scholarly journals A simple pressure-assisted method for cryo-EM specimen preparation

2019 ◽  
Author(s):  
J. Zhao ◽  
H. Xu ◽  
M. Carroni ◽  
H. Lebrette ◽  
K. Wallden ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Pressure Gradient ◽  
Structural Biology ◽  
Specimen Preparation ◽  
Single Particles ◽  
Simple Method ◽  
Cryo Electron Microscopy ◽  
Low Concentrations ◽  
Wide Range ◽  
Major Bottleneck

AbstractCryo-electron microscopy (cryo-EM) has made great impacts on structural biology. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing cryo-EM specimens, named Preassis, in which the excess liquid is removed by introducing a pressure gradient through the EM grid. We show the unique advantages of Preassis in handling samples with low concentrations of protein single particles and micro-crystals in a wide range of buffer conditions.

scholarly journals A basic introduction to single particles cryo-electron microscopy

2021 ◽  
Vol 9 (1) ◽  
pp. 5-20
Author(s):  
Vittoria Raimondi ◽  
◽  
Alessandro Grinzato ◽  
◽  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Protein Complexes ◽  
Computational Power ◽  
Single Particles ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
First Choice ◽  
New Generation

<abstract> <p>In the last years, cryogenic-electron microscopy (cryo-EM) underwent the most impressive improvement compared to other techniques used in structural biology, such as X-ray crystallography and NMR. Electron microscopy was invented nearly one century ago but, up to the beginning of the last decades, the 3D maps produced through this technique were poorly detailed, justifying the term “blobbology” to appeal to cryo-EM. Recently, thanks to a new generation of microscopes and detectors, more efficient algorithms, and easier access to computational power, single particles cryo-EM can routinely produce 3D structures at resolutions comparable to those obtained with X-ray crystallography. However, unlike X-ray crystallography, which needs crystallized proteins, cryo-EM exploits purified samples in solution, allowing the study of proteins and protein complexes that are hard or even impossible to crystallize. For these reasons, single-particle cryo-EM is often the first choice of structural biologists today. Nevertheless, before starting a cryo-EM experiment, many drawbacks and limitations must be considered. Moreover, in practice, the process between the purified sample and the final structure could be trickier than initially expected. Based on these observations, this review aims to offer an overview of the principal technical aspects and setups to be considered while planning and performing a cryo-EM experiment.</p> </abstract>

An Improved Holey Carbon Film for Cryo-Electron Microscopy

2007 ◽  
Vol 13 (5) ◽  
pp. 365-371 ◽  
Author(s):  
Joel Quispe ◽  
John Damiano ◽  
Stephen E. Mick ◽  
David P. Nackashi ◽  
Denis Fellmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Particle Distribution ◽  
Carbon Film ◽  
Soft Materials ◽  
Carbon Films ◽  
Water Soluble ◽  
Specimen Preparation ◽  
Dramatic Improvement ◽  
Single Particles ◽  
Cryo Electron Microscopy

Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and minimize their impact during specimen preparation, an improved holey carbon film has been developed. Rather than using a consumable template based on soft materials that must be removed prior to grid assembly, a method was developed that uses a hard template and a water-soluble release layer to replicate the template pattern into the carbon films. The advantages of this method are the improved purity and flatness of the carbon films, and these attributes are shown to have a dramatic improvement on the distribution of single particles embedded in vitreous ice suspended across the holes. Improving particle distribution is an enabling factor toward increasing the throughput of data collection for cryoEM.

scholarly journals A simple pressure-assisted method for MicroED specimen preparation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jingjing Zhao ◽  
Hongyi Xu ◽  
Hugo Lebrette ◽  
Marta Carroni ◽  
Helena Taberman ◽  
...  
Keyword(s):  
High Viscosity ◽  
Specimen Preparation ◽  
Universal Method ◽  
X Ray Diffraction ◽  
Simple Method ◽  
X Ray ◽  
Wide Range ◽  
Major Bottleneck ◽  
Crystal Electron

AbstractMicro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate carbon hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal concentrations. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED.

scholarly journals Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age

2019 ◽  
Vol 75 (1) ◽  
pp. 3-11 ◽  
Author(s):  
Alok K. Mitra
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particles ◽  
Cryo Electron Microscopy ◽  
Electron Microscopes ◽  
Resolution Single ◽  
Resolution Structure

Structural biology is going through a revolution as a result of transformational advances in the field of cryo-electron microscopy (cryo-EM) driven by the development of direct electron detectors and ultrastable electron microscopes. High-resolution cryo-EM images of isolated biomolecules (single particles) suspended in a thin layer of vitrified buffer are subjected to powerful image-processing algorithms, enabling near-atomic resolution structures to be determined in unprecedented numbers. Prior to these advances, electron crystallography of two-dimensional crystals and helical assemblies of proteins had established the feasibility of atomic resolution structure determination using cryo-EM. Atomic resolution single-particle analysis, without the need for crystals, now promises to resolve problems in structural biology that were intractable just a few years ago.

scholarly journals A simple pressure-assisted method for MicroED specimen preparation

2021 ◽  
Author(s):  
Jingjing Zhao ◽  
Hongyi Xu ◽  
Hugo Lebrette ◽  
Marta Carroni ◽  
Helena Taberman ◽  
...  
Keyword(s):  
High Viscosity ◽  
Specimen Preparation ◽  
Universal Method ◽  
X Ray Diffraction ◽  
Simple Method ◽  
X Ray ◽  
Wide Range ◽  
Major Bottleneck ◽  
Crystal Electron

Abstract Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal contents. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Cryo-EM Is a Powerful Tool, But Helical Applications Can Have Pitfalls

Soft Matter ◽  
2021 ◽  
Author(s):  
Edward Egelman ◽  
Fengbin Wang
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Macromolecular Complexes ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Main Technique

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct...

scholarly journals Single-particle cryo-EM—How did it get here and where will it go

Science ◽  
2018 ◽  
Vol 361 (6405) ◽  
pp. 876-880 ◽  
Author(s):  
Yifan Cheng
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Single Particle ◽  
Electron Crystallography ◽  
The Past ◽  
Cryo Electron Microscopy ◽  
Technological Advances ◽  
The Future ◽  
Electron Cryotomography

Cryo–electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.

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