About this Research Topic
Cryogenic electron microscopy (cryo-EM) has emerged as a major structural biology technique during the last decade. In practice, the sample is embedded in a thin layer of vitreous ice before being imaged directly in a transmission electron microscope (TEM) while maintained at liquid nitrogen temperature. A three-dimensional reconstruction can then be calculated from the images acquired. Driven by technological breakthroughs in TEM optics and cameras, automation and optimization of data collection and improvement in image processing procedures, many parts of the workflow are now streamlined and allow for high-resolution structure determination.
Despite these advances, sample preparation remains a major bottleneck for many projects. To obtain a three-dimensional structure, a thin film of an aqueous solution containing the biological macromolecule of interest must be cooled rapidly so that water molecules do not crystallize but form amorphous ice. Within this layer, ideally as thin as possible, the particles must be well spread and partitioned in random orientations. The prevailing method for obtaining such thin specimens has not changed substantially since being first reported several decades ago: a relatively large sample volume (~3 μL) is applied to a cryo-EM grid, the excess liquid is blotted away with filter paper and then the grid is rapidly plunged into a liquid cryogen.
Although undeniably successful, this method also has problems: reproducibility and ice thickness uniformity are generally poor, preferential orientation of the particles within the ice layer is very common and exposure to the large air-water interface formed can lead to partial or apparent total denaturation of the complex. Successful cryo-EM sample preparation and optimisation through empirical testing of parameters can be a long and tedious process. Fortunately, recent advancements have led to a greater awareness of conditions that can be changed to alter the distribution and behaviour of particles on a cryo-EM grid and new techniques and devices are also emerging allowing better control of the whole pipeline.
This Research Topic aims to collate original research, (mini-) review, method and perspective articles relating to sample preparation toward high-resolution cryo-EM, with a great emphasis on the aspects often eluded or only succinctly mentioned in papers. Areas to be covered in this Research Topic may include, but are not limited to:
· Sample biochemistry: sample modification, chemical treatment, buffer optimization, imaging scaffolds
· Grid technologies: chemical and physical treatment, support films etc.
· Vitrification: new devices, multi-sample deposition, automation, etc
· Advanced screening: automated pipeline, quality assessment, etc
Despite these advances, sample preparation remains a major bottleneck for many projects. To obtain a three-dimensional structure, a thin film of an aqueous solution containing the biological macromolecule of interest must be cooled rapidly so that water molecules do not crystallize but form amorphous ice. Within this layer, ideally as thin as possible, the particles must be well spread and partitioned in random orientations. The prevailing method for obtaining such thin specimens has not changed substantially since being first reported several decades ago: a relatively large sample volume (~3 μL) is applied to a cryo-EM grid, the excess liquid is blotted away with filter paper and then the grid is rapidly plunged into a liquid cryogen.
Although undeniably successful, this method also has problems: reproducibility and ice thickness uniformity are generally poor, preferential orientation of the particles within the ice layer is very common and exposure to the large air-water interface formed can lead to partial or apparent total denaturation of the complex. Successful cryo-EM sample preparation and optimisation through empirical testing of parameters can be a long and tedious process. Fortunately, recent advancements have led to a greater awareness of conditions that can be changed to alter the distribution and behaviour of particles on a cryo-EM grid and new techniques and devices are also emerging allowing better control of the whole pipeline.
This Research Topic aims to collate original research, (mini-) review, method and perspective articles relating to sample preparation toward high-resolution cryo-EM, with a great emphasis on the aspects often eluded or only succinctly mentioned in papers. Areas to be covered in this Research Topic may include, but are not limited to:
· Sample biochemistry: sample modification, chemical treatment, buffer optimization, imaging scaffolds
· Grid technologies: chemical and physical treatment, support films etc.
· Vitrification: new devices, multi-sample deposition, automation, etc
· Advanced screening: automated pipeline, quality assessment, etc
Keywords: Cryo-EM, biochemistry, sample preparation, vitrification, high-resolution
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