Phoebe Stewart - US grants

This proposal is for matching funds to purchase a new cryo-electron microscope equipped with a slow-scan CCD camera. This system will allow direct acquisition of digital images suitable for computer image reconstruction and structural analysis of macromolecular complexes. Although the UCLA campus has several electron microscopes, none are suitable for state-of-the-art cryo techniques. Each of the major users will contribute different areas of expertise to the shared facility, including cryo sample preparation, classical electron microscopy, site-specific antibody and gold labeling techniques, two-dimensional crystal growth, and three-dimensional image reconstruction. Proposed research projects involve a diverse array of biological assemblies: human adenovirus particles with bound cellular receptors; retroviral capsid protein assemblies; the spliceosome, a complex particle involved in mRNA processing; the vault organelle, a ubiquitous cytosolic ribonucleoprotein particle; cell-to-cell channels, which directly connect the cytoplasms of adjacent cells; and GABAA receptors. The proposed facility will complement the existing strengths at UCLA in imaging and biological structure. The Nuclear Medicine Division has leaders in the fields of PET scanning and medical image reconstruction. The Molecular Biology Institute has several renown x-ray crystallographers. One goal of the proposed facility is to bridge the resolution gap between x-ray crystallography and electron microscopy by combining atomic structural information of component molecules with image Reconstructions of macromolecular complexes. The Department of Molecular and Medical Pharmacology will provide suitable renovated space for the cryo-electron microscope and funds for half of the instrument's cost. Sufficient computational power for image processing already exists at UCLA. Additional computer graphics workstations will be purchased from the Principal Investigator' s start-up funds. After the initial one ye ar warranty period, the ongoing maintenance and operating expenses for the cryo-electron microscope and CCD cameras will be covered jointly by the major users' grants and departmental funds. The administration of the proposed cryo-electron microscopy facility will be carried out by an inter-departmental committee at UCLA. Dr. Nigel Unwin, a recognized leader in the field, has agreed to serve as an external consultant and provide both technical advice and overall scientific guidance.

This proposal is for funds to purchase a new cryo-electron microscope equipped with a slow-scan CCD camera. This system will allow direct acquisition of digital images suitable for computer image reconstruction and structural analysis of macromolecular complexes. Although the UCLA campus has several electron microscopes, most are more than seven years old and none are suitable for state-of-the-art cryo techniques. Each of the major users will contribute different areas of expertise to the shared facility, including cryo sample preparation, classical electron microscopy, site-specific antibody and gold labeling techniques, two- dimensional crystal growth, and three-dimensional image reconstruction. Proposed research projects involve a diverse array of biological assemblies: human adenovirus particles with bound cellular receptors; retroviral capsid protein assemblies; the spliceosome, a complex particle involved in mRNA processing; the vault organelle, a ubiquitous cytosolic ribonucleoprotein particle; cell-to-cell channels, which directly connect the cytoplasms of adjacent cells; and GABAA receptors. The proposed facility will complement the existing strengths at UCLA in imaging and biological structure. The Nuclear Medicine Division has leaders in the fields of PET scanning and medical image reconstruction. The Molecular Biology Institute has several renown x-ray crystallographers. One goal of the proposed facility is to bridge the resolution gap between x-ray crystallography and electron microscopy by combining atomic structural information of component molecules with image reconstructions of macromolecular complexes. The Department of Molecular and Medical Pharmacology will provide suitable renovated space for the cryo-electron microscope. Sufficient computational power for image processing already exists at UCLA. Additional computer graphics workstations will be purchased from the Principal Investigator's start-up funds. After the initial one year warranty period, the ongoing maintenance and operating expenses for the cryo-electron microscope and CCD cameras will be covered jointly by the major users' grants and departmental funds. The administration of the proposed cryo-electron microscopy facility will be carried out by an inter-departmental committee at UCLA. Dr. Nigel Unwin, a recognized leader in the field, has agreed to serve as an external consultant and provide both technical advice and overall scientific guidance.

