank website to disseminate the results of our investigations and to provide a platform for community engagement and discussions. Our approach will continue to build on the decades of experience of our team in developing tools for 3DEM map generation and modeling, analyzing large assemblies especially those containing nucleic acids, and working with the community to create standards for structure validation.

? DESCRIPTION (provided by applicant): We propose to acquire a FEI Krios 300 kV transmission electron cryo-microscope equipped with a counting mode electron detector, a phase plate holder, a Gatan post-column energy filter with a K2-XP camera and automated data acquisition software. This facility will be administered by the National Center for Macromolecular Imaging (NCMI), a NIGMS supported Biomedical Technology Resource Center at Baylor College of Medicine. In addition to technology development, one of the NCMI's primary missions is to collaborate with and provide service to researchers who need Cryo-electron microscopy (CryoEM) and Cryo-electron tomography (CryoET) for structure determinations from macromolecules to cells at atomic to nanometer resolutions. We have identified 7 major users and 10 minor users in this proposal who are already active NCMI users both in and outside the Houston Area. This instrument facility is aimed for our users who have projects with one or more of the following objectives: (1) atomic resolution structure determination of molecular machines, (2) single particles structures with conformational and/or compositional heterogeneity, (3) subcellular and macromolecular studies of cells in normal and pathological states. All of these users' projects require high throughput and high quality data which will lead to 3-D structures yielding new structural insights of molecular and cellular components and biological processes related to cancer, infectious and neurological diseases. Due to the large quantity of data needed for each of these projects, we will manage our facility similar to an X-ray synchrotron beamline to provide 24 hours per day and 6 days per week (24/6) operation. None of the existing NCMI electron microscopes can deliver comparable data quality with the high throughput needed by the current state of art of CryoEM/ET projects. The actual microscope scheduling will be made quarterly by considering users' preference and the microscope availability. The proposed instrument will be maintained and operated by two NCMI staff who will assist users with data collection. We will provide regular training courses to users and their lab members to enable them to collect data with minimal assistance from our staff. We plan to make the proposed instrument available to other NCMI users after the first 18 months of operation. Then, an external user project evaluation committee made of CryoEM/ET experts appointed by the PI will evaluate and rank the proposals submitted by existing and new users. Two advisory committees will be set up to monitor the operation and productivity of the proposed instrument. One local committee will be appointed by the Senior Vice President of Research at Baylor College of Medicine; while an external committee is set up by the PI as mandated by the NIGMS supported P41 Center. The total cost of the instrument is ~$6 million. Baylor College of Medicine is committed to provide matching funds of $4 million towards the purchase of the instrument and the cost for the laboratory renovation. We have the adequate NIH support for the operation of this proposed instrument via the existing P41 Center grant funding the NCMI, now in its 30th year, extending at least until January 1, 2020. We expect to seek competitive renewal of this grant to continue the operation of the proposed facility beyond 2020. Should this prove unsuccessful, we plan for the proposed instrument to become a Core Lab under the management of the Advanced Technology Core Laboratories (ATCL) at Baylor College of Medicine, which will provide half of the instrument operational costs (service contract, personnel and supplies) and use established ATCL cost recharge mechanisms to recover the remaining operational costs.

