New York Structural Biology Center Inc

NCCAT: NATIONAL CENTER FOR CRYOEM ACCESS AND TRAINING

NCCAT: NATIONAL CENTER FOR CRYOEM ACCESS AND TRAINING - PROJECT SUMMARY/ABSTRACT THE NATIONAL CENTER FOR CRYOEM ACCESS AND TRAINING (NCCAT) ENABLES USERS TO SOLVE MACROMOLECULAR STRUCTURES RAPIDLY AND TO THE HIGHEST POSSIBLE RESOLUTION USING CRYO-ELECTRON MICROSCOPY (CRYOEM) METHODS. WE PROPOSE TO CONTINUE TO: (1) PROVIDE ACCESS TO STATE-OF-THE-ART EQUIPMENT, (2) MAINTAIN A HIGHLY QUALIFIED TECHNICAL TEAM OFFERING DIRECT SUPPORT, GUIDANCE, AND ASSISTANCE, (3) OPERATE A COMPREHENSIVE CROSS-TRAINING PROGRAM FOR USERS ACROSS DIVERSE SKILL LEVELS AND CAREER GOALS, AND (4) ENSURE EQUAL OPPORTUNITY NATIONWIDE ACCESS THROUGH AN OPEN AND TRANSPARENT APPLICATION PROCESS. NCCAT IS HOUSED AT THE SIMONS ELECTRON MICROSCOPY CENTER (SEMC) WITHIN THE NEW YORK STRUCTURAL BIOLOGY CENTER (NYSBC) IN NEW YORK CITY. NCCAT’S ESTABLISHED OPERATIONS AT SEMC AND NYSBC BENEFIT FROM THE VIBRANT ENVIRONMENT AND EXPERTISE OF EXISTING HIGHLY TRAINED STAFF. NCCAT OCCUPIES CUSTOM-BUILT SPACE THAT PROVIDES OPTIMAL, ENVIRONMENTALLY STABLE CONDITIONS. THE PRIMARY INSTRUMENTATION CONSISTS OF 4 DEDICATED TITAN KRIOS TEMS, EACH EQUIPPED WITH DIRECT DETECTORS AND ENERGY FILTERS; 1 (SOON TO BE 3) DEDICATED GLACIOS TEMS, EACH EQUIPPED WITH DIRECT DETECTORS, AND 3 SIDE ENTRY SCREENING MICROSCOPES (1 OF WHICH IS DEDICATED TO NCCAT) EQUIPPED WITH CMOS CAMERAS. THESE ARE AUGMENTED BY ALL ANCILLARY EQUIPMENT NEEDED FOR THE CRYOEM WORKFLOW, INCLUDING SEVERAL CLASSES OF VITRIFICATION AND SAMPLE PREPARATION EQUIPMENT. NCCAT PROVIDES ON-SITE, REMOTE-ACCESS, AND MAIL-IN DATA COLLECTION SERVICES WITH FAIR AND EQUAL ACCESS TO A NATIONWIDE USER BASE, AND WE GUIDE AND ASSIST USERS WITH ALL ASPECTS OF CRYOEM, INCLUDING SAMPLE PREPARATION, DATA COLLECTION, DATA PROCESSING, DATA ANALYSIS, AND STRUCTURE REFINEMENT. BY PROVIDING USERS WITH ACCESS TO STREAMLINED WORKFLOWS AND RESOURCES, NCCAT ALLOWS RESEARCHERS TO COLLECT HIGH-QUALITY DATA QUICKLY AND EFFICIENTLY, WHICH WILL LEAD TO FASTER PROGRESS IN STRUCTURAL BIOLOGY RESEARCH. NCCAT’S TRAINING PROGRAM ENCOMPASSES A FULL RANGE OF EXPECTATIONS, FROM THE BASICS REQUIRED TO ENSURE THAT USERS PRODUCE VALID STRUCTURES, THROUGH MORE DETAILED TRAINING FOR USERS SEEKING INDEPENDENCE, TO THOSE EXPECTED TO RUN CRYOEM RESEARCH LABORATORIES. A WELL-ESTABLISHED GRID PREPARATION AND SCREENING SERVICE PROGRAM SUPPORTS NEW PRACTITIONERS OF CRYOEM METHODS AND/OR LABORATORIES THAT LACK LOCAL FACILITIES AND RESOURCES TO OPTIMIZE SAMPLE AND GRID PREPARATION CONDITIONS AND, WHEN POSSIBLE, OBTAIN PRELIMINARY CRYOEM PROCESSED DATA. WE PROPOSE TO BE TRANSFORMATIVE BY SETTING THE STANDARD FOR ACCESS AND TRAINING TO CRYOEM TECHNOLOGY. BY FACILITATING THE SHIFT FROM THE CURRENT MODEL OF ISOLATED AND EXPENSIVE FACILITIES TO A UNIFIED AND MORE EFFICIENT CENTER, WE WILL ENABLE SCIENCE OF THE HIGHEST QUALITY AND VALUE AND THEREFORE PROVIDE ESSENTIAL FOUNDATIONAL KNOWLEDGE NEEDED TO DEVELOP NEW THERAPIES, DIAGNOSTICS, AND CLINICAL PRACTICES TO BETTER HUMAN HEALTH.

