EMD-51744
Cryo-EM map conventional T20S proteasome
EMD-51744
Single-particle2.6 Å
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Map released: 11/12/2024
Last modified: 11/12/2024
Sample Organism:
Thermoplasma acidophilum
Sample: T20S Proteasome
Deposition Authors: Straub MS
,
Harder OF
,
Mowry NJ
,
Barrass SV
,
Hruby J
,
Drabbels M
,
Lorenz UJ
Sample: T20S Proteasome
Deposition Authors: Straub MS
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Laser Flash Melting Cryo-EM Samples to Overcome Preferred Orientation.
Abstract:
Sample preparation remains a bottleneck for protein structure determination by cryo-electron microscopy. A frequently encountered issue is that proteins adsorb to the air-water interface of the sample in a limited number of orientations. This makes it challenging to obtain high-resolution reconstructions or may even cause projects to fail altogether. We have previously observed that laser flash melting and revitrification of cryo samples reduces preferred orientation for large, symmetric particles. Here, we demonstrate that our method can in fact be used to scramble the orientation of proteins of a range of sizes and symmetries. The effect can be enhanced for some proteins by increasing the heating rate during flash melting or by depositing amorphous ice onto the sample prior to revitrification. This also allows us to shed light onto the underlying mechanism. Our experiments establish a set of tools for overcoming preferred orientation that can be easily integrated into existing workflows.
Sample preparation remains a bottleneck for protein structure determination by cryo-electron microscopy. A frequently encountered issue is that proteins adsorb to the air-water interface of the sample in a limited number of orientations. This makes it challenging to obtain high-resolution reconstructions or may even cause projects to fail altogether. We have previously observed that laser flash melting and revitrification of cryo samples reduces preferred orientation for large, symmetric particles. Here, we demonstrate that our method can in fact be used to scramble the orientation of proteins of a range of sizes and symmetries. The effect can be enhanced for some proteins by increasing the heating rate during flash melting or by depositing amorphous ice onto the sample prior to revitrification. This also allows us to shed light onto the underlying mechanism. Our experiments establish a set of tools for overcoming preferred orientation that can be easily integrated into existing workflows.