---
_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'
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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
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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'
...
---
_id: '8284'
abstract:
- lang: eng
text: Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit
Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven
proton pumps, such as respiratory complex I. The mechanism of coupling between
ion or electron transfer and proton translocation in this large protein family
is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus
flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit
protomers with 50 trans-membrane helices each. Surface charge distribution within
each monomer is remarkably asymmetric, revealing probable proton and sodium translocation
pathways. On the basis of the structure we propose a mechanism where the coupling
between sodium and proton translocation is facilitated by a series of electrostatic
interactions between a cation and key charged residues. This mechanism is likely
to be applicable to the entire family of redox proton pumps, where electron transfer
to substrates replaces cation movements.
acknowledged_ssus:
- _id: EM-Fac
- _id: LifeSc
acknowledgement: This research was supported by the Scientific Service Units (SSU)
of IST Austria through resources provided by the Electron Microscopy Facility (EMF),
the Life Science Facility (LSF) and the IST high-performance computing cluster.
We thank Dr Victor-Valentin Hodirnau and Daniel Johann Gütl from IST Austria for
assistance with collecting cryo-EM data. We thank Prof. Masahiro Ito (Graduate School
of Life Sciences, Toyo University, Japan) for a kind provision of plasmid DNA encoding
Mrp from A. flavithermus WK1. JS is a recipient of a DOC Fellowship of the Austrian
Academy of Sciences at the Institute of Science and Technology, Austria.
article_number: e59407
article_processing_charge: No
article_type: original
author:
- first_name: Julia
full_name: Steiner, Julia
id: 3BB67EB0-F248-11E8-B48F-1D18A9856A87
last_name: Steiner
orcid: 0000-0003-0493-3775
- 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: Steiner J, Sazanov LA. Structure and mechanism of the Mrp complex, an ancient
cation/proton antiporter. eLife. 2020;9. doi:10.7554/eLife.59407
apa: Steiner, J., & Sazanov, L. A. (2020). Structure and mechanism of the Mrp
complex, an ancient cation/proton antiporter. ELife. eLife Sciences Publications.
https://doi.org/10.7554/eLife.59407
chicago: Steiner, Julia, and Leonid A Sazanov. “Structure and Mechanism of the Mrp
Complex, an Ancient Cation/Proton Antiporter.” ELife. eLife Sciences Publications,
2020. https://doi.org/10.7554/eLife.59407.
ieee: J. Steiner and L. A. Sazanov, “Structure and mechanism of the Mrp complex,
an ancient cation/proton antiporter,” eLife, vol. 9. eLife Sciences Publications,
2020.
ista: Steiner J, Sazanov LA. 2020. Structure and mechanism of the Mrp complex, an
ancient cation/proton antiporter. eLife. 9, e59407.
mla: Steiner, Julia, and Leonid A. Sazanov. “Structure and Mechanism of the Mrp
Complex, an Ancient Cation/Proton Antiporter.” ELife, vol. 9, e59407, eLife
Sciences Publications, 2020, doi:10.7554/eLife.59407.
short: J. Steiner, L.A. Sazanov, ELife 9 (2020).
date_created: 2020-08-24T06:24:04Z
date_published: 2020-07-31T00:00:00Z
date_updated: 2023-09-07T13:14:08Z
day: '31'
ddc:
- '570'
department:
- _id: LeSa
doi: 10.7554/eLife.59407
external_id:
isi:
- '000562123600001'
pmid:
- '32735215'
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content_type: application/pdf
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date_created: 2020-08-24T13:31:53Z
date_updated: 2020-08-24T13:31:53Z
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file_name: 2020_eLife_Steiner.pdf
file_size: 7320493
relation: main_file
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file_date_updated: 2020-08-24T13:31:53Z
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intvolume: ' 9'
isi: 1
language:
- iso: eng
month: '07'
oa: 1
oa_version: Published Version
pmid: 1
project:
- _id: 26169496-B435-11E9-9278-68D0E5697425
grant_number: '24741'
name: Revealing the functional mechanism of Mrp antiporter, an ancestor of complex
I
publication: eLife
publication_identifier:
eissn:
- 2050084X
publication_status: published
publisher: eLife Sciences Publications
quality_controlled: '1'
related_material:
link:
- description: News on IST Homepage
relation: press_release
url: https://ist.ac.at/en/news/mystery-of-giant-proton-pump-solved/
record:
- id: '8353'
relation: dissertation_contains
status: public
scopus_import: '1'
status: public
title: Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 9
year: '2020'
...
---
_id: '8353'
abstract:
- lang: eng
text: "Mrp (Multi resistance and pH adaptation) are broadly distributed secondary
active antiporters that catalyze the transport of monovalent ions such as sodium
and potassium outside of the cell coupled to the inward translocation of protons.
Mrp antiporters are unique in a way that they are composed of seven subunits (MrpABCDEFG)
encoded in a single operon, whereas other antiporters catalyzing the same reaction
are mostly encoded by a single gene. Mrp exchangers are crucial for intracellular
pH homeostasis and Na+ efflux, essential mechanisms for H+ uptake under alkaline
environments and for reduction of the intracellular concentration of toxic cations.