STRUCTURAL GENOMICS ON MEMBRANE PROTEINS

NATIONAL RESOURCE FOR AUTOMATED MOLECULAR MICROSCOPY

THE NATIONAL CENTER FOR IN-SITU TOMOGRAPHIC ULTRAMICROSCOPY (NCITU)

CENTER ON MACROMOLECULAR DYNAMICS BY NMR SPECTROSCOPY

CENTER ON MEMBRANE PROTEIN PRODUCTION AND ANALYSIS (COMPPAA)

RM1 CENTER ON MACROMOLECULAR DYNAMICS BY NMR SPECTROSCOPY AT THE NEW YORK STRUCTURAL BIOLOGY CENTER (COMD/NMR) - THIS PROPOSAL DESCRIBES THE MISSION AND STRATEGIC PLANS FOR OF A BIOMEDICAL TECHNOLOGY DEVELOPMENT AND DISSEMINATION CENTER (RM1) ENTITLED CENTER ON MACROMOLECULAR DYNAMICS BY NMR SPECTROSCOPY (COMD/NMR). LOCATED AT THE NEW YORK STRUCTURAL BIOLOGY CENTER, COMD/NMR HAS DEVELOPED NEW EXPERIMENTAL AND COMPUTATIONAL TECHNIQUES IN NMR SPECTROSCOPY, INCLUDING BOTH STRUCTURE AND DYNAMICS METHODS FOR BOTH SOLUTION AND SOLID STATE NMR. HERE WE FOCUS MAINLY ON SPIN RELAXATION METHODS, WHICH HAVE ALREADY HAD GREAT IMPACT FOR CHARACTERIZING PROTEIN AND NUCLEIC ACID CONFORMATIONAL DYNAMICS DURING BIOLOGICAL PROCESSES INCLUDING LIGAND RECOGNITION, ALLOSTERISM, OLIGOMERIZATION, CATALYSIS, AND FOLDING. THE CENTRAL CHALLENGE ADDRESSED BY COMD/NMR IS TO BREAK DOWN THE HIGH ACTIVATION BARRIER FOR NEW USERS TO APPLY ADVANCED NMR SPECTROSCOPIC AND COMPUTATIONAL METHODS AND THEREBY MAKE SOPHISTICATED NMR APPROACHES AVAILABLE TO A WIDE BIOLOGICAL RESEARCH COMMUNITY. TO DO SO, COMD/NMR ADDRESSES FOUR PRIMARY OBSTACLES. (I) BECAUSE DYNAMICS PROBLEMS FOR BIOPOLYMERS ARE FREQUENTLY UNDERDETERMINED, WE DEVELOP INCISIVE NEW EXPERIMENTS, PULSE SEQUENCES AND COMPUTATIONAL METHODS. (II) BECAUSE ACCESS TO ADVANCED NMR INSTRUMENTS CAN BE A LIMITATION FOR MANY USERS, WE PROVIDE ACCESS TO A RANGE OF ENABLING NMR INSTRUMENTATION, INCLUDING NMR SPECTROMETERS AT MULTIPLE STATIC MAGNETIC FIELDS, A RANGE OF MODERN PROBES, DYNAMIC NUCLEAR POLARIZATION, RAPID-FREEZE-QUENCH, HIGH-PRESSURE EQUIPMENT, AND FIELD CYCLING RELAXOMETRY, ALL IN A WELL MAINTAINED, STAFF SUPPORTED, AND MULTIUSER ENVIRONMENT. (III) BECAUSE TRANSFER OF TECHNOLOGY TO BIOLOGISTS (AND EVEN TO NMR SPECTROSCOPISTS) HAS BEEN HINDERED BY THE COMPLEXITY OF THE METHODS, AND SINCE TIME EFFICIENT USE OF THE INSTRUMENTS IS CRUCIAL, WE DEVELOP ROBUST EFFICIENT PIPELINES TO FACILITATE EXPERIMENTAL PLANNING, DATA ACQUISITION AND ANALYSIS BY NON- SPECIALISTS. (IV) BECAUSE BIOLOGISTS WITH RESEARCH PROGRAMS THAT WOULD BENEFIT FROM THESE METHODS ARE UNAWARE OF THE POTENTIAL, WE ENGAGE IN COMMUNITY OUTREACH AND EDUCATION. A UNIQUE STRENGTH OF OUR PROGRAM IS THE INTEGRATION OF FOREFRONT SOLID STATE NMR, SOLUTION NMR AND COMPUTATIONAL EXPERTS, AND THIS PROPOSAL HIGHLIGHTS SYNERGY ACROSS THESE APPROACHES. ADDITIONALLY, BEING SITUATED WITHIN THE NYSBC, WE HAVE EXCELLENT PARTNERSHIPS WITH X-RAY DIFFRACTION, CRYO EM, AND MEMBRANE PROTEIN PRODUCTION TECHNOLOGIES. GIVEN THE DEMANDING NATURE OF THE THREE TECHNOLOGY DEVELOPMENT PROJECTS (TDPS) PROPOSED HEREIN, WE HAVE IDENTIFIED A NUMBER OF TECHNOLOGY PARTNERSHIP PROJECTS (TPPS), ENGAGING WORLD LEADERS IN ASPECTS OF NMR WHO HAVE INDICATED THEIR EAGERNESS TO PARTNER WITH US. TECHNOLOGY DEVELOPMENT PROCEEDS MOST EFFECTIVELY WHEN DRIVEN BY EXCITING AND CHALLENGING APPLICATIONS; ACCORDINGLY, COMD/NMR WILL WORK CLOSELY WITH OUTSTANDING LOCAL AND NATIONAL INVESTIGATORS THROUGH COLLABORATIVE AND SERVICE PROJECTS (CSP), DRIVING BIOMEDICAL PROJECTS (DBB) AND COMMUNITY ENGAGEMENT (CE) ACTIVITIES, INCLUDING EXTENSIVE TRAINING AND DISSEMINATION PROGRAMS. THROUGH ITS VARIOUS COMPONENTS, COMD/NMR WILL IMPACT A DIVERSE RANGE OF BIOLOGICAL RESEARCH WITH HUMAN HEALTH RELATEDNESS, INCLUDING DEGENERATIVE DISEASES, METABOLIC DISORDERS, AND CANCER.