DESCRIPTION: Cryo-electron microscopy (cryo-EM) combined with three-dimensional image reconstruction techniques and newly developed time-resolved methods provide a powerful way to examine virus-host cell interactions as well as the dynamic structural changes that occur during viral cell entry. In fact these methods often represent the only feasible structural approach for large (>100 million Dalton) macromolecular complexes and virus/receptor complexes with highly mobile receptor binding sites, as has been postulated for adenovirus. These proposed studies will elucidate the structural events during adenovirus/receptor binding at thle cell surface, intemalization into coatedpits, and escape ofthe virus particle from the endosome. Specific aims include imaging the adenovirus particle complexed with a Fab fragment of a monoclonal antibody that blocks cell entry in order to localize the Arg-Gly-Asp integrin binding site on the penton base viral component. In addition, the structure of the virus complexed with a soluble form of the internalization receptor, the integrins avb3 and avb5, will be solved in order to evaluate possible structural changes that occur upon receptor binding. We will also examine conformational changes in the virus and virus/receptor complexes as a consequence of varying the pH value to mimic both the extracellular (pH 8) and early endosomal (pH 6.0 - 6.5) environments. Rapid freezing (within milliseconds) after low pH treatment allows the trapping of structural intermediates. A further objective involves calculating an image reconstruction of the adenovirus temperature sensitive mutant, tsl, which is defective for cell entry. The structural differences between the tsl mutant and wild type will be analyzed to isolate elements essential for productive adenoviral cell entry. These cryo-EM structural studies will provide a greater understanding of the cell entry process of adenovirus, a promising vector for in vivo gene therapy.

9722353 Stewart Cryo-electron microscopy (cryo-EM) combined with three-dimensional image reconstruction techniques provides a powerful way to examine the structure of large macromolecular assemblies. Specific research objectives include cryo-EM studies of the ribonucleoprotein vault particle and alpha-crystallin, a molecular chaperone. Vault studies will include a three-dimensional reconstruction of the intact particle, difference imaging analysis with ribonuclease treated particles to reveal the location of the internal RNA, and structural mapping using truncated forms of the major vault protein. Alpha-crystallin studies will include reconstruction of native and cross-linked recombinant human (B crystallin multimers and (B crystallin complexed with lactalbumin, a large target protein. Specific educational objectives include: mentoring graduate students and postdoctoral fellows, strengthening multi-departmental graduate education at UCLA, and volunteering in science outreach programs for minority and high school students. Cryo-EM often represents the only feasible structural approach for large (1-400 million Dalton) complexes if the sample has proven resistant to crystallization, as have both the vault and alpha-crystallin. Thus these studies will enable structure/function analysis of a ribonucleoprotein particle conserved across species as diverse as humans and the slime mold Dictyostelium, and provide insight into the quaternary structure of alpha-crystallin and its mechanism of action in binding partially denatured proteins. Integration of the research and educational goals will be accomplished by teaching novel structural methods such as cryo-EM and image reconstruction to upper level undergraduates and graduate students, as well as mentoring graduate students in the laboratory. ***

The goal of this project is to examine the functional organization of human primary motor cortex controlling hand and arm movements. By using multiple imaging modalities we will determine the relationship between activations in primary Motor cortex. as measured by functional magnetic resonance imaging (fMRI) and optical intrinsic signaling (OIS), and the motor map obtained by cortical stimulation. By studying a group of patients undergoing awake craniotomy we will have a unique opportunity to validate non-invasive mapping techniques with direct cortical measurements. This will serve to establish the role of fMRI in presurgical mapping and lay the foundation for studies of recovery of human motor function in neurological disease. Specific Aims Hypothesis 1: Human primary motor cortex controlling arm and hand movement is characterized by functionally related overlapping regions, which will be demonstrable by fMR.1 and optical intrinsic signaling. Hypothesis 2: Discrepancies between the maps of primary motor cortex obtained by direct cortical stimulation, fMR1 and OIS will give rise to a better understanding of the physiologic properties underlying these signals and ultimately to better localization of cortical function in surgical patients. Hypothesis 3: Primary motor cortex is activated during imagined movements.

