---
_id: '9205'
abstract:
- lang: eng
text: Cryo-EM grid preparation is an important bottleneck in protein structure determination,
especially for membrane proteins, typically requiring screening of a large number
of conditions. We systematically investigated the effects of buffer components,
blotting conditions and grid types on the outcome of grid preparation of five
different membrane protein samples. Aggregation was the most common type of problem
which was addressed by changing detergents, salt concentration or reconstitution
of proteins into nanodiscs or amphipols. We show that the optimal concentration
of detergent is between 0.05 and 0.4% and that the presence of a low concentration
of detergent with a high critical micellar concentration protects the proteins
from denaturation at the air-water interface. Furthermore, we discuss the strategies
for achieving an adequate ice thickness, particle coverage and orientation distribution
on free ice and on support films. Our findings provide a clear roadmap for comprehensive
screening of conditions for cryo-EM grid preparation of membrane proteins.
acknowledged_ssus:
- _id: EM-Fac
acknowledgement: We thank the Electron Microscopy Facilities at the Institute of Science
and Technology Austria and at the Vienna Biocenter for providing access and training
for the electron microscopes. This project has received funding from the European
Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie
Grant Agreement no. 665385 .
article_number: '102139'
article_processing_charge: No
article_type: original
author:
- first_name: Domen
full_name: Kampjut, Domen
id: 37233050-F248-11E8-B48F-1D18A9856A87
last_name: Kampjut
- first_name: Julia
full_name: Steiner, Julia
id: 3BB67EB0-F248-11E8-B48F-1D18A9856A87
last_name: Steiner
- first_name: Leonid A
full_name: Sazanov, Leonid A
id: 338D39FE-F248-11E8-B48F-1D18A9856A87
last_name: Sazanov
orcid: 0000-0002-0977-7989
citation:
ama: Kampjut D, Steiner J, Sazanov LA. Cryo-EM grid optimization for membrane proteins.
iScience. 2021;24(3). doi:10.1016/j.isci.2021.102139
apa: Kampjut, D., Steiner, J., & Sazanov, L. A. (2021). Cryo-EM grid optimization
for membrane proteins. IScience. Elsevier. https://doi.org/10.1016/j.isci.2021.102139
chicago: Kampjut, Domen, Julia Steiner, and Leonid A Sazanov. “Cryo-EM Grid Optimization
for Membrane Proteins.” IScience. Elsevier, 2021. https://doi.org/10.1016/j.isci.2021.102139.
ieee: D. Kampjut, J. Steiner, and L. A. Sazanov, “Cryo-EM grid optimization for
membrane proteins,” iScience, vol. 24, no. 3. Elsevier, 2021.
ista: Kampjut D, Steiner J, Sazanov LA. 2021. Cryo-EM grid optimization for membrane
proteins. iScience. 24(3), 102139.
mla: Kampjut, Domen, et al. “Cryo-EM Grid Optimization for Membrane Proteins.” IScience,
vol. 24, no. 3, 102139, Elsevier, 2021, doi:10.1016/j.isci.2021.102139.
short: D. Kampjut, J. Steiner, L.A. Sazanov, IScience 24 (2021).
date_created: 2021-02-28T23:01:24Z
date_published: 2021-03-19T00:00:00Z
date_updated: 2023-08-07T13:54:06Z
day: '19'
ddc:
- '570'
department:
- _id: LeSa
doi: 10.1016/j.isci.2021.102139
ec_funded: 1
external_id:
isi:
- '000631646000012'
pmid:
- '33665558'
file:
- access_level: open_access
checksum: 50585447386fe5842f07ab9b3a66e7e9
content_type: application/pdf
creator: dernst
date_created: 2021-03-03T07:38:14Z
date_updated: 2021-03-03T07:38:14Z
file_id: '9219'
file_name: 2021_iScience_Kampjut.pdf
file_size: 7431411
relation: main_file
success: 1
file_date_updated: 2021-03-03T07:38:14Z
has_accepted_license: '1'
intvolume: ' 24'
isi: 1
issue: '3'
language:
- iso: eng
month: '03'
oa: 1
oa_version: Published Version
pmid: 1
project:
- _id: 2564DBCA-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '665385'
name: International IST Doctoral Program
publication: iScience
publication_identifier:
eissn:
- '25890042'
publication_status: published
publisher: Elsevier
quality_controlled: '1'
scopus_import: '1'
status: public
title: Cryo-EM grid optimization for membrane proteins
tmp:
image: /images/cc_by_nc_nd.png
legal_code_url: https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode
name: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
(CC BY-NC-ND 4.0)
short: CC BY-NC-ND (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 24
year: '2021'
...