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- PDB-7b03: Cryo-EM structure of the green-light absorbing proteorhodopsin -

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Basic information

Entry
Database: PDB / ID: 7b03
TitleCryo-EM structure of the green-light absorbing proteorhodopsin
ComponentsProteorhodopsin
KeywordsPROTON TRANSPORT / Light-driven proton pump / microbial rhodopsin / retinal
Function / homology
Function and homology information


light-activated monoatomic ion channel activity / : / photoreceptor activity / phototransduction / membrane => GO:0016020
Similarity search - Function
Proteorhodopsin / Bacterial rhodopsins signature 1. / Rhodopsin, retinal binding site / Bacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein
Similarity search - Domain/homology
RETINAL / Proteorhodopsin
Similarity search - Component
Biological speciesuncultured Gammaproteobacteria bacterium (environmental samples)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsHirschi, S. / Kalbermatter, D. / Fotiadis, D.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin.
Authors: Stephan Hirschi / David Kalbermatter / Zöhre Ucurum / Thomas Lemmin / Dimitrios Fotiadis /
Abstract: The green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important ...The green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.
History
DepositionNov 18, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 16, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 14, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Proteorhodopsin
B: Proteorhodopsin
C: Proteorhodopsin
D: Proteorhodopsin
E: Proteorhodopsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)129,48110
Polymers128,0595
Non-polymers1,4225
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12060 Å2
ΔGint-95 kcal/mol
Surface area42830 Å2
MethodPISA

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Components

#1: Protein
Proteorhodopsin


Mass: 25611.842 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Details: Expressed without signal sequence.
Source: (gene. exp.) uncultured Gammaproteobacteria bacterium (environmental samples)
Production host: Escherichia coli (E. coli) / References: UniProt: Q6J4G7
#2: Chemical
ChemComp-RET / RETINAL


Mass: 284.436 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C20H28O / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Pentamer of the green-light absorbing proteorhodopsin / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: uncultured Gammaproteobacteria bacterium (environmental samples)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 1.36 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4.1.13CTF correction
9RELION3.1initial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 717107
Details: Final map was obtained after applying density modification using Resolve Cryo-EM in Phenix.
Symmetry type: POINT

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