DESCRIPTION (provided by applicant): This proposal is for funds to purchase a 300kV FEC liquid helium cryo-electron microscope for single particle analysis. Cryo-electron microscopy (cryo-EM) has emerged as a powerful approach for visualizing the structures of macromolecular assemblies. Two technological advances, the field emission gun (FEG) electron source and a liquid helium-cooled specimen stage, provide the potential of atomic, or near atomic, resolution. Several ongoing NIH-funded biomedical research projects will benefit greatly from access to a liquid helium cryo-microscope. These include imaging of adenovirus vectors and adenovirus-receptor complexes; DNA-dependent protein kinase (DNA-PK) DNA double-strand break repair complexes; and wild-type and knock-out mouse vaults and recombinant vault-like particles. The vault is a ubiquitous and highly conserved cellular component that has a role in multidrug resistance in cancer cells. The new microscope will ultimately be housed in the California NanoSystems Institute (CSNI) Court of Sciences building at UCLA, which is in the architectural planning phase and is scheduled for completion in 2004. A temporary location for the requested microscope has been identified and evaluated by the FEI Company in the Crump Institute for Molecular Imaging at UCLA, adjacent to UCLA's current 120kV liquid nitrogen cryo-electron microscope. The CNSI seeks to foster interdisciplinary interactions between researchers in the UCLA School of Medicine, College of Letters and Science, School of Engineering, and between UCLA and UC Santa Barbara. Imaging at the molecular, cellular, and organismal levels is an important facet of the CNSI. The CNSI is one of three research efforts selected to receive $100 million in support from the state of California. Nearly 30 corporate partners have already pledged support for the CNSI. If the liquid helium cryo-microscope is funded, CNSI will seek a corporate partner, such as a pharmaceutical company, to help fund the long term operating costs of the microscope. The financial plan for the long term operation of the microscope includes a significant additional commitment from UCLA for microscope operating expenses.

A Vanderbilt University-led collaboratory proposes to develop a Research and Education Data Depot Network (REDDnet) in response to the need for a shared, wide-area storage infrastructure where data flows can be buffered for collaborative processing, including analysis, reduction, visualization, exploration. The general "data logistics" problem arises when quantities of scientific or other data become large, at the level of 1TB or more per day. High performance networks help users move their data faster, but users routinely have nowhere to put their terabyte down to work on it or share it with someone. REDDnet will consist of a set of eight large storage nodes, strategically positioned across the nation's high performance research networks, and configured with a distributed storage management technology, called Logistical Networking (LN), expressly designed to attack major problems of data logistics. With more than 320 TB of projected capacity, it will be used by a diver set of researchers and educators with large datasets to manage and an established need for distributed collaboration. The initial group of REDDnet users includes two "petascale" particle physics experiments with approximately 5000 collaborators world-wide, (ATLAS and CMS), a nationwide community (AmericaView) mining information from satellite-based sensors, a consortium of eight universities and Oak Ridge National Laboratory doing large scale simulations of supernovas (the Terascale Supernova Initiative), and a collaborative effort between a Vanderbilt and Lawrence Berkeley National Laboratory investigating the structure of biological molecules (Image Reconstruction of Large Macromolecular Assemblies). The two main organizational collaborators in REDDnet, Vanderbilt's Advanced Computing Center for Research and Education (ACCRE) and AmericaView (AV), have educational missions and ongoing activities that are well positioned to exploit the significant potential of REDDnet for education and outreach.

