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)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-BST-J (40))
Program Officer
Flicker, Paula F
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Harvard University
Schools of Medicine
United States
Zip Code
Tran, Timothy H; Hsiao, Yu-Shan; Jo, Jeanyoung et al. (2015) Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase. Nature 518:120-4
Luque, Daniel; Gómez-Blanco, Josué; Garriga, Damiá et al. (2014) Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage. Proc Natl Acad Sci U S A 111:7641-6
Rohou, Alexis; Grigorieff, Nikolaus (2014) Frealix: model-based refinement of helical filament structures from electron micrographs. J Struct Biol 186:234-44
Svidritskiy, Egor; Brilot, Axel F; Koh, Cha San et al. (2014) Structures of yeast 80S ribosome-tRNA complexes in the rotated and nonrotated conformations. Structure 22:1210-8
Szyk, Agnieszka; Deaconescu, Alexandra M; Spector, Jeffrey et al. (2014) Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase. Cell 157:1405-15
Anthony, Kelsey C; You, Changjiang; Piehler, Jacob et al. (2014) High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies. Structure 22:628-35
Bigalke, Janna M; Heuser, Thomas; Nicastro, Daniela et al. (2014) Membrane deformation and scission by the HSV-1 nuclear egress complex. Nat Commun 5:4131
Kuang, Yi; Long, Marcus J C; Zhou, Jie et al. (2014) Prion-like nanofibrils of small molecules (PriSM) selectively inhibit cancer cells by impeding cytoskeleton dynamics. J Biol Chem 289:29208-18
Koh, Cha San; Brilot, Axel F; Grigorieff, Nikolaus et al. (2014) Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA-like structural element in the ribosomal decoding center. Proc Natl Acad Sci U S A 111:9139-44
Eisele, Dörthe M; Arias, Dylan H; Fu, Xiaofeng et al. (2014) Robust excitons inhabit soft supramolecular nanotubes. Proc Natl Acad Sci U S A 111:E3367-75

Showing the most recent 10 out of 123 publications