STRUCTURAL GENOMICS OF MEMBRANE PROTEINS

STRUCTURE, FUNCTION, AND DYNAMICS OF VIRAL RNAS AND RNA-CONTAINING COMPLEXES

NYX BEAMLINE COMPLETION

MECHANISMS OF VIRAL RNA MATURATION BY CO-OPTING CELLULAR EXONUCLEASES

DEVELOPMENT OF A SYNCHROTRON BEAMLINE FOR HIGH-RESOLUTION MICRODIFFRACTION ANALYSIS OF BIOLOGICAL MACROMOLECULES

DEVELOPMENT OF X-RAY DETECTORS FOR A NEW UNDULATOR BEAMLINE AT THE NATIONAL SYNCHROTRON LIGHT SOURCE-II

DYNAMIC NUCLEAR POLARIZATION SOLID STATE NMR SPECTROMETER FOR BIOMOLECULAR STUDIE

ACQUISITION OF AN 800 MHZ NMR SPECTROMETER CONSOLE AND PROBES - FUNDING IS REQUESTED TO OBTAIN A BRUKER BIOSPIN NMR AVANCE NEO 800 MHZ CONSOLE AND ASSOCIATED COMPONENTS, INCLUDING BMPC-2 MAGNET CONTROL SYSTEM, CRYOPLATFORM/5, MASIII PNEUMATIC CONTROL UNIT, TCI-H&F TRIPLE-RESONANCE CRYOPROBE, MAS TRI-GAMMA 1.9 MM SOLID-STATE PROBE, AND MAS 3.2 MM SOLID-STATE CRYOPROBE, TO BE INSTALLED ON AN EXISTING BRUKER BIOSPIN 800 MHZ NMR MAGNET SYSTEM. THE PRESENT NMR AVANCE I CONSOLE AND TCI CRYOPROBE/1 WERE INSTALLED 2003 AND 2004 AND WERE DECLARED BY BRUKER BIOSPIN TO HAVE REACHED “END OF SERVICE” (EOS) IN 2018 AND 2019, RESPECTIVELY. THE REQUESTED INSTRUMENTATION PROVIDES ENHANCED SENSITIVITY AND CAPABILITIES FOR MODERN NMR EXPERIMENTS, INCLUDING TEMPERATURE-LOCKING, NON-UNIFORM SAMPLING, COMPLEX WAVEFORM GENERATION, MAGIC-ANGLE SPINNING (MAS) SOLID-STATE NMR SPECTROSCOPY, AND ADVANCED CRYOGENIC PROBE DESIGN FOR BOTH SOLUTION AND SOLID-STATE MAS APPLICATIONS. THE REQUESTED INSTRUMENTATION IS ESSENTIAL TO THE OVER-RIDING PURPOSE OF THE NEW YORK STRUCTURAL BIOLOGY CENTER (NYSBC), A CONSORTIUM OF NINE RESEARCH INSTITUTIONS IN NEW YORK STATE, TO SUPPORT THE SPECIFIC AIMS OF THE NIH-FUNDED RESEARCH OF THE MAJOR AND MINOR USERS OF THE SPECTROMETER. RESEARCH CONDUCTED ON THE 800 MHZ NMR SPECTROMETER RANGES FROM FUNDAMENTAL STUDIES OF STRUCTURE/FUNCTION OF BIOMOLECULES, TO ELUCIDATION OF MECHANISMS OF PATHOGENESIS, TO DISCOVERY OF NOVEL LEAD COMPOUNDS TO PHARMACEUTICAL APPLICATIONS. THE REQUESTED INSTRUMENTATION WILL DRAMATICALLY ENHANCE THE CAPACITY OF THE 800 MHZ NMR SPECTROMETER TO CONDUCT THIS RESEARCH.