Circadian clocks are self-sustained biochemical oscillators that underlie daily rhythms of sleep/waking, metabolic activity, gene expression, and many other biological processes. Their properties include temperature compensation, a time constant of approximately 24 hours, and high precision. These properties are difficult to explain by known biochemical reactions. The ultimate explanation for the mechanism of these unusual oscillators will require characterizing the structures, functions, and interactions of the molecular components of circadian clocks. The simplest cells that are known to exhibit circadian phenomena are the prokaryotic cyanobacteria. Genetic and biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC in the cyanobacterium Synechococcus elongatus. These three proteins plus ATP are competent to reconstitute a phosphorylation / dephosphorylation cycle in vitro that parallels the 24 hour cycles observed for global gene regulation in vivo. This in vitro circadian oscillator is the best available system for structural and biophysical analyses of a circadian clockwork. Preliminary electron microscopy (EM) data suggests that numerous and large conformational changes occur within the KaiA-KaiB-KaiC molecular oscillator, thus three-dimensional EM is well suited for structural analysis of this system. The specific aims of this proposal are 1) perform a cryoEM evaluation of three forms of the KaiB/KaiC complex with mutated forms of KaiC, and 2) determine a subnanometer resolution (<10[unreadable]) cryoEM structure of the chosen KaiB/KaiC complex. Characterizing the molecular mechanisms that allow a circadian clockwork to oscillate with a 24 hour cycle is essential for understanding circadian rhythms in cyanobacteria to humans. Detailed structural analysis of the core oscillator from cyanobacteria will provide the mechanisms underlying the controlled protein-protein associations that appear to drive this unique clockwork. The long-term goal of this proposal is to apply hybrid methods including three-dimensional EM to characterize the structure and function of supramolecular protein complexes formed during the cyanobacterial circadian oscillation cycle.

DESCRIPTION (provided by applicant): Nonhomologous end joining (NHEJ) serves as the primary pathway for repairing DNA double-strand breaks (DSBs) in humans. Repairing DNA damage that occurs from oxidative damage and exposure to ionizing radiation is vital for genetic stability and for suppression of oncogenesis. NHEJ is also essential for V-D-J recombination in lymphocytes, which generates a functional adaptive immune system. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates repair the NHEJ pathway along with other key components Ku and Artemis. The Ku70/80 heterodimer is the first protein to recognize and bind DNA ends at double strand breaks and recruits DNA-PKcs to the damage sites. Artemis in complex with DNA-PKcs performs the endonucleolytic activity necessary for the hairpin-opening step of V-D-J recombination and DNA end processing in NHEJ. Mutations in any of these three components results in radiosensitivity and severe combined immunodeficiency in humans. The lack of high resolution structural information on DNA- PKcs and NHEJ complexes has prevented a mechanistic understanding of their critical DNA repair activity and regulation. CryoEM single particle image reconstruction is well suited for studying DNA-PKcs and large NHEJ complexes. The specific aims of this proposal are to determine subnanometer (<10E) resolution cryoEM structures of DNA-PKcs/Artemis/DNA, DNA-PKcs/Artemis, DNA-PKcs/dsDNA, and DNA- PKcs/Ku/DNA complexes, as well as perform an atomic level structural analysis of these NHEJ complexes with emerging tools from the protein structure prediction field. The structural analysis will include docking of available atomic resolution structures and comparative models, as well as application of hybrid cryoEM de novo protein structure prediction methods. These studies will be highly complementary to ongoing biochemical, genetics, and x-ray crystallographic studies. Detailed knowledge of the molecular geometry of these complexes will provide insight into the kinase activation and endonuclease phases of NHEJ and will enable generation of testable hypotheses on molecular mechanisms underlying DNA break repair by the NHEJ pathway. Ultimately our ability to therapeutically treat cancer and immunodeficiency diseases will be enhanced by a molecular understanding of the underlying biological processes that are improperly regulated in the disease state. PUBLIC HEALTH RELEVANCE: The proposed studies are biomedically relevant in that structural information on NHEJ complexes will help to answer key questions on how these complexes assemble at DNA damage sites, how the repair and recombination processes are guided, and what triggers the choice between multiple parallel pathways and outcomes. Ultimately this information will be helpful in understanding and treating cancer, severe combined immune deficiency (SCID), and sensitivity to ionizing radiation (RS-SCID).