Mrp displays no homology to any other monovalent Na+(K+)/H+ antiporters but Mrp
subunits have primary sequence similarity to essential redox-driven proton pumps,
such as respiratory complex I and membrane-bound hydrogenases. This similarity
reinforces the hypothesis that these present day redox-driven proton pumps are
descended from the Mrp antiporter. The Mrp structure serves as a model to understand
the yet obscure coupling mechanism between ion or electron transfer and proton
translocation in this large group of proteins. In the thesis, I am presenting
the purification, biochemical analysis, cryo-EM analysis and molecular structure
of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å
resolution. Numerous conditions were screened to purify Mrp to high homogeneity
and to obtain an appropriate distribution of single particles on cryo-EM grids
covered with a continuous layer of ultrathin carbon. A preferred particle orientation
problem was solved by performing a tilted data collection. The activity assays
showed the specific pH-dependent\r\nprofile of secondary active antiporters. The
molecular structure shows that Mrp is a dimer of seven-subunit protomers with
50 trans-membrane helices each. The dimer interface is built by many short and
tilted transmembrane helices, probably causing a thinning of the bacterial membrane.
The surface charge distribution shows an extraordinary asymmetry within each monomer,
revealing presumable proton and sodium translocation pathways. The two largest\r\nand
homologous Mrp subunits MrpA and MrpD probably translocate one proton each into
the cell. The sodium ion is likely being translocated in the opposite direction
within the small subunits along a ladder of charged and conserved residues. Based
on the structure, we propose a mechanism were the antiport activity is accomplished
via electrostatic interactions between the charged cations and key charged residues.
The flexible key TM helices coordinate these\r\nelectrostatic interactions, while
the membrane thinning between the monomers enables the translocation of sodium
across the charged membrane. The entire family of redox-driven proton pumps is
likely to perform their mechanism in a likewise manner."
acknowledged_ssus:
- _id: LifeSc
- _id: EM-Fac
- _id: ScienComp
acknowledgement: "I acknowledge the scientific service units of the IST Austria for
providing resources by the Life Science Facility, the Electron Microscopy Facility
and the high-performance computer cluster. Special thanks to the cryo-EM specialists
Valentin Hodirnau and Daniel Johann Gütl for spending many hours with me in front
of the microscope and for supporting me to collect the data presented here. I also
want to thank Professor Masahiro Ito for providing plasmid DNA\r\nencoding Mrp from
Anoxybacillus flavithermus WK1. I am a recipient of a DOC Fellowship of the Austrian
Academy of Sciences."
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Julia
full_name: Steiner, Julia
id: 3BB67EB0-F248-11E8-B48F-1D18A9856A87
last_name: Steiner
orcid: 0000-0003-0493-3775
citation:
ama: Steiner J. Biochemical and structural investigation of the Mrp antiporter,
an ancestor of complex I. 2020. doi:10.15479/AT:ISTA:8353
apa: Steiner, J. (2020). Biochemical and structural investigation of the Mrp
antiporter, an ancestor of complex I. Institute of Science and Technology
Austria. https://doi.org/10.15479/AT:ISTA:8353
chicago: Steiner, Julia. “Biochemical and Structural Investigation of the Mrp Antiporter,
an Ancestor of Complex I.” Institute of Science and Technology Austria, 2020.
https://doi.org/10.15479/AT:ISTA:8353.
ieee: J. Steiner, “Biochemical and structural investigation of the Mrp antiporter,
an ancestor of complex I,” Institute of Science and Technology Austria, 2020.
ista: Steiner J. 2020. Biochemical and structural investigation of the Mrp antiporter,
an ancestor of complex I. Institute of Science and Technology Austria.
mla: Steiner, Julia. Biochemical and Structural Investigation of the Mrp Antiporter,
an Ancestor of Complex I. Institute of Science and Technology Austria, 2020,
doi:10.15479/AT:ISTA:8353.
short: J. Steiner, Biochemical and Structural Investigation of the Mrp Antiporter,
an Ancestor of Complex I, Institute of Science and Technology Austria, 2020.
date_created: 2020-09-09T14:27:01Z
date_published: 2020-09-09T00:00:00Z
date_updated: 2023-09-07T13:14:09Z
day: '09'
ddc:
- '572'
degree_awarded: PhD
department:
- _id: LeSa
doi: 10.15479/AT:ISTA:8353
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grant_number: '24741'
name: Revealing the functional mechanism of Mrp antiporter, an ancestor of complex
I
publication_identifier:
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publication_status: published
publisher: Institute of Science and Technology Austria
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status: public
supervisor:
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full_name: Sazanov, Leonid A
id: 338D39FE-F248-11E8-B48F-1D18A9856A87
last_name: Sazanov
orcid: 0000-0002-0977-7989
title: Biochemical and structural investigation of the Mrp antiporter, an ancestor
of complex I
type: dissertation
user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1
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...