ACQUISITION OF A FAST SHUTTLE SYSTEM APPARATUS - FUNDING IS REQUESTED TO OBTAIN A BRUKER BIOSPIN FAST SHUTTLE SYSTEM (FSS) APPARATUS TO BE INSTALLED ON AN EXISTING 700 MHZ NMR SPECTROMETER AT THE NEW YORK STRUCTURAL BIOLOGY CENTER (NYSBC), A CONSORTIUM OF NINE RESEARCH INSTITUTIONS IN NEW YORK STATE. THE REQUESTED INSTRUMENTATION IS ESSENTIAL TO THE OVER-RIDING PURPOSE OF NYSBC AND ITS NIH-FUNDED RM-1 CENTER FOR MACROMOLECULAR DYNAMICS BY NMR SPECTROSCOPY (COMD/NMR) TO SUPPORT THE SPECIFIC AIMS OF THE NIH-FUNDED RESEARCH OF THE MAJOR AND MINOR USERS OF THE REQUESTED INSTRUMENTATION. THE FSS APPARATUS IS A TECHNOLOGICAL BREAKTHROUGH THAT ENABLES FOR THE VERY FIRST TIME MEASUREMENTS OF SPIN-LATTICE RELAXATION RATE CONSTANTS FOR 1H, 15N, 13C AND OTHER SPINS IN BIOLOGICAL MACROMOLECULES, INCLUDING PROTEINS AND NUCLEIC ACIDS OVER THE FULL RANGE OF MAGNETIC FIELDS FROM 100 ΜT TO 16.4 T (TERMED R1 RELAXATION DISPERSION OR RELAXOMETRY) THE FSS APPARATUS CONSISTS OF A FAST SHUTTLE SYSTEM, A MAGNETIC TUNNEL INSERTED INTO THE BORE OF THE NMR DEWAR, AND AN EXTERNAL ELECTROMAGNET FIELD CYCLING COIL (FCC) MOUNTED ON TOP OF THE NMR MAGNET DEWAR. THE KEY PART OF THE FSS APPARATUS IS THE MAGNETIC TUNNEL, WHICH MAINTAINS THE MINIMUM FIELD IN THE BORE OF THE MAGNET >1 T IN ORDER TO PREVENT EXTREMELY LARGE INCREASES IN R1 DURING TRANSFER FROM HIGH FIELD (IN THE SUPERCONDUCTING COIL) TO LOW FIELD (IN THE FCC) AND BACK. CONSEQUENTLY, SENSITIVITY IS DRAMATICALLY INCREASED, RELAXATION EFFECTS DURING SHUTTLING OF THE SAMPLE ARE MINIMIZED, AND LOWER MAGNETIC FIELDS CAN BE ACCESSED, COMPARED TO EXISTING SHUTTLE SYSTEMS LACKING A MAGNETIC TUNNEL OR FCC (RELYING ONLY ON THE FRINGE FIELD OF THE SUPERCONDUCTING COIL). THE FCC IS DESIGNED TO OPERATE IN THE RANGE 100 ΜT TO 0.8 T, WHILE THE FRINGE FIELD OF THE SPECTROMETER IS USED TO MEASURE RELAXATION RATES AT FIELDS > 0.8 T. BOTH THEORETICAL CALCULATIONS AND PRELIMINARY MEASUREMENTS OF 15N AND 13C R1 RELAXATION DISPERSION DEMONSTRATE THAT CONFORMATIONAL DYNAMICS OF BIOLOGICAL MACROMOLECULES CAN BE CHARACTERIZED IN UNPRECEDENTED DETAIL USING THE FSS, COMPARED WITH HIGH-FIELD MEASUREMENTS (>10 T) OR RELAXOMETRY USING ONLY THE FRINGE FIELD (>0.5 T). THE REQUESTED FSS APPARATUS WILL BE THE THIRD ONE IN THE WORLD AND FIRST IN THE UNITED STATES; THUS, A KEY GOAL OF COMD/NMR IS TO MAKE THIS GROUNDBREAKING TECHNOLOGY AVAILABLE TO THE MAJOR AND MINOR USERS, BUT ALSO TO THE NATIONAL RESEARCH COMMUNITY. THE RESEARCH CONDUCTED USING THIS TRANSFORMATIVE TECHNOLOGY RANGES FROM FUNDAMENTAL STUDIES OF STRUCTURE/DYNAMICS/FUNCTION OF BIOMOLECULES, TO ELUCIDATION OF MECHANISMS OF PATHOGENESIS, TO DISCOVERY OF NOVEL LEAD COMPOUNDS, AND TO PHARMACEUTICAL APPLICATIONS.

