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 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 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 High-resolution cryo-EM using beam-image shift at 200 keV

2020 ◽  
Author(s):  
Jennifer N. Cash ◽  
Sarah Kearns ◽  
Yilai Li ◽  
Michael A. Cianfrocco
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Data Collection ◽  
Data Quality ◽  
Single Particle ◽  
Aberration Correction ◽  
Cryo Electron Microscopy ◽  
Beam Image ◽  
Recent Advances ◽  
Shift Data

ABSTRACTRecent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilizes beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image shift data collection affects data quality on 200 keV instruments and how much aberrations can be computationally corrected. To test this, we collected and analyzed a cryo-EM dataset of aldolase at 200 keV using beam-image shift. This analysis shows that beam tilt on the instrument initially limited the resolution of aldolase to 4.9Å. After iterative rounds of aberration correction and particle polishing in RELION, we were able to obtain a 2.8Å structure. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV.

scholarly journals High-resolution cryo-EM using beam-image shift at 200 keV

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 1179-1187 ◽  
Author(s):  
Jennifer N. Cash ◽  
Sarah Kearns ◽  
Yilai Li ◽  
Michael A. Cianfrocco
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Data Collection ◽  
Single Particle ◽  
Particle Motion ◽  
Data Set ◽  
Cryo Electron Microscopy ◽  
Beam Image ◽  
Shift Data ◽  
Resolution Structure

Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200 keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200 keV using beam-image shift and analyzed. This analysis shows that the instrument beam tilt and particle motion initially limited the resolution to 4.9 Å. After particle polishing and iterative rounds of aberration correction in RELION, a 2.8 Å resolution structure could be obtained. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV.

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

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 High-Resolution Insights into Nascent-Chain Conformation by Single-Particle Cryo-Electron Microscopy

2021 ◽  
Vol 120 (3) ◽  
pp. 296a
Author(s):  
Meranda Masse ◽  
Christopher Morgan ◽  
Wanting Wei ◽  
Edward W. Yu ◽  
Silvia Cavagnero
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Single Particle ◽  
Chain Conformation ◽  
Cryo Electron Microscopy ◽  
Nascent Chain

scholarly journals Approaches to altering particle distributions in cryo-electron microscopy sample preparation

2018 ◽  
Vol 74 (6) ◽  
pp. 560-571 ◽  
Author(s):  
Ieva Drulyte ◽  
Rachel M. Johnson ◽  
Emma L. Hesketh ◽  
Daniel L. Hurdiss ◽  
Charlotte A. Scarff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Sample Preparation ◽  
Single Particle ◽  
Structural Information ◽  
Particle Analysis ◽  
Diverse Range ◽  
Sample Distribution ◽  
High Resolution Data ◽  
Cryo Electron Microscopy

Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the sample-preparation stage; obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the sample itself (buffer components, labile complexes), sample distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid-making process within and between batches. This review outlines a number of methodologies used within the electron-microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high-resolution data collection.

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