2015
DOI: 10.3791/52311
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Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Abstract: Citation: Cabra, V., Samsó, M. Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction. J. Vis. Exp. (95), e52311, doi:10.3791/52311 (2015). AbstractCryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of ch… Show more

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Cited by 14 publications
(18 citation statements)
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“…Consequently, the extracted free-energy profile should be a representation of the system at that temperature. However, freezing takes on the order of s 55 to complete, so all relaxation processes faster than this timescale are lost. Since vitrification is not instantaneous, cooling might depopulate the barrier and cause the estimated barrier to be artificially large.…”
Section: Discussionmentioning
confidence: 99%
“…Consequently, the extracted free-energy profile should be a representation of the system at that temperature. However, freezing takes on the order of s 55 to complete, so all relaxation processes faster than this timescale are lost. Since vitrification is not instantaneous, cooling might depopulate the barrier and cause the estimated barrier to be artificially large.…”
Section: Discussionmentioning
confidence: 99%
“…This protocol will go through the required microscope settings to image a vitrified protein sample and minimize exposure to a dose of ~ 20 electrons/Å 2 . For sample grid transfer and loading the holder in the microscope refer to the following cryo-EM resources (42, 43). …”
Section: Methodsmentioning
confidence: 99%
“…Solubilized RyRs became a very good test specimen for the new “single particle” 3D reconstruction techniques whereby thousands of TEM images of the protein in different views are computationally combined to form a 3D reconstruction . Single particle techniques reach their maximum potential when used in combination with cryo‐electron microscopy (cryoEM), which by imaging the specimen without any staining and under frozen‐hydrated conditions, gives access to the structure of the protein fully hydrated and in physiological buffer . The first 3D reconstruction from negatively stained RyR using the “random conical” algorithm obtained in 1989 was soon improved by cryo EM; RyR1 was also the showcase for the “angular reconstitution” algorithm .…”
Section: Ryr As a Showcase Protein For Cryoemmentioning
confidence: 99%
“…31 Single particle techniques reach their maximum potential when used in combination with cryoelectron microscopy (cryoEM), 32,33 which by imaging the specimen without any staining and under frozen-hydrated conditions, gives access to the structure of the protein fully hydrated and in physiological buffer. 34,35 The first 3D reconstruction from negatively stained RyR using the "random conical" algorithm obtained in 1989 36 was soon improved by cryo EM; 37 RyR1 was also the showcase for the "angular reconstitution" algorithm. 38 This was followed by 3D difference mapping to localize several important domains: divergent regions between RyR isoforms, sequence regions, and biological ligands that modulate RyR's channel properties.…”
Section: Ryr As a Showcase Protein For Cryoemmentioning
confidence: 99%