DUAL-BEAM SCANNING ELECTRON MICROSCOPE FOR NEW YORK STRUCTURAL BIOLOGY CENTER

AN EIGER2 XE 9M DETECTOR FOR THE NYSBC-OPERATED NYX BEAMLINE AT NSLS-II - PROJECT SUMMARY THE NEW YORK STRUCTURAL BIOLOGY CENTER (NYSBC) OWNS AND OPERATES THE NYX BEAMLINE AT THE NATIONAL SYNCHROTRON LIGHT SOURCE II (NSLS-II) WHICH IS LOCATED AT THE BROOKHAVEN NATIONAL LABORATORY (BNL) AND OPERATED FOR THE BENEFIT OF THE MEMBER INSTITUTIONS OF THE NYSBC AND THE COMMUNITY OF NON-CONSORTIUM-MEMBER STRUCTURAL BIOLOGISTS. WE SEEK FUNDS TO ACQUIRE A DECTRIS EIGER2 XE 9M X-RAY DETECTOR FOR MACROMOLECULAR CRYSTALLOGRAPHY. THIS ADVANCED, HIGH-SPEED READOUT OF THE DETECTOR WILL SIGNIFICANTLY INCREASE THE THROUGHPUT OF SAMPLES AND THEREFORE INCREASE THE NUMBER OF PROJECTS THAT CAN BE PROSECUTED IN A SET AMOUNT OF TIME. THE HIGH FRAME RATE WILL ALLOW US TO PERFORM ADVANCED CRYSTALLOGRAPHIC METHODS THAT ARE NOT POSSIBLE WITH OUR CURRENT EXPERIMENTAL SETUP, SUCH AS SERIAL CRYSTALLOGRAPHY, ULTRA-FAST RASTERING AND POTENTIALLY OUTRUNNING RADIATION DAMAGE DURING DATA COLLECTION. NYX HAS BEEN OPERATING WITH A PROTOTYPE PIXEL ARRAY DETECTOR FROM AREA DETECTOR SYSTEMS CORPORATION (HF-4M) SINCE ITS INCEPTION AT NSLS-II. WHILE THE DATA QUALITY FROM THIS DETECTOR HAS BEEN COMPARABLE WITH OTHER AVAILABLE DETECTORS, ADSC CEASED TO OPERATE SEVERAL YEARS AGO WITH THE RESULT THAT THE DETECTOR IS NO LONGER SERVICEABLE AND IS STARTING TO FAIL. IN ORDER TO REMAIN VALUABLE TO USERS, IT IS IMPERATIVE THAT WE REPLACE THE ADSC HF-4M WITH A MODERN PIXEL ARRAY DETECTOR WITH THE EXPECTATION THAT IT WILL REMAIN USEFUL AND SERVICEABLE FOR THE NEXT 8-10 YEARS. OF PARTICULAR IMPORTANCE IS PERFORMING THIS UPGRADE PRIOR TO THE TEMPORARY SHUTDOWN OF THE ADVANCED PHOTON SOURCE FOR A FACILITY UPGRADE IN 2022 WHEN THE DEMAND FOR SYNCHROTRON ACCESS FOR NIH-FUNDED PROJECTS IS EXPECTED TO INCREASE DRAMATICALLY. HAVING THIS DETECTOR IN PLACE IN 2021 WILL ALLOW NYX TO SIGNIFICANTLY ENHANCE THE NIH-FUNDED RESEARCH PROGRAMS OF THE NYSBC CONSORTIUM MEMBERS, NON- CONSORTIUM MEMBERS AS WELL AS BE FULLY PREPARED TO PROVIDE UNINTERRUPTED ACCESS TO NIH-FUNDED PROJECTS DUE TO THE TEMPORARY CLOSURE OF THE APS.

