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| Title | Laser Flash Melting Cryo-EM Samples to Overcome Preferred Orientation. |
|---|---|
| Journal, issue, pages | bioRxiv, Year 2024 |
| Publish date | Nov 21, 2024 |
 Authors | Monique S Straub / Oliver F Harder / Nathan J Mowry / Sarah V Barrass / Jakub Hruby / Marcel Drabbels / Ulrich J Lorenz /  |
| PubMed 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 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. |
 External links | bioRxiv / PubMed:39605560 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 2.5 - 4.1 Å |
| Structure data |  EMDB-51744: Cryo-EM map conventional T20S proteasome  EMDB-51745: Cryo-EM map of revitrified T20S proteasome  EMDB-51746: Cryo-EM map of deposited and revitrified T20S proteasome  EMDB-51747: Cryo-EM map of conventional 50S ribosomal subunit  EMDB-51748: Cryo-EM map of revitrified 50S ribosomal subunit  EMDB-51749: Cryo-EM map of deposited and revitrified 50S ribosomal subunit  EMDB-51750: Cryo-EM map of conventional 50S ribosomal subunit used as control for shaped pulses  EMDB-51751: Cryo-EM map of shaped pulse revitrified 50S ribosomal subunit  EMDB-51752: Cryo-EM map of conventional HIV-1 envelope protein  EMDB-51753: Cryo-EM map of revitrified HIV-1 envelope protein  EMDB-51754: Cryo-EM map of conventional Hemagglutinin  EMDB-51755: Cryo-EM map of revitrified Hemagglutinin  EMDB-51756: Cryo-EM map of deposited and revitrified Hemagglutinin  EMDB-51757: Cryo-EM map of shaped pulse revitrified Hemagglutinin |
| Source |
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Thermoplasma acidophilum (acidophilic)
E.coli K12 strain BW25113 (bacteria)
Human immunodeficiency virus 1
Influenza A virus