The NCMI is dedicated to the advancement of cryo-electron microscopy and tomography methodology for structure determination of macromolecules, molecular machines and cells in their various functional states at the highest possible resolutions. Having completed 10 C-alpha backbone traceable cryo-EM Structures of molecular machines during the current grant period, we are well poised to tackle the next set of challenging structural biology problems. Our technology research development will focus around optimization of cutting edge instrumentation, data collection strategy, data management, image processing, modeling and visualization from electron images recorded from two unique combinations of instrumentation: (i) a 300 kV electron microscope with a direct electron detector and an in-column energy filter and (ii) a 200 kV electron microscope with a direct electron detector, an in-column energy filter and a Zernike phase contrast optics. Our technology development is led by 10 driving biological projects and also synergizes with 10 user projects that together include animal, plant and bacterial viruses, apoptosis-causing protein machine, chaperonin-substrate complex, membrane ion channels, nuclear receptor-coactivator complex, small RNA, oncogene protein complex, lipoproteins, amyloid protein aggregates, neuronal cells, virus-infected cells and mammalian cells related to cancer and eye diseases. Specifically, our Center will focus on 3 technology research and development projects: (i) Characterize and determine the optimal utilization of direct electron detectors and Zernike phase optics; (ii) Extend the structural determinations of biochemically purified molecular machines beyond current resolution limits; (iii) Develop novel methodologies for analyzing subcellular structures in cells by cryo- ET. We will proactively identify new projects from NIH-funded investigators across the US. We will continue our rigor in disseminating our software, experimental and computational protocols via workshops and web seminars. We will maintain an engaging advisory board to critique our progress and guide our strategic planning annually. Our efforts will extend cryo-EM/cryo-ET capabilities to fill the information gaps between x-ray crystallography, NMR and optical microscopy from nanometer to atomic resolutions.
Our proposed cryo-electron microscopy and tomography methodology is targeted to study structures of biologically active macromolecules, molecular machines and cells, which are potential drug targets for treating or preventing diseases. Our projects cover specimens relevant to infectious diseases (viruses and bacteria), neurodegenerate diseases (chaperonins and amyloid), eye disease (rod cell), cancer (complexes involved in gene expression and signaling); cardiovascular diseases (lipoprotein and ion channels).
|Wensel, Theodore G; Zhang, Zhixian; Anastassov, Ivan A et al. (2016) Structural and molecular bases of rod photoreceptor morphogenesis and disease. Prog Retin Eye Res 55:32-51|
|Marabini, Roberto; Ludtke, Steven J; Murray, Stephen C et al. (2016) The Electron Microscopy eXchange (EMX) initiative. J Struct Biol 194:156-63|
|Darrow, Michele C; Zhang, Yujin; Cinquin, Bertrand P et al. (2016) Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography. J Cell Sci 129:3511-7|
|Hecksel, Corey W; Darrow, Michele C; Dai, Wei et al. (2016) Quantifying Variability of Manual Annotation in Cryo-Electron Tomograms. Microsc Microanal 22:487-96|
|Li, Hui; Zhang, Kaiming; Pi, Fengmei et al. (2016) Controllable Self-Assembly of RNA Tetrahedrons with Precise Shape and Size for Cancer Targeting. Adv Mater 28:7501-7|
|Chen, Muyuan; Baldwin, Philip R; Ludtke, Steven J et al. (2016) De Novo modeling in cryo-EM density maps with Pathwalking. J Struct Biol :|
|Bell, James M; Chen, Muyuan; Baldwin, Philip R et al. (2016) High resolution single particle refinement in EMAN2.1. Methods 100:25-34|
|Gorzelnik, Karl V; Cui, Zhicheng; Reed, Catrina A et al. (2016) Asymmetric cryo-EM structure of the canonical Allolevivirus QÎ² reveals a single maturation protein and the genomic ssRNA in situ. Proc Natl Acad Sci U S A 113:11519-11524|
|Khisamutdinov, Emil F; Jasinski, Daniel L; Li, Hui et al. (2016) Fabrication of RNA 3D Nanoprisms for Loading and Protection of Small RNAs and Model Drugs. Adv Mater 28:10079-10087|
|Roh, Soung-Hun; Kasembeli, Moses M; Galaz-Montoya, JesÃºs G et al. (2016) Chaperonin TRiC/CCT Recognizes Fusion Oncoprotein AML1-ETO through Subunit-Specific Interactions. Biophys J 110:2377-85|
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