scholarly journals High-resolution structure determination by continuous-rotation data collection in MicroED

2014 ◽  
Vol 11 (9) ◽  
pp. 927-930 ◽  
Author(s):  
Brent L Nannenga ◽  
Dan Shi ◽  
Andrew G W Leslie ◽  
Tamir Gonen
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
Structure Determination ◽  
Continuous Rotation ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

FEBS Letters ◽  
2010 ◽  
Vol 584 (12) ◽  
pp. 2539-2547 ◽  
Author(s):  
Yo Sonoda ◽  
Alex Cameron ◽  
Simon Newstead ◽  
Hiroshi Omote ◽  
Yoshinori Moriyama ◽  
...  
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Structure Determination ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals Multifunctional graphene supports for electron cryomicroscopy

2019 ◽  
pp. 201904766 ◽  
Author(s):  
Katerina Naydenova ◽  
Mathew J. Peet ◽  
Christopher J. Russo
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Monolayer Graphene ◽  
Electron Cryomicroscopy ◽  
Support Design ◽  
Modification System ◽  
Technological Advances ◽  
High Resolution Structure ◽  
Resolution Imaging ◽  
Resolution Structure

With recent technological advances, the atomic resolution structure of any purified biomolecular complex can, in principle, be determined by single-particle electron cryomicroscopy (cryoEM). In practice, the primary barrier to structure determination is the preparation of a frozen specimen suitable for high-resolution imaging. To address this, we present a multifunctional specimen support for cryoEM, comprising large-crystal monolayer graphene suspended across the surface of an ultrastable gold specimen support. Using a low-energy plasma surface modification system, we tune the surface of this support to the specimen by patterning a range of covalent functionalizations across the graphene layer on a single grid. This support design reduces specimen movement during imaging, improves image quality, and allows high-resolution structure determination with a minimum of material and data.

scholarly journals High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

2018 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Drug Target ◽  
Phase Plate ◽  
Biological Macromolecules ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
High Resolution Structure ◽  
Resolution Structure

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

scholarly journals Devitrification reduces beam-induced movement in cryo-EM

IUCrJ ◽  
2021 ◽  
Vol 8 (2) ◽  
pp. 186-194
Author(s):  
Jan-Philip Wieferig ◽  
Deryck J. Mills ◽  
Werner Kühlbrandt
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
High Energy ◽  
Full Potential ◽  
High Energy Electrons ◽  
High Resolution Structure ◽  
Image Blurring ◽  
Particle Images ◽  
Resolution Structure

As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent problems has been beam-induced movement, which occurs when the specimen is irradiated with high-energy electrons. Beam-induced movement results in image blurring and loss of high-resolution information. It is particularly severe for biological samples in unsupported thin films of vitreous water. By controlled devitrification of conventionally plunge-frozen samples, the suspended film of vitrified water was converted into cubic ice, a polycrystalline, mechanically stable solid. It is shown that compared with vitrified samples, devitrification reduces beam-induced movement in the first 5 e Å−2 of an exposure by a factor of ∼4, substantially enhancing the contribution of the initial, minimally damaged frames to a structure. A 3D apoferritin map reconstructed from the first frames of 20 000 particle images of devitrified samples resolved undamaged side chains. Devitrification of frozen-hydrated specimens helps to overcome beam-induced specimen motion in single-particle cryo-EM, as a further step towards realizing the full potential of cryo-EM for high-resolution structure determination.

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