scholarly journals Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

2017 ◽  
Author(s):  
Alex J. Noble ◽  
Venkata P. Dandey ◽  
Hui Wei ◽  
Julia Brasch ◽  
Jillian Chase ◽  
...  
Keyword(s):  
Single Particle ◽  
Particle Distribution ◽  
De Novo ◽  
Protein Denaturation ◽  
Electron Tomography ◽  
Particle Collection ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Air Water Interface ◽  
Particle Alignment

AbstractSingle particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

scholarly journals Routine single particle CryoEM sample and grid characterization by tomography

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Alex J Noble ◽  
Venkata P Dandey ◽  
Hui Wei ◽  
Julia Brasch ◽  
Jillian Chase ◽  
...  
Keyword(s):  
Single Particle ◽  
Particle Distribution ◽  
De Novo ◽  
Protein Denaturation ◽  
Electron Tomography ◽  
Particle Collection ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Air Water Interface ◽  
Particle Alignment

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

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 The advent of structural biology in situ by single particle cryo-electron tomography

2017 ◽  
Vol 3 (1-3) ◽  
pp. 17-35 ◽  
Author(s):  
Jesús G. Galaz-Montoya ◽  
Steven J. Ludtke
Keyword(s):  
Structural Biology ◽  
Single Particle ◽  
Electron Tomography ◽  
Cryo Electron Tomography

Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

2013 ◽  
Vol 126 ◽  
pp. 33-39 ◽  
Author(s):  
Alexander G. Myasnikov ◽  
Zhanna A. Afonina ◽  
Bruno P. Klaholz
Keyword(s):  
Single Particle ◽  
Electron Tomography ◽  
Molecular Assembly ◽  
Cryo Electron Tomography ◽  
Assembly Analysis

scholarly journals Phase Contrast Cryo-Electron Tomography and Single Particle Analysis with a New Phase Plate

2014 ◽  
Vol 20 (S3) ◽  
pp. 232-233
Author(s):  
Maryam Khoshouei ◽  
Radostin Danev ◽  
Günther Gerisch ◽  
Maria Ecke ◽  
Juergen Plitzko ◽  
...  
Keyword(s):  
Single Particle ◽  
Phase Contrast ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Phase Plate ◽  
Single Particle Analysis ◽  
Cryo Electron Tomography ◽  
New Phase

scholarly journals Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography

2016 ◽  
Vol 194 (3) ◽  
pp. 383-394 ◽  
Author(s):  
Jesús G. Galaz-Montoya ◽  
Corey W. Hecksel ◽  
Philip R. Baldwin ◽  
Eryu Wang ◽  
Scott C. Weaver ◽  
...  
Keyword(s):  
Single Particle ◽  
Electron Tomography ◽  
Alignment Algorithms ◽  
Cryo Electron Tomography
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