ACQUISITION OF A 900 MHZ NMR SPECTROMETER CONSOLE AND PROBES - FUNDING IS REQUESTED TO OBTAIN A BRUKER BIOSPIN NMR AVANCE CONSOLE AND PROBES TO BE INSTALLED ON AN EXISTING BRUKER BIOSPIN 900 MHZ NMR MAGNET SYSTEM. THE EXISTING NMR CONSOLE AND PROBES ARE MORE THAN 14 YEARS OLD AND HAVE REACHED THE END OF SERVICE LIFETIME, AS DESIGNATED BY BRUKER BIOSPIN. THE REQUESTED INSTRUMENTATION PROVIDES ENHANCED SENSITIVITY AND CAPABILITIES FOR MODERN NMR EXPERIMENTS, INCLUDING NON-UNIFORM SAMPLING, COMPLEX WAVEFORM GENERATION, AND 1H MAGIC- ANGLE SPINNING SOLID-STATE NMR SPECTROSCOPY. THE REQUESTED INSTRUMENTATION IS ESSENTIAL TO THE OVER- RIDING PURPOSE OF THE NEW YORK STRUCTURAL BIOLOGY CENTER (NYSBC), A CONSORTIUM OF NINE RESEARCH INSTITUTIONS IN NEW YORK STATE, TO SUPPORT THE PARTICULAR AIMS OF THE NIH-FUNDED RESEARCH OF THE MAJOR AND MINOR USERS OF THE SPECTROMETER. RESEARCH CONDUCTED ON THE 900 MHZ NMR SPECTROMETER RANGES FROM FUNDAMENTAL STUDIES OF STRUCTURE/FUNCTION OF BIOMOLECULES, TO ELUCIDATION OF MECHANISMS OF PATHOGENESIS, TO DISCOVERY OF NOVEL LEAD COMPOUNDS TO PHARMACEUTICAL APPLICATIONS. THE REQUESTED INSTRUMENTATION WILL DRAMATICALLY ENHANCE THE CAPACITY OF THE 900 MHZ NMR SPECTROMETER TO CONDUCT THIS RESEARCH.

HIGH THRUPUT EM SAMPLE PREPARATION FOR STRUCTURAL BIOLOGY

CONTINUED DEVELOPMENT AND MAINTENANCE OF APPION SOFTWARE

ACQUISITION OF 700 MHZ NMR SPECTROMETER

MEMBRANE PROTEIN STRUCTURE BY ELECTRON CRYSTALLOGRAPHY

ACQUISITION OF DETECTOR FOR JEOL 3200 FSC ELECTRON MICROSCOPE

UPGRADE OF 800 MHZ SPECTROMETER

ARCHITECTURE OF A DIVERGENT BIPARTITE INTERNAL RIBOSOME ENTRY SITE FROM GIARDIAVIRUS - PROJECT SUMMARY TRANSLATION INITIATION IS THE MOST REGULATED STEP IN PROTEIN SYNTHESIS. IN EUKARYOTES, CANONICAL TRANSLATION INITIATION BEGINS WITH RECOGNITION OF A CAP STRUCTURE ON THE MRNA 5’ END, AND IS A VERY COMPLEX PROCESS REQUIRING MANY PROTEIN FACTORS AND INTERMEDIATES. TO FAVOR TRANSLATION OF THEIR OWN PROTEINS, RNA VIRUSES HAVE EVOLVED SIMPLER WAYS TO INITIATE PROTEIN SYNTHESIS. ONE SUCH VIRAL TOOL IS AN INTERNAL RNA ENTRY SITE, OR IRES, A STRUCTURED RNA THAT DRIVES NONCANONICAL, CAP-INDEPENDENT TRANSLATION. SOME IRES ELEMENTS SHOW GREAT PROMISE, EITHER AS NEW TARGETS FOR THERAPIES DIRECTED AGAINST PATHOGENIC RNA VIRUSES, OR AS TOOLS FOR OPTIMIZING PROTEIN EXPRESSION IN MRNA-BASED VACCINES. THE THREE-DIMENSIONAL IRES STRUCTURES DETERMINED TO DATE HAVE COME ALMOST EXCLUSIVELY FROM JUST TWO OF THE MANY IRES CLASSES. MORE STRUCTURES OF DIVERSE IRES ELEMENTS ARE NEEDED TO DETERMINE EXISTING IRES MECHANISMS, AND ULTIMATELY TO LAY THE GROUNDWORK FOR THE DEVELOPMENT OF IMPROVED IRES TOOLS FOR NOVEL THERAPIES. DESPITE THEIR SIGNIFICANT STRUCTURAL AND MECHANISTIC DIFFERENCES, ALMOST ALL IRES ELEMENTS SHARE ONE NOTABLE FEATURE: LITTLE IF ANY OF THE IRES IS LOCATED IN THE CODING REGION. REMARKABLY, A IRES FROM GIARDIA LAMBLIA VIRUS (GLV) VIOLATES THIS PARADIGM. THE GLV IRES IS BIPARTITE, WITH ONE CANONICAL DOMAIN 5’ TO THE START CODON, AND A SECOND LARGE, HIGHLY STRUCTURED DOMAIN LOCATED ENTIRELY IN ITS CODING REGION. THE SECONDARY AND THREE-DIMENSIONAL STRUCTURES OF THE GLV IRES ARE UNKNOWN. WE PROPOSE TO DETERMINE THE ARCHITECTURE OF THE GLV IRES BOUND TO ITS HOST RIBOSOME. THIS WORK WILL PROVIDE FOUNDATIONAL KNOWLEDGE ABOUT THE ROLE OF AN UNEXPLORED CLASS OF IRES DOMAIN IN VIRAL INFECTION OF A HUMAN PATHOGEN, GIARDIA LAMBLIA. GIARDIASIS IS THE MOST COMMON PARASITIC HUMAN DIARRHEAL DISEASE WORLDWIDE, AND AN INCREASINGLY SERIOUS NEGLECTED DISEASE. OUR RESULTS WILL PAVE THE WAY FOR LONG-TERM STUDIES OF THIS DIVERGENT IRES MECHANISM, AND POTENTIALLY REVEAL PATHWAYS THAT CAN BE TARGETED IN DIVERSE VIRUSES, HELPING THE LONG-TERM GOAL OF DEVELOPING NEW THERAPIES FOR ONE OF THE WORLD’S LEADING INTESTINAL DISEASES.