Project Summary/Abstract Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the Huntingtin (HTT) protein. A hallmark of HD found in the brains of patients is the presence of intracellular deposits containing the N- terminal fragment of mutant HTT (mHTT) with an expanded poly-glutamine sequence. Mutant HTT is prone to misfold and form aberrant species, including oligomers, fibrils and bundles of fibrils; one or more of these species can be toxic, particularly to neurons. Mutant HTT species is modulated by the protein homeostasis (or proteostasis) status of the cell. The 1 MDa, ring-shaped hetero-oligomeric chaperonin TRiC/CCT, essential for proteostasis, has emerged as a central regulator of mHTT aggregation and toxicity. TRiC is a chaperone that. Our preliminary data showed that TRiC binds to wild type (WT) and mHTT monomers, oligomers and fibers suppressing both aggregation and toxicity. Further, we find that overexpression of TRiC or small TRiC-derived domains remodels mHTT aggregates in vivo and in vitro and can protect from mHTT toxicity in HD models. We hypothesize that understanding the action of TRiC on mHTT can provide a therapeutic avenue for HD treatment. This Project will provide a deeper mechanistic and structural understanding of how TRiC and TRiC- derived domains (hereafter TRiC reagents) interact with mHTT species at the different stages of the aggregation pathway, including monomers, oligomers and fibrils, and how they influence the misfolding and aggregation landscape of mHTT. Aim 1 will use biochemical, functional and cryo-electron tomographic analyses to characterize mHTT aberrant species and their interactions with TRiC reagents using recombinant proteins. Aim 2 will examine the effects of TRiC reagents on the formation and remodeling of mHTT aberrant species in the cellular context, using neuronal lysates from HD cell models, including primary neurons from R6/2 and BACHD mice. Aim 3 will use structural and biochemical assays to examine how mHTT expression affects selected subcellular components such as axonal microtubules and mitochondria, and TRiC proteostasis in HD cell models with and without TRiC reagent treatment. We will establish a scoring system indicative of the effects of the presence of TRiC reagents on our biochemical and structural assays of mHTT species both ex vivo and in vitro. Our experiments are highly synergistic with Projects 2 and 3, which assess TRiC's role in ameliorating pathogenesis in HD neuronal and HD animal models. The results from this Project will provide a conceptual framework for these more physiological and translational studies by defining new, optimized TRiC reagents and which cellular loci and processes to examine, in preparation for engaging in studies that could establish proof-of-concept TRiC-based therapeutics for Huntington's disease.

? DESCRIPTION (provided by applicant): The National Center for Macromolecular Imaging (NCMI) at Baylor College of Medicine is the host laboratory offering to provide a portion of the time on their JEM3200FSC and JEM2200FS electron microscopes for a cryo-electron microscopy (cryoEM) and tomography (cryoET) data acquisition facility Consortium. Both instruments are equipped with a field emission gun and an in-column energy filter, while the JEM2200FS also has a Zernike phase plate attachment. Both instruments are currently equipped with DE20 and DE12 cameras operated in integrating mode. These microscopes have been used productively for a variety of biological specimens, including macromolecules, molecular machines and cells, at state-of-the-art resolution. Our facilities will be allocated for he proposed Consortium while taking into account the ongoing research in the host institution. Our lead PI, Wah Chiu is a recognized expert in pushing cryoEM and cryoET beyond current boundaries and has decades of experience in leading several NIH funded research centers and academic training programs. There are 11 participating institutions across the USA, led by investigators who have a research track record in cryoEM and/or cryoET. Day-to-day operation will be carried out by a part-time cryoEM/ET scientist and IT staff. Both in person and remote data collection protocols will be used for distant users. The Consortium will provide travel funds for the distant users. We request funding for a Direct Detection Device (DDD) operated in counting mode to meet the needs of all types of specimens. The host Institution will provide an administrative assistant to support user visits and various Consortium activities, and matching funds to purchase the DDD and adequate hard drives for short-term data storage. The participating PIs will be involved in formulating the specific policies for Consortium governance and developing training activities. Scheduling will be allocated evenly in the first year among participants with some flexibility based on specimen readiness. Allocation of microscope times and priorities will be assigned in the second year and beyond based on recommendations of an external microscope allocation committee appointed by all the PIs to avoid conflict of interest. Al PIs will meet quarterly on WebEx to discuss technical and administrative issues, and we will hold an annual user meeting. An annual review of all aspects of the Consortium operation and the data outcomes of the users will be conducted by an advisory committee to assure the highest productivity and usage of the proposed facility. The membership of the participating institutions will be dynamically reviewed by all the PIs with the inputs from the external advisory committee after the second year of our operation.

