A number of new technologies have been developed from our work over the last few years that are now at the core of our discovery efforts in the areas of HIV/AIDS and cancer biology. The development of the technological infrastructure for 3D imaging in our laboratory has enabled the analysis, over the last year, of numerous HIV-1/SIV spike and spike complex structures. Major improvements in speed and resolution allow us to operate round the clock, remotely, and with average instrument uptimes of 85% for data collection. We have further refined this pipeline by adding tools for separating distinct conformations using in-line, unsupervised classification procedures. We have reported new data processing strategies that allow clear separation of the closed and open states, as well as unliganded and antibody liganded states of trimeric Env when they are present in mixtures. Improvements in processing single particle images have led to the first sub-nanometer ( 9 Angstrom) resolution structure of the trimeric HIV-1 envelope glycoprotein in its activated state, at a vulnerable stage prior to entry. Similarly, new methods we have developed for imaging whole cells have revolutionized the type of information we can glean about the nature of cell-cell contacts relevant to HIV transmission between infected and uninfected cells. The 3D images we now obtain routinely at resolutions that are two orders of magnitude higher than that possible by light microscopy have led to paradigm-shifting insights into virus transmission and the cellular mechanisms that are relevant to mediating effective cell-cell transfer of HIV.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010826-06
Application #
8552848
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2012
Total Cost
$905,109
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Patwardhan, Ardan; Brandt, Robert; Butcher, Sarah J et al. (2017) Building bridges between cellular and molecular structural biology. Elife 6:
Merk, Alan; Bartesaghi, Alberto; Banerjee, Soojay et al. (2016) Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery. Cell 165:1698-1707
Ognjenovi?, Jana; Wu, Jiang; Matthies, Doreen et al. (2016) The crystal structure of human GlnRS provides basis for the development of neurological disorders. Nucleic Acids Res 44:3420-31
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Subramaniam, Sriram; Kühlbrandt, Werner; Henderson, Richard (2016) CryoEM at IUCrJ: a new era. IUCrJ 3:3-7
Bartesaghi, Alberto; Merk, Alan; Banerjee, Soojay et al. (2015) 2.2 Å resolution cryo-EM structure of ?-galactosidase in complex with a cell-permeant inhibitor. Science 348:1147-51
Narayan, Kedar; Danielson, Cindy M; Lagarec, Ken et al. (2014) Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology. J Struct Biol 185:278-84
Patwardhan, Ardan; Ashton, Alun; Brandt, Robert et al. (2014) A 3D cellular context for the macromolecular world. Nat Struct Mol Biol 21:841-5

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