Version 1
: Received: 16 August 2023 / Approved: 17 August 2023 / Online: 21 August 2023 (09:52:18 CEST)
Version 2
: Received: 21 September 2023 / Approved: 22 September 2023 / Online: 22 September 2023 (13:10:51 CEST)
Vénien-Bryan, C.; Fernandes, C.A.H. Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis. Int. J. Mol. Sci.2023, 24, 14785.
Vénien-Bryan, C.; Fernandes, C.A.H. Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis. Int. J. Mol. Sci. 2023, 24, 14785.
Vénien-Bryan, C.; Fernandes, C.A.H. Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis. Int. J. Mol. Sci.2023, 24, 14785.
Vénien-Bryan, C.; Fernandes, C.A.H. Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis. Int. J. Mol. Sci. 2023, 24, 14785.
Abstract
Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in the last years, it was observed a huge increase in the number of MPs solved by cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB). However, sample preparation remains a significant challenge in the field. Here, we evaluated in the MPs solved by cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å the most critical parameters for sample preparation: i) the surfactant used for protein extraction from the membrane, ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, iii) the vitrification method employed, and iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs, but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures.
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received:
22 September 2023
Commenter:
Carlos Fernandes
Commenter's Conflict of Interests:
Author
Comment:
In the revision version of the manuscript, we have incorporated the majority of the modifications recommended by the reviewers. Notably, we have introduced a new section (Section 2) that offers additional analysis of the size and of the membrane protein families identified in high-resolution reports obtained by cryo-EM (resolution better than 3.0). Furthermore, we have expanded the discussion on strategies to address sample heterogeneity and excess detergent on the protein samples, with a focus on the application of SEC-MALS and transmission electron microscopy analysis of negative-stained samples. Additionally, we have addressed the use of binding partners (Fabs, nanobodies and megabodies) to increase the particle size of small membrane proteins, making then suitable for cryo-EM single-particles. We have thoroughly restructured the Conclusion and Perspectives section (Section 6) aiming to provide valuable guidance for researchers and offer a comprehensive perspective on the future direction of the field.
Commenter: Carlos Fernandes
Commenter's Conflict of Interests: Author