Electronic Bio Imaging Center

Didcot, United Kingdom

Electronic Bio Imaging Center

Didcot, United Kingdom
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Perilla J.R.,University of Illinois at Urbana - Champaign | Zhao G.,University of Pittsburgh | Lu M.,University of Pittsburgh | Lu M.,University of Delaware | And 10 more authors.
Journal of Physical Chemistry B | Year: 2017

Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging. Here, we present a novel integrated strategy to combine the highly complementary experimental data from cryoEM and NMR computationally by molecular dynamics simulations to derive an atomistic model, which is not attainable by either approach alone. We use the HIV-1 capsid protein (CA) C-terminal domain as well as the large capsid assembly to demonstrate the feasibility of this approach, termed NMR CS-biased cryoEM structure refinement. © 2017 American Chemical Society.


Thompson R.F.,University of Leeds | Walker M.,MLW Consulting | Siebert C.A.,Electronic Bio Imaging Center | Muench S.P.,University of Leeds | Ranson N.A.,University of Leeds
Methods | Year: 2016

Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins >150. kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a 'resolution revolution', owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM. © 2016 The Authors.

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