ACQUISITION OF AN 800 MHZ TXO CRYOPROBE

STRUCTURAL STUDIES OF TYPE II CADHERINS ON MEMBRANES BY CRYO-ELECTRON TOMOGRAPHY

FUNCTIONAL IMPLICATIONS OF STRUCTURAL HETEROGENEITY IN A VIRAL RNA TRANSLATION INITIATION ELEMENT - PROJECT SUMMARY RNA VIRUSES ARE AN EMERGING HEALTH THREAT, EVOLVING RAPIDLY TO PROLIFERATE AND SPREAD. WITHIN THESE VIRUSES ARE STRUCTURED RNA ELEMENTS THAT FACILITATE INFECTION AND VIRUS SURVIVAL. INTERNAL RIBOSOME ENTRY SITES (IRESS) ARE A TYPE OF RNA STRUCTURE FOUND WITHIN RNA VIRUSES THAT FACILITATE INFECTION BY ENABLING INITIATING TRANSLATION ON THE VIRAL GENOME WHEN CELLULAR TRANSLATION IS REPRESSED DURING INFECTION. THE HEPATITIS C VIRUS (HCV) CONTAINS AN IRES THAT HAS BEEN EXTENSIVELY STUDIED, DEMONSTRATING A MECHANISM OF INITIATION INTIMATELY LINKED TO RNA ARCHITECTURE. THE HCV IRES CONSISTS OF A LARGE, MULTI-DOMAIN STRUCTURE THAT INTERACTS DIRECTLY WITH THE SMALL RIBOSOMAL SUBUNIT (40S) AND EUKARYOTIC INITIATION FACTOR 3 (EIF3) TO FORM AN INITIATION COMPLEX. IN TANDEM WITH MECHANISTIC AND STRUCTURAL STUDIES OF THE HCV IRES, A HANDFUL OF OTHER VIRUSES WITH A SIMILAR STRUCTURE– TERMED HCV-LIKE IRESS – HAVE BEEN SPORADICALLY IDENTIFIED THROUGH SEQUENCE HOMOLOGY. DESPITE DEMONSTRATING NOTABLE STRUCTURAL DIVERSITY, HCV-LIKE IRESS ARE INFERRED TO SHARE THE SAME MECHANISM. I HYPOTHESIZE THAT THERE ARE DISTINCT STRUCTURAL SUBGROUPS WITHIN THE HCV CLASS OF IRESS AND BETWEEN THESE SUBGROUPS THERE ARE DIFFERENCES IN THEIR INITIATION COMPLEX COMPONENTS AND INTERMOLECULAR INTERACTIONS. IN MY FIRST AIM, I WILL DEFINE THE STRUCTURAL DIVERSITY OF HCV-LIKE IRESS AS A CLASS USING COMPUTATIONAL AND BIOCHEMICAL METHODS. I HAVE PERFORMED A STRUCTURE ALIGNMENT-BASED SEARCH TO MINE THE NCBI VIRUS DATABASE, REVEALING 178 UNIQUE PUTATIVE IRESS. I WILL USE COMPUTATIONAL METHODS TO STRUCTURALLY CHARACTERIZE DISCRETE REGIONS OF THESE PUTATIVE IRESS THAT DIFFER BETWEEN SUBGROUPS. PREDICTED IRES ARCHITECTURE AND FUNCTION WILL BE VALIDATED BIOCHEMICALLY TO LINK STRUCTURAL HETEROGENEITY WITH TRANSLATION INITIATION EFFICIENCY. FOR AIM 2, I WILL UNCOVER THE MECHANISTIC BASIS UNDERLYING THE STRUCTURAL AND FUNCTIONAL DIFFERENCES AMONG HCV-LIKE IRES BY INVESTIGATING INITIATION COMPLEX FORMATION. I WILL DETERMINE BOTH THE COMPOSITION OF THESE IRES INITIATION COMPLEXES AND THE AFFINITY OF THE IRES FOR EACH COMPONENT WITHIN THE COMPLEX. SELECT IRES INITIATION COMPLEXES WILL BE VISUALIZED USING CRYO-ELECTRON MICROSCOPY, POTENTIALLY REVEALING NOVEL INTERMOLECULAR INTERACTIONS. THE WORK OUTLINED IN THIS PROPOSAL WILL ENHANCE UNDERSTANDING OF THE RELATIONSHIP BETWEEN HCV-LIKE IRES STRUCTURE AND FUNCTION. FURTHER, THIS RESEARCH WILL PROVIDE INSIGHT INTO MECHANISMS OF EUKARYOTIC INITIATION THAT COULD BE LEVERAGED FOR USE IN VACCINES AND THERAPEUTICS TO COMBAT RNA VIRUSES.

