The technological novelty is three-fold, i) Establishing a correlative multi-mode imaging system using live cell single particle imaging, super-resolution single molecule imaging, and cryoET imaging advances our previously established correlative live-cell and cryoET methodology to determine HIV-i protein interactions with host cell factors. At one end of the imaging spectrum, the dynamic behavior of a specific target in the context of a living biological system can be characterized with compromised resolution, while at the other end, high resolution structural snapshots of regions of interest can be obtained. Super-resolution molecular imaging bridges these methods and allows identification and localization of specific target proteins associated with cryoET structures. 2) Creating cell lamella in vitreously frozen cells, similar to yo-ultramicrotomy, but without sectioning artifacts, is a novel approach to generate suitably thin vitreous biological specimens and enhances the applicability of cryoET for high resolution 3D structural analysis of native cells and tissues. 3) Using a short fusion peptide with high affinity to a gold surface constitutes a novel approach to overcome some of the difficulties in developing a GFP-equivalent taggable EM label. Ultimately the innovation of the technology program relies on the integration of several approaches developed by us (correlative light microscopy/cryoEM imaging) and by others (super resolution imaging). Our proposed studies build on our prior work and develop new capabilities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center (P50)
Project #
5P50GM082251-07
Application #
8546406
Study Section
Special Emphasis Panel (ZRG1-AARR-K)
Project Start
Project End
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
7
Fiscal Year
2013
Total Cost
$269,010
Indirect Cost
$91,446
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Varlakhanova, Natalia V; Alvarez, Frances J D; Brady, Tyler M et al. (2018) Structures of the fungal dynamin-related protein Vps1 reveal a unique, open helical architecture. J Cell Biol 217:3608-3624
Ning, Jiying; Zhong, Zhou; Fischer, Douglas K et al. (2018) Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid. J Virol 92:
Himes, Benjamin A; Zhang, Peijun (2018) emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging. Nat Methods 15:955-961
Balasubramaniam, Muthukumar; Zhou, Jing; Addai, Amma et al. (2018) PF74 Inhibits HIV-1 Integration by Altering The Composition of the Preintegration Complex. J Virol :
Lu, Manman; Sarkar, Sucharita; Wang, Mingzhang et al. (2018) 19F Magic Angle Spinning NMR Spectroscopy and Density Functional Theory Calculations of Fluorosubstituted Tryptophans: Integrating Experiment and Theory for Accurate Determination of Chemical Shift Tensors. J Phys Chem B 122:6148-6155
Kraus, Jodi; Gupta, Rupal; Yehl, Jenna et al. (2018) Chemical Shifts of the Carbohydrate Binding Domain of Galectin-3 from Magic Angle Spinning NMR and Hybrid Quantum Mechanics/Molecular Mechanics Calculations. J Phys Chem B 122:2931-2939
Quinn, Caitlin M; Wang, Mingzhang; Polenova, Tatyana (2018) NMR of Macromolecular Assemblies and Machines at 1 GHz and Beyond: New Transformative Opportunities for Molecular Structural Biology. Methods Mol Biol 1688:1-35
Hadden, Jodi A; Perilla, Juan R (2018) All-atom virus simulations. Curr Opin Virol 31:82-91
Yan, Junpeng; Shun, Ming-Chieh; Hao, Caili et al. (2018) HIV-1 Vpr Reprograms CLR4DCAF1 E3 Ubiquitin Ligase to Antagonize Exonuclease 1-Mediated Restriction of HIV-1 Infection. MBio 9:
Dick, Robert A; Zadrozny, Kaneil K; Xu, Chaoyi et al. (2018) Inositol phosphates are assembly co-factors for HIV-1. Nature 560:509-512

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