? DESCRIPTION (provided by applicant): The National Center for Macromolecular Imaging (NCMI) at Baylor College of Medicine is the host laboratory offering to provide a portion of the time on their JEM3200FSC and JEM2200FS electron microscopes for a cryo-electron microscopy (cryoEM) and tomography (cryoET) data acquisition facility Consortium. Both instruments are equipped with a field emission gun and an in-column energy filter, while the JEM2200FS also has a Zernike phase plate attachment. Both instruments are currently equipped with DE20 and DE12 cameras operated in integrating mode. These microscopes have been used productively for a variety of biological specimens, including macromolecules, molecular machines and cells, at state-of-the-art resolution. Our facilities will be allocated for he proposed Consortium while taking into account the ongoing research in the host institution. Our lead PI, Wah Chiu is a recognized expert in pushing cryoEM and cryoET beyond current boundaries and has decades of experience in leading several NIH funded research centers and academic training programs. There are 11 participating institutions across the USA, led by investigators who have a research track record in cryoEM and/or cryoET. Day-to-day operation will be carried out by a part-time cryoEM/ET scientist and IT staff. Both in person and remote data collection protocols will be used for distant users. The Consortium will provide travel funds for the distant users. We request funding for a Direct Detection Device (DDD) operated in counting mode to meet the needs of all types of specimens. The host Institution will provide an administrative assistant to support user visits and various Consortium activities, and matching funds to purchase the DDD and adequate hard drives for short-term data storage. The participating PIs will be involved in formulating the specific policies for Consortium governance and developing training activities. Scheduling will be allocated evenly in the first year among participants with some flexibility based on specimen readiness. Allocation of microscope times and priorities will be assigned in the second year and beyond based on recommendations of an external microscope allocation committee appointed by all the PIs to avoid conflict of interest. Al PIs will meet quarterly on WebEx to discuss technical and administrative issues, and we will hold an annual user meeting. An annual review of all aspects of the Consortium operation and the data outcomes of the users will be conducted by an advisory committee to assure the highest productivity and usage of the proposed facility. The membership of the participating institutions will be dynamically reviewed by all the PIs with the inputs from the external advisory committee after the second year of our operation.

Core 3: CryoEM Summary Since the CRNA?s inception, cryo-electron microscopy (cryoEM) has been a key structural biology technology provided through the CryoEM Core. During CRNA 1.0 we made many advances in developing cryoEM procedures for studying the structures of small RNAs, initiated CryoEM projects with several CRNA investigators, and held cryoEM workshops to inspire CRNA members and others to use this powerful structure technique. Our most notable achievements are reaching a major technical milestone in determining a subnanometer resolution cryoEM structure of the 30 kDa HIV-1 dimerization initiation site (DIS) RNA dimer and revising the current Rev response element (RRE) structure with rigorous cryoEM structure validation protocols. These studies were done in collaboration with multiple CRNA groups, including Marchant, Summers and Case, to integrate structural information from cryoEM, NMR and molecular dynamics. The DIS structure represents the smallest macromolecule structure determined to date by cryoEM. Moving into CRNA 2.0, we have extended our team to include investigators with complementary expertise from Baylor College of Medicine, the University of Virginia and NIH. We have set three aims: (i) to utilize state-of-the-art cryoEM as a tool for determining the highest possible resolution structures of RNAs, RNA-protein complexes and HIV- related protein complexes, in collaboration with investigators from the CRNA projects and throughout the CRNA; (ii) to develop new experimental and computational protocols in cryoEM for characterizing challenging specimens of RNA and RNA-protein complexes, which are either small, compositionally or conformationally variable, or both; and (iii) to integrate measurements from NMR, X-ray crystallography, cryoEM, molecular dynamics simulation, and biochemistry for partial or intact biological complexes relevant to the CRNA research missions in order to understand their structure and function. In addition, we will continue to offer systematic, hands-on cryoEM training to CRNA students and postdoctoral fellows so that they will become more versatile structural biologists in the coming decades.