CHARACTERIZING THE STRUCTURE AND FUNCTION OF A VIRAL RNA ELEMENT IN INHIBITING AN ANTIVIRAL PROTEIN - PROJECT SUMMARY RNA VIRUSES ARE A CONSTANT THREAT TO GLOBAL HEALTH AND UNDERSTANDING THEIR INTERACTIONS WITH HOST CELLS AT THE MOLECULAR LEVEL IS NECESSARY FOR IDENTIFYING THERAPEUTIC TARGETS. VIRUSES CONTAIN DIVERSE TYPES OF STRUCTURED RNA ELEMENTS WITHIN THEIR GENOMES WITH A VARIETY OF FUNCTIONS THAT ARE CRITICAL FOR SUCCESSFUL INFECTION. ONE SUCH STRUCTURED RNA ELEMENT WAS IDENTIFIED WITHIN POLIOVIRUS AND PROTECTS THE GENOMIC RNA FROM DEGRADATION BY THE MAMMALIAN ENDONUCLEASE, RIBONUCLEASE L (RNASE L). THIS RNA WAS FOUND TO BE A COMPETITIVE INHIBITOR OF RNASE L, PREVENTING DEGRADATION OF VIRAL GENOMIC RNA. PREVIOUS EXPERIMENTS HAVE CONFIRMED THE SECONDARY STRUCTURE OF THE POLIOVIRUS COMPETITIVE INHIBITOR RNA (CIRNA) AS WELL AS KEY TERTIARY CONTACTS REQUIRED FOR INHIBITION FUNCTION; HOWEVER, THE 3D STRUCTURE OF THE POLIOVIRUS CIRNA AND ITS SPECIFIC INTERMOLECULAR INTERACTIONS WITH RNASE L THAT ENABLE INHIBITION REMAIN UNRESOLVED. ADDITIONALLY, THE CIRNA ELEMENT WAS IDENTIFIED IN THE POLIOVIRUS GENOME BUT ITS PRESENCE IN DIVERGENT RNA VIRUSES HAS NOT BEEN EXPLORED OR CLASSIFIED. I HYPOTHESIZE THE CIRNA STRUCTURE DIRECTLY INHIBITS RNASE L BY PHYSICALLY BLOCKING ITS ACTIVE SITE. THE CIRNA RELIES ON CRUCIAL SECONDARY AND TERTIARY CONTACTS TO MAINTAIN ITS INHIBITORY PROPERTIES. I WILL IDENTIFY CIRNA ELEMENTS IN ADDITIONAL VIRAL GENOMES AND DEFINE THE PHYLOGENETIC DISTRIBUTION OF THIS CLASS OF RNAS. TO TEST THESE HYPOTHESES, I WILL USE A COMBINATION OF BIOPHYSICAL TECHNIQUES, STRUCTURAL BIOLOGY, BIOCHEMICAL ASSAYS, AND BIOINFORMATICS. AIM 1. I WILL USE A BIOINFORMATIC APPROACH TO QUERY A VIRAL GENOMIC DATABASE FOR CIRNA ELEMENTS AND TEST NEW PUTATIVE HITS FOR RNASE L INHIBITION ACTIVITY USING A FLUORESCENCE-BASED ASSAY. I WILL USE BIOCHEMICAL METHODS TO MAP THE BINDING SITE OF RNASE L ON CIRNA TO FIND THE CONSERVED BINDING SITE AMONG ALL EXAMPLES. AIM 2. I WILL CHARACTERIZE THE INTERACTION BETWEEN THE CIRNA AND RNASE L (AND THEIR MUTANTS) USING MICROSCALE THERMOPHORESIS TO QUANTIFY THE BINDING AFFINITY AND MEASURE THE STOICHIOMETRY BETWEEN THESE TWO MOLECULES AND BIOCHEMICALLY RELEVANT MUTANTS. TO FULLY CHARACTERIZE THIS INTERACTION ON A MOLECULAR LEVEL, I WILL USE CRYO-ELECTRON MICROSCOPY TO SOLVE THE STRUCTURE OF THE CIRNA-RNASE L COMPLEX. THE WORK OUTLINED IN THIS PROPOSAL WILL PROVIDE INSIGHT INTO HOW STRUCTURED RNA ELEMENTS DIRECTLY INTERACT WITH HOST PROTEIN FACTORS, AND HOW THESE HAVE EVOLVED IN THE VIRAL WORLD.