scholarly journals A simple and robust procedure for preparing graphene-oxide cryo-EM grids

2018 ◽  
Author(s):  
Eugene Palovcak ◽  
Feng Wang ◽  
Shawn Q. Zheng ◽  
Zanlin Yu ◽  
Sam Li ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
High Resolution ◽  
Motion Correction ◽  
Biological Macromolecules ◽  
Robust Method ◽  
Partial Protection ◽  
Air Water Interface ◽  
Induced Motion ◽  
Resolution Single ◽  
Substrate Film

AbstractGraphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited its widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids are suitable for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and providing partial protection from the air-water interface. Additionally, the signal from images of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.

scholarly journals Amino and PEG-Amino Graphene Oxide Grids Enrich and Protect Samples for High-resolution Single Particle Cryo-electron Microscopy

2019 ◽  
Author(s):  
Feng Wang ◽  
Zanlin Yu ◽  
Miguel Betegon ◽  
Melody Campbell ◽  
Tural Aksel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
High Resolution ◽  
Single Particle ◽  
Particle Distribution ◽  
Water Interface ◽  
Amino Groups ◽  
Cryo Electron Microscopy ◽  
Air Water Interface ◽  
Resolution Single

AbstractCryo-EM samples prepared using the traditional methods often suffer from too few particles, poor particle distribution, or strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.

scholarly journals Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM

2021 ◽  
Author(s):  
Nan Liu ◽  
Liming Zheng ◽  
Jie Xu ◽  
Jia Wang ◽  
Cuixia Hu ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
High Resolution ◽  
Reduced Graphene Oxide ◽  
Interface Problem ◽  
Specimen Preparation ◽  
Simple Method ◽  
Reduced Graphene ◽  
Two Dimensional Materials ◽  
Air Water Interface ◽  
Oxide Membrane

AbstractAlthough single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air-water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100 kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

scholarly journals Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy

2020 ◽  
Vol 209 (2) ◽  
pp. 107437 ◽  
Author(s):  
Feng Wang ◽  
Zanlin Yu ◽  
Miguel Betegon ◽  
Melody G. Campbell ◽  
Tural Aksel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
High Resolution ◽  
Single Particle ◽  
Cryo Electron Microscopy ◽  
Resolution Single

scholarly journals Graphene-Oxide Substrate for High-Resolution Single Particle Cryo-EM

2018 ◽  
Vol 114 (3) ◽  
pp. 11a
Author(s):  
Eugene Palovcak ◽  
David Bulkley ◽  
Shawn Zheng ◽  
Feng Wang ◽  
David Agard ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
High Resolution ◽  
Single Particle ◽  
Oxide Substrate ◽  
Resolution Single

scholarly journals Simplified Approach for Preparing Graphene Oxide TEM Grids for Stained and Vitrified Biomolecules

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 643
Author(s):  
Anil Kumar ◽  
Nayanika Sengupta ◽  
Somnath Dutta
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
High Resolution ◽  
Biological Macromolecules ◽  
Contrast Transfer Function ◽  
Discharge System ◽  
Simplified Approach ◽  
Heavy Equipment ◽  
Peristaltic Pumps ◽  
High Resolution Structure

In this manuscript, we report the application of graphene oxide (GO) in the preparation of cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) grids. We treated GO with water and organic solvents, such as, methanol, ethanol and isopropanol separately to isolate significantly large GO monolayer flake to fabricate the grids for cryo-EM and TEM study. We implemented a simplified approach to isolate flakes of GO monolayer for constructing the TEM grids, independent of expensive heavy equipment (Langmuir–Blodgett trough, glow-discharge system, carbon-evaporator or plasma-cleaner or peristaltic pumps). We employed confocal microscopy, SEM and TEM to characterize the flake size, stability and transparency of the GO monolayer and atomic force microscopy (AFM) to probe the depth of GO coated grids. Additionally, GO grids are visualized at cryogenic condition for suitability of GO monolayer for cryo-EM study. In addition, GO-Met-H2O grids reduce the effect of preferred orientation of biological macromolecules within the amorphous ice. The power-spectrum and contrast-transfer-function unequivocally suggest that GO-Met-H2O fabricated holey grids have excellent potential for application in high-resolution structural characterization of biomolecules. Furthermore, only 200 movies and ~8000 70S ribosome particles are selected on GO-coated grids for cryo-EM reconstruction to achieve high-resolution structure.

scholarly journals Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

2013 ◽  
Vol 10 (6) ◽  
pp. 584-590 ◽  
Author(s):  
Xueming Li ◽  
Paul Mooney ◽  
Shawn Zheng ◽  
Christopher R Booth ◽  
Michael B Braunfeld ◽  
...  
Keyword(s):  
Single Particle ◽  
Motion Correction ◽  
Atomic Resolution ◽  
Electron Counting ◽  
Induced Motion ◽  
Resolution Single
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