Summary  Stanford University and the SLAC National Accelerator Laboratory (SLAC) propose to host a National Center  for Cryoelectron Microscopy ? The Stanford-­SLAC CryoEM Center (S2C2) ? to meet the emerging national  need for cryoEM as a tool for atomic-­resolution structural biology. The proposed S2C2 will (1) establish, and  keep at the forefront, state-­of-­the-­art cryo-­electron microscopes to satisfy users? needs for atomic-­resolution  image data, (2) archive image data with appropriate metadata, (3) provide computing resources to assess data  quality in real time, (4) inform and recruit potential users across the U.S. about the public accessibility of the  Center, (5) establish an open, fair, transparent and efficient process to select user proposals based on  scientific impact and specimen readiness, regardless of users? geographic locations or affiliations, (6) help  users to overcome technical hurdles and enable them to obtain high-­resolution cryoEM structures quickly with  a rigorous validation protocol, (7) form and facilitate a user network to exchange information seamlessly, (8)  train users to become independent cryoEM investigators, (9) integrate user and trainee feedback into a  continuous loop of facility enhancement, and (10) optimize operations to achieve the above tasks effectively  and efficiently. Dr. Wah Chiu, the Contact PI, has decades of experience developing high-­resolution cryoEM  technologies and directing research and training enterprises across multiple institutions. Drs. Michael Schmid,  Georgios Skiniotis , and Britt Hedman, also PIs of the Center, bring complementary expertise in cryoEM,  structural biology, and management and operation of large-­scale national user facilities. A Center Director of  Operations, three skilled cryoEM specialists, a technical assistant, and a user facility administrator will carry  out the S2C2?s day-­to-­day operations, including data collection, quality assessment, and cross-­training. This  S2C2 will reside in a new building at SLAC. It will leverage SLAC?s expertise in serving thousands of users each  year at its renowned X-­ray synchrotron and free electron laser user facilities. An Advisory Committee  comprised of external expert scientists as well as user representatives will provide advice regarding S2C2  operations and development. Our Center will build on the unique strengths and support from Stanford and  SLAC, and will create a unique environment that combines high throughput cryoEM data generation with  training the next generation of cryoEM scientists. 

Project Summary Stanford University and the SLAC National Accelerator Laboratory (SLAC) propose to host a National Center for Cryoelectron Microscopy ? The Stanford-SLAC CryoEM Center (S2C2) ? to meet the emerging national need for cryoEM as a tool for atomic-resolution structural biology. The proposed S2C2 will (1) establish and keep at the forefront, state-of-the-art cryo-electron microscopes to satisfy users? needs for atomic-resolution image data, (2) archive image data with appropriate metadata, (3) provide computing resources to assess data quality in real time, (4) inform and recruit potential users across the U.S. about the public accessibility of the Center, (5) establish an open, fair, transparent and efficient process to select user proposals based on scientific impact and specimen readiness, regardless of users? geographic locations or affiliations, (6) help users to overcome technical hurdles and enable them to obtain high-resolution cryoEM structures quickly with a rigorous validation protocol, (7) form and facilitate a user network to exchange information seamlessly, (8) train users to become independent cryoEM investigators, (9) integrate user and trainee feedback into a continuous loop of facility enhancement, and (10) optimize operations to achieve the above tasks effectively and efficiently. Dr. Wah Chiu, the Contact PI, has decades of experience developing high-resolution cryoEM technologies and directing research and training enterprises across multiple institutions. Drs. Michael Schmid, Georgios Skiniotis, and Britt Hedman, also PIs of the Center, bring complementary expertise in cryoEM, structural biology, and management and operation of large-scale national user facilities. A Center Director of Operations, three skilled cryoEM specialists, a technical assistant, and a user facility administrator will carry out the S2C2?s day-to-day operations, including data collection, quality assessment, and cross-training. This S2C2 will reside in a new building at SLAC. It will leverage SLAC?s expertise in serving thousands of users each year at its renowned X-ray synchrotron and free electron laser user facilities. An Advisory Committee comprised of external expert scientists as well as user representatives will provide advice regarding S2C2 operations and development. Our Center will build on the unique strengths and support from Stanford and SLAC, and will create a unique environment that combines high throughput cryoEM data generation with training the next generation of cryoEM scientists.

