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 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 Reducing effects of particle adsorption to the air-water interface in cryoEM

2018 ◽  
Author(s):  
Alex J. Noble ◽  
Hui Wei ◽  
Venkata P. Dandey ◽  
Zhening Zhang ◽  
Clinton S. Potter ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Dwell Time ◽  
Liquid Films ◽  
Thin Liquid Films ◽  
Water Interface ◽  
Cryo Electron Microscopy ◽  
Air Water Interface ◽  
Protein Particles ◽  
Particle Adsorption

AbstractMost protein particles prepared in vitreous ice for single particle cryo-electron microscopy are adsorbed to air-water or substrate-water interfaces, potentially causing particles to adopt preferred orientations. Using the Spotiton robot and nanowire grids, we can significantly reduce air-water interface issues by decreasing the dwell time of particles in thin liquid films. We demonstrate this by using single particle cryoEM and cryoET on three biological samples.

scholarly journals Collection, pre-processing and on-the-fly analysis of data for high-resolution, single-particle cryo-electron microscopy

2018 ◽  
Vol 14 (1) ◽  
pp. 100-118 ◽  
Author(s):  
Rebecca F. Thompson ◽  
Matthew G. Iadanza ◽  
Emma L. Hesketh ◽  
Shaun Rawson ◽  
Neil A. Ranson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Single Particle ◽  
Cryo Electron Microscopy ◽  
Resolution Single

scholarly journals A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein

2021 ◽  
Author(s):  
Hongcheng Fan ◽  
Bo Wang ◽  
Yan Zhang ◽  
Yun Zhu ◽  
Bo Song ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Electrostatic Interactions ◽  
Preferred Orientation ◽  
Particle Orientation ◽  
Quality Data ◽  
Water Interface ◽  
Small Proteins ◽  
Cryo Electron Microscopy ◽  
Air Water Interface

Cryo-electron microscopy (cryo-EM) has become the most powerful tool to resolve the high-resolution structures of biomacromolecules in solution. However, the air-water interface induced preferred orientation, dissociation or denaturation of biomacromolecules during cryo-vitrification is still a major limitation factor for many specimens. To solve this bottleneck, we developed a new type of cryo-EM support film using the 2D crystal of hydrophobin I (HFBI) protein. The HFBI-film utilizes its hydrophilic side to adsorb protein particles via electrostatic interactions and keep air-water interface away, allowing thin-enough ice and high-quality data collection. The particle orientation distribution can be optimized by changing the buffer pH. We, for the first time, solved the cryo-EM structure of catalase (2.28 Å) that exhibited strong preferred orientation using conventional cryo-vitrification protocol. We further proved the HFBI-film is suitable to solve the high-resolution structures of small proteins including aldolase (150 kDa, 3.34 Å) and hemoglobin (64 kDa, 3.6 Å). Our work implied that the HFBI-film will have a wide application in the future to increase the successful rate and efficiency of cryo-EM.

scholarly journals High resolution single particle cryo-electron microscopy using beam-image shift

2018 ◽  
Vol 204 (2) ◽  
pp. 270-275 ◽  
Author(s):  
Anchi Cheng ◽  
Edward T. Eng ◽  
Lambertus Alink ◽  
William J. Rice ◽  
Kelsey D. Jordan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Single Particle ◽  
Cryo Electron Microscopy ◽  
Beam Image ◽  
Resolution Single

scholarly journals A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hongcheng Fan ◽  
Bo Wang ◽  
Yan Zhang ◽  
Yun Zhu ◽  
Bo Song ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Electrostatic Interactions ◽  
Particle Orientation ◽  
Quality Data ◽  
Water Interface ◽  
Small Proteins ◽  
Cryo Electron Microscopy ◽  
Air Water Interface ◽  
2D Crystals

AbstractCryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.

How Good Can Single-Particle Cryo-EM Become? What Remains Before It Approaches Its Physical Limits?

2019 ◽  
Vol 48 (1) ◽  
pp. 45-61 ◽  
Author(s):  
Robert M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Single Particle ◽  
Energy Use ◽  
Optimal Choice ◽  
Structural Basis ◽  
Detective Quantum Efficiency ◽  
Quantum Metrology ◽  
Cryo Electron Microscopy ◽  
Induced Motion

Impressive though the achievements of single-particle cryo–electron microscopy are today, a substantial gap still remains between what is currently accomplished and what is theoretically possible. As is reviewed here, twofold or more improvements are possible as regards ( a) the detective quantum efficiency of cameras at high resolution, ( b) converting phase modulations to intensity modulations in the image, and ( c) recovering the full amount of high-resolution signal in the presence of beam-induced motion of the specimen. In addition, potential for improvement is reviewed for other topics such as optimal choice of electron energy, use of aberration correctors, and quantum metrology. With the help of such improvements, it does not seem to be too much to imagine that determining the structural basis for every aspect of catalytic control, signaling, and regulation, in any type of cell of interest, could easily be accelerated fivefold or more.

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
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