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).
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|Heymann, J Bernard; Marabini, Roberto; Kazemi, Mohsen et al. (2018) The first single particle analysis Map Challenge: A summary of the assessments. J Struct Biol 204:291-300|
|Sun, Stella Y; Kaelber, Jason T; Chen, Muyuan et al. (2018) Flagellum couples cell shape to motility in Trypanosoma brucei. Proc Natl Acad Sci U S A 115:E5916-E5925|
|Walsh Jr, Richard M; Roh, Soung-Hun; Gharpure, Anant et al. (2018) Structural principles of distinct assemblies of the human ?4?2 nicotinic receptor. Nature 557:261-265|
|Fan, Guizhen; Baker, Mariah R; Wang, Zhao et al. (2018) Cryo-EM reveals ligand induced allostery underlying InsP3R channel gating. Cell Res 28:1158-1170|
|Jin, Jing; Galaz-Montoya, Jesús G; Sherman, Michael B et al. (2018) Neutralizing Antibodies Inhibit Chikungunya Virus Budding at the Plasma Membrane. Cell Host Microbe 24:417-428.e5|
|Su, Zhaoming; Wu, Chao; Shi, Liuqing et al. (2018) Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly. Cell 172:966-978.e12|
|Pintilie, Grigore; Chiu, Wah (2018) Assessment of structural features in Cryo-EM density maps using SSE and side chain Z-scores. J Struct Biol 204:564-571|
|Qi, Xiaodong; Zhang, Fei; Su, Zhaoming et al. (2018) Programming molecular topologies from single-stranded nucleic acids. Nat Commun 9:4579|
|Dai, Wei; Chen, Muyuan; Myers, Christopher et al. (2018) Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography. J Mol Biol 430:4156-4167|
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