The broad goals of this Project, now entering its tenth year, are to develop the experimental and computational tools of electron cryomicroscopy (cryoEM) in the context of a widening range of biological applications. We seek in particular to connect electron microscopy (EM) with x-ray crystallography and to move molecular EM toward becoming a high-resolution tool. One of several advances during the past funding period has been to reach near-atomic resolution (<4A) for several virus structures, fulfilling a conjecture made 15 years ago by Henderson that it would be possible to image biological assemblies by cryoEM at this level of detail. We propose three principal themes for the coming project period. (1) Continued methods development. We will extend computational methods for near-atomic resolution structures to include images of multi-state single particles and helical assemblies (Grigorieff, Harrison);we will improve sample preparation for uniformity and homogeneity, building on the development of Affinity Grids during the last grant period (Walz);we will explore methods to reduce beam-induced movement (Grigorieff);resolution improvement for cellular imaging (Nicastro). (2) Electron cryotomography (cryo-ET) as a bridge between visualizing near-atomic resolution structures and studying their intracellular dynamics by optical microscopy and live-cell imaging (Nicastro, Harrison, Grigorieff). Rotavirus entry and clathrin-coat dynamics are two specific projects for which structures determined as part of this Project and results from live cell fluorescence microscopy raise mechanistic questions best answered by frontier methods in cryo-ET. (3) Analysis of transient and multi-state assemblies, including enhancements made possible by the methods developed as part of theme 1 (all four projects). In pursuit of this theme, we will focus especially on the large-scale organization of dynamic structures such as kinetochores, cilia, and transport-vesicle tethering complexes.

Public Health Relevance

Large macromolecular assemblies are the working machineries of a cell. For understanding the normal functions of complex assemblies in healthy cells and their malfunctions in disease, molecular electron microscopy is a critical bridge between their atomic structures and their dynamics in living cells. The project will develop new methods and apply them to problems such as viral infection, amyloid formation, and intracellular transport.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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Special Emphasis Panel (ZRG1-BST-J (40))
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Flicker, Paula F
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Harvard University
Schools of Medicine
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Loveland, Anna B; Demo, Gabriel; Grigorieff, Nikolaus et al. (2017) Ensemble cryo-EM elucidates the mechanism of translation fidelity. Nature 546:113-117
Liu, Yuhang; Pan, Junhua; Jenni, Simon et al. (2017) CryoEM Structure of an Influenza Virus Receptor-Binding Site Antibody-Antigen Interface. J Mol Biol 429:1829-1839
Abeyrathne, Priyanka D; Koh, Cha San; Grant, Timothy et al. (2016) Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome. Elife 5:
Schmidt, Andreas; Annamalai, Karthikeyan; Schmidt, Matthias et al. (2016) Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril. Proc Natl Acad Sci U S A 113:6200-5
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Loveland, Anna B; Bah, Eugene; Madireddy, Rohini et al. (2016) Ribosomeā€¢RelA structures reveal the mechanism of stringent response activation. Elife 5:
Baytshtok, Vladimir; Fei, Xue; Grant, Robert A et al. (2016) A Structurally Dynamic Region of the HslU Intermediate Domain Controls Protein Degradation and ATP Hydrolysis. Structure 24:1766-1777
Laxmikanthan, Gurunathan; Xu, Chen; Brilot, Axel F et al. (2016) Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction. Elife 5:
Ha, Jun Yong; Chou, Hui-Ting; Ungar, Daniel et al. (2016) Molecular architecture of the complete COG tethering complex. Nat Struct Mol Biol 23:758-60

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