Herpesviruses are pathogens of medical and economic significance that cause a range of diseases in humans and animals. Varicella-zoster virus (VZV) is an important human alpha herpesvirus that causes varicella and zoster after reactivation from latency in sensory ganglia. The morphogenesis of varicella-zoster virus (VZV), like all herpesviruses, involves egress of DNA-containing capsids from the nucleus to the trans- Golgi network for secondary envelopment by viral glycoprotein-enriched membranes followed by transport in intracellular vesicles to the cell surface. Although purified herpesvirus structures have been described, much less is known at the structural level about virus particle morphology within infected cells and this dynamic process, which takes place at different spatial locations and temporal order. Cryogenic electron tomography (cryo-ET) has the promise to uncover 3D structures of assembly intermediates, providing structural details of each molecular component of the virion during this dynamic process. Recent advances in cryo-specimen preparation, data collection strategy, electron optics, electron detector and data processing methods make this type of study tractable. First, we will characterize the structure of VZV complete or light (L; lacking capsids) particles at the cell surface (Aim 1), based on preliminary data showing the feasibility of visualizing these particles by cryo-ET. The cryo-ET dataset will be used to derive capsid structures as a benchmark in the initial protocol development to define the attainable resolution of our data collection and image processing strategy. Next, we aim to determine the structures of the spike densities visible in our dataset, at the resolution attained for capsids. These analyses will put the glycoprotein structures in context to define their distribution, interaction with each other and possibly, structural rearrangement upon interacting with the cell surface. Second, we will characterize the structure of VZV particles inside infected cells (Aim 2). Our well- characterized VZV recombinant expressing the ORF23 capsid protein tagged with RFP will be used for correlative cryo-fluorescence confocal microscopy of vitrified cells with our new cryo-FIB/SEM instrument to prepare thin lamellae of VZV infected cells at sites of an RFP signal in the nucleus or cytoplasm. Milled lamellae will be used for cryo-ET and sub tomogram averaging to generate structures of viral and associated cellular components. This approach will be used to derive the structure of VZV capsids and associated proteins at intracellular sites, to be followed by generating de novo structures of glycoproteins on VZV particles at intracellular sites. This work will address gaps in structural knowledge of herpesvirus morphogenesis within infected cells, using VZV as a model, and advance the application of cryo-ET techniques and data analytics to the study of virus-host cell interactions, which has broad relevance including for SARS-CoV-2, and the molecular biology of human cells. These structure discoveries have the potential to inspire the development of novel drug or prophylactic strategies for the human herpesviruses.

In response to the NIH RFA-RM-19-009, we propose to establish the Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC) to accomplish four specific aims: (1) establish a platform, which is available to the scientific community at large, to streamline the preparation of samples suitable for downstream cryogenic electron tomography (cryoET) data collection; (2) provide access to advanced cryo-specimen preparation techniques for a wide range of samples: macromolecular complexes, microcrystals of biological materials, cell lysates, organelles, cells and tissues; (3) generate a training curriculum for new users on the preparation of frozen, hydrated biological specimens, with the option of carrying out correlative cryo-fluorescence light microscopy (cryoFLM), cryo-focused ion beam scanning electron microscopy (cryoFIB), and cryoET; and (4) adopt new methods for sample preparation using innovative technologies developed elsewhere. In this proposal, we will leverage our existing facilities and in-house expertise in each of the following imaging modalities and their integration into a correlated sample preparation workflow: cryoFLM, cryoFIB, and cryoET. We will establish all the necessary equipment dedicated to the proposed service and training activities. We will prepare video curriculum materials to train new users in the aforementioned protocols. We will adapt our existing infrastructure to manage project administration and resource allocations. Administrative support is in place to help users with lodging arrangements, as well as laboratory on-boarding and specimen biosafety approvals. We have a well set- up communication infrastructure for remote users to participate in actual experiments, particularly for cryoFLM, cryoFIB, and cryoET. We will provide access to existing Talos Arctica and/or Titan Krios microscopes to evaluate whether the prepared samples are ready for data collection in the associated Hub, which will be set up at another institution with separate NIH support. We will make our existing Stanford-SLAC cryo-specimen preparation equipment available on a limited basis immediately after this proposal is funded. We anticipate serving ~40 users per year once the Center is fully operational in early Year 2 of the award period. We will also offer regular hands- on workshops to train ~10-12 new users per year. We will disseminate our resources to the broad community via a web portal and booths at professional societies? annual conferences. The number of users will increase in subsequent years as we and our recurring users optimize the use of the Center?s resources. In Years 3-6, we will work closely with collaborators pioneering advanced protocols such as semi-automated and automated cryoFIB and ?lift-out? for tissue samples to implement them in the proposed Center. Lastly, we will assemble a Scientific Advisory Committee of experts to guide the practices of our Service Center and the ongoing implementation of cutting-edge technologies.