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- PDB-8cqd: Cryo-EM structure of hexameric proteorhodopsin A18L mutant -

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Basic information

Entry
Database: PDB / ID: 8cqd
TitleCryo-EM structure of hexameric proteorhodopsin A18L mutant
ComponentsGreen-light absorbing proteorhodopsin
KeywordsPROTON TRANSPORT / Membrane protein / Light-driven proton pump / Proteorhodopsin
Function / homology
Function and homology information


light-activated monoatomic ion channel activity / photoreceptor activity / phototransduction / plasma membrane
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 / Green-light absorbing proteorhodopsin
Similarity search - Component
Biological speciesuncultured Gammaproteobacteria bacterium (environmental samples)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsHirschi, S. / Lemmin, T. / Fotiadis, D.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Commun / Year: 2024
Title: Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging.
Authors: Stephan Hirschi / Thomas Lemmin / Nooraldeen Ayoub / David Kalbermatter / Daniele Pellegata / Zöhre Ucurum / Jürg Gertsch / Dimitrios Fotiadis /
Abstract: Microbial ion-pumping rhodopsins (MRs) are extensively studied retinal-binding membrane proteins. However, their biogenesis, including oligomerisation and retinal incorporation, remains poorly ...Microbial ion-pumping rhodopsins (MRs) are extensively studied retinal-binding membrane proteins. However, their biogenesis, including oligomerisation and retinal incorporation, remains poorly understood. The bacterial green-light absorbing proton pump proteorhodopsin (GPR) has emerged as a model protein for MRs and is used here to address these open questions using cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations. Specifically, conflicting studies regarding GPR stoichiometry reported pentamer and hexamer mixtures without providing possible assembly mechanisms. We report the pentameric and hexameric cryo-EM structures of a GPR mutant, uncovering the role of the unprocessed N-terminal signal peptide in the assembly of hexameric GPR. Furthermore, certain proteorhodopsin-expressing bacteria lack retinal biosynthesis pathways, suggesting that they scavenge the cofactor from their environment. We shed light on this hypothesis by solving the cryo-EM structure of retinal-free proteoopsin, which together with mass spectrometry and MD simulations suggests that decanoate serves as a temporary placeholder for retinal in the chromophore binding pocket. Further MD simulations elucidate possible pathways for the exchange of decanoate and retinal, offering a mechanism for retinal scavenging. Collectively, our findings provide insights into the biogenesis of MRs, including their oligomeric assembly, variations in protomer stoichiometry and retinal incorporation through a potential cofactor scavenging mechanism.
History
DepositionMar 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 3, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Green-light absorbing proteorhodopsin
B: Green-light absorbing proteorhodopsin
C: Green-light absorbing proteorhodopsin
E: Green-light absorbing proteorhodopsin
F: Green-light absorbing proteorhodopsin
D: Green-light absorbing proteorhodopsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,37212
Polymers168,6656
Non-polymers1,7076
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18600 Å2
ΔGint-144 kcal/mol
Surface area52900 Å2

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Components

#1: Protein
Green-light absorbing proteorhodopsin / GPR


Mass: 28110.854 Da / Num. of mol.: 6 / Mutation: A18L
Source method: isolated from a genetically manipulated source
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.: 6 / 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: Hexameric proteorhodopsin A