DESCRIPTION (provided by applicant): Adenovirus vectors (Ad) are the second most frequently used vectors in clinical trials in the US to treat numerous inborn and acquired human diseases, including cancer. Although no cure so far is found for disseminated metastatic tumor disease, it is currently accepted that disseminated metastases can potentially be treated through a systemic delivery routes, such as vasculature, to allow for access to all body sites were metastatic tumors may reside. However, upon using this route to achieve systemic adenovirus delivery, over 90% of the administered vector dose is rapidly sequestered by the liver, leading to virus inactivation, reducing the efficacy of extra-hepatic gene transfer, and triggering systemic innate immune and inflammatory responses. Although the in vitro-derived model of Ad cell infection postulates key roles for Ad fiber and penton proteins in mediating virus entry into cells, our in vivo analyses demonstrate that after intravascular delivery, the major Ad capsid protein - hexon - plays the principal mechanistic role in driving virus sequestration in the liver and hepatocyte transduction. Importantly, our preliminary studies strongly suggest that specific interactions of circulating antibodies with solvent-exposed hyper-variable hexon loops mechanistically define virus interaction with Kupffer cells, leading to virus trapping in the liver and inactivation. Furthermore, our preliminary studies also demonstrated that only simultaneous inactivation of adenovirus interactions with hepatocytes, sinusoid endothelial cells, and Kupffer cells allows for virus escape from being sequestered in the liver after intravascular delivery. Although Ad vectors that are attenuated at either hepatocyte transduction or interaction with Kupffer cells have been described, to date, there are no studies published that provide direct and definitive evidence that such vectors escape liver sequestration shortly after intravascular injection. Based on the novel concept of equifunctional role of different hepatocellular compartments in sequestering Ad from the blood, in this proposal we will fill the gap in our knowledge of the role of Ad hexon in guiding virus bio- distribution an infectivity after intravascular delivery. Through a combination of structural cryo-electron- microscopy (cryo-EM) and computational methods of analysis and site-directed mutagenesis, in this proposal we will 1) determine the surface regions of adenovirus hexon that interact with low affinity natural antibodies (IgM) and high affinity mouse and human antibodies (IgG). We will also 2) determine the role of the hexon HVR1 loop variation in virus infection, replication, and Kupffer cell trapping. Finally, using a set of unique vectors with modified pentons and hexons, we will 3) develop novel hexon-mutated viruses that will avoid Kupffer cell trapping and resist neutralization with virus-specific antibodies after intravascular delivery. Our hypothesis and data-driven studies proposed in this application will greatly advance our understanding of Ad hexon - host cell and factor interactions in vivo and should ultimately lead to the experimental validation of novel strategies to prevent Ad sequestration from the blood. Conceptual and experimental validation of these strategies would represent a major step toward the development of safe and effective systemically-applicable Ad vectors for numerous therapeutic applications in humans.

Abstract The Southeastern Center for Microscopy of MacroMolecular Machines (SECM4) is a consortium of 15 Universities/Medical Centers with a total of 19 investigators throughout the Eastern United States studying a wide range of important biomedical projects as variable as high resolution virus structure, membrane protein structure, macromolecular complexes of various types, some isolated in active form from cells, bacterial ultrastructure, muscle filaments, spliceosomes, ribosomes complexes all of which will benefit from ready access to a high resolution electron microscope such as a Titan Krios equipped with a direct electron detector (DED). Human health implications extend from virus and bacterial pathogens to the understanding of diseases resulting form genetic mutations. The basic biology of cancer and heart disease is being studied in several member laboratories. The Titan Krios at Florida State University has been in operation since 2009 and recently has had its image recording device upgraded from CCD camera to a Direct Electron LLC, DE-20 direct electron detector positioned ahead of an existing imaging filter which removes inelastically scattered electrons thereby improving the image quality. Although we propose a robust plan to enable members to come to Florida State University, we propose creating a facility based on the synchrotron template currently in use at multiple sites X-ray crystallography beam lines around the country whereby users ship specimens to us and watch the data being collected as it comes off the microscope from the familiar confines of their own laboratories. We will provide sufficient preprocessing that consortium members can evaluate the prospects for obtaining a final high resolution structure from damage and motion corrected ?movie? images of their samples. The result will be a model for high throughput structure determination utilizing high-end instrumentation that can reveal the inner workings of complex macromolecules and subcellular structures.

? DESCRIPTION (provided by applicant): This application seeks funding to establish the West/Midwest Consortium for High-Resolution Cryo Electron Microscopy (cryoEM). The goal of the consortium is to offer 19 cryoEM users from 10 regional institutes free access to a high-end cryoEM facility with proven high-resolution capabilities located in the California NanoSystems Institute (CNSI) at University of California, Los Angeles (UCLA). UCLA will act as host institute and provide investigators in these cryoEM laboratories access to its highly productive Titan Krios cryo electron microscope recently upgraded with a Volta phase plate, a Gatan imaging filter (GIF), and pre- and post-GIF direct electron detectors. A highly experienced staff with proven records of cryoEM-derived atomic structures will provide on-site assistance to collect data and provide streamlined movie pre-processing for our consortium user laboratories. The critical need for this consortium for recording atomic-resolution cryoEM images of a broad range of bio-medically significant macromolecular complexes is justified by the immediate benefits to the 19 consortium users. Of particular note, we have demonstrated that, once trained by our staff, users no longer have to be present at the microscope and can remotely control the microscope and perform high-resolution cryoEM imaging after our staff have aligned the instruments for top performance, thus significantly improving accessibility to the high-end instrument. A three-member supervisory committee representing non-consortium users, regional consortium users and the UCLA facility staff will provide evaluation and approval of user projects, adding and dropping users. Under our typical operation, a user group with approved projects requests research instrument time with an existing professionally-designed online reservation system, sends cryoEM grids by overnight courier (such as FedEx) in a liquid nitrogen-cooled dry shipper, and, after their grids are loaded by our staff, image their sample remotely for 3-7 days on average, and up to 2-3 weeks if necessary. The 10 institutes are concentrated on the West coast (University of Washington in Seattle, University of California at San Diego, Scripps) and Midwest (Case Western Reserve University, Purdue University, The University of Kansas, University of Alabama at Birmingham) except for three (University of Texas at El Paso, University of Texas Medical Branch in Galveston, and University of Florida at Gainesville). With the exception of three new investigators, all these users are funded by the NIH or other federal agencies to pursue biomedical research, ranging from biology, biochemistry, virology and microbiology. The establishment of this consortium will immediately empower our users with high-resolution cryoEM capabilities for a broad range of biological samples, enabling them to understand mechanisms of action and identify new targets for the development of new therapeutics.

? 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.

ABSTRACT The Southeastern Consortium for Microscopy of MacroMolecular Machines (SECM4) comprises 10 Universities/Medical Centers throughout the Eastern United States with a total of 13 cryoEM investigators studying a wide range of important biomedical problems as variable as high resolution virus structure, membrane protein structure, macromolecu- lar complexes of various types, some isolated in active form from cells, bacterial ultrastructure, spliceosomes, ribosome complexes all of which benefit from access to Florida State Universities (FSU) Titan Krios and its DE-64 direct electron detector. Recently the FSU Titan Krios was upgraded through the addition of a FEI Volta phase plate and a Gatan BioQuantum/K3 imaging filter which facilitate imaging of small molecules using single particle methods as well as thicker specimens that are imaged using cryoelectron tomography. The upgrades expanded the range of medically related structural biology problems to which SECM4 members can contribute. These upgrades also have made the FSU Titan Krios, which was one of the earliest ones installed in the US, comparable to recently installed Titan Krios microscopes, except for one feature. Newer Titan Krios microscopes have a more robust Autoloader than the early version currently operating on The FSU microscope. The Autoloader is the device that facilitates exchange of frozen hydrated specimens from the outside world into the high, contamination free environment of the Titan Krios. The current Autoloader, installed in August 2011, is currently responsible for more than 50% of the operational down time due to instrument failure. This Administrative Supplement seeks funds to replace the current Autoloader with the most recent version with the goal of reducing the greatest cause of instrument down time. SECM4 operates on the synchrotron template currently in use at sites having X-ray crystallography beam lines around the country. SECM4 members ship specimens to FSU and watch the data being collected as it comes off the microscope from the familiar confines of their own laboratories. SECM4 provides sufficient preprocessing that consortium members can evaluate the prospects for obtaining a final high-resolution structure from damage and motion corrected ?movie? images of their samples. SECM4 will become a model for high throughput structure determination utilizing high-end instrumentation to reveal the inner workings of complex macromolecules and subcellular structures.

ABSTRACT Intravascular administration of adenovirus (Ad) vectors holds great promise for improving survival of patients with disseminated metastatic cancer and ameliorating numerous genetic diseases through permanent correction of the hematopoietic stem cell compartment. Although potentially advantageous, intravascular delivery makes therapeutic Ads vulnerable to humoral components of the innate and adaptive arms of the immune system. Extensive previous analyses by us and others showed that binding of coagulation FX to HAdv-5-based vectors in the blood facilitates highly efficient hepatocyte transduction, triggering hepatotoxicity. Furthermore, a relatively high prevalence of HAdv-5-neutralizing antibodies (NAbs) in the human population, prompted active development of therapeutic vectors based on alternate Ad serotypes that don't interact with FX and exhibit low NAb prevalence. Our preliminary studies indicate however, that both pre-existing neutralizing and non- neutralizing humoral immunity can significantly exacerbate the host inflammatory antiviral response. We found that the majority of human sera that lack HAdv-5-neutralizing activity are still able to trigger complement fixation on the virus, leading to potentiated inflammatory cytokine production by immune cells. Moreover, we found that immunization of mice with adenovirus HAdv-11 triggers generation of non-neutralizing antibodies, which are highly efficient at complement fixation on phylogenetically-distant HAdv-5 virus. Based on these findings, we propose a novel concept of cryptic opsonizing non-neutralizing antibodies, or CON-Abs, which bind to Ad after systemic delivery, trigger complement fixation on the virus, and target Ad-immune complexes (Ad-ICs) to immune phagocytic cells, leading to drastically potentiated systemic inflammation. The molecular mechanisms mediating the immune-stimulatory properties of pre-existing non-neutralizing immunity and its effect on the safety of systemic Ad delivery are virtually unknown. Therefore, in Specific Aim 1 of this project we will analyze how phylogenetically-distant HAdv serotypes trigger generation of CON-Abs to HAdv-5. In Specific Aim 2, we will determine the structural bases for CON-Ab-mediated complement fixation on the virus and complement- mediated virus neutralization. In Specific Aim 3 we will determine specific cell types and their receptors that sequester Ad-ICs in vivo; and in Specific Aim 4 we will determine specific signaling pathways responsible for the exacerbated inflammatory response to Ad-ICs and develop targeted pharmacological approaches to improve the safety of systemic Ad delivery in gene transfer and cancer therapy models. The proposed studies will provide new mechanistic insights into fundamental functions of innate and adaptive immunity and host anti-viral defense, as well as allow for the development of novel patient stratification tools and pharmacological approaches to improve the safety of clinical interventions that rely on systemic delivery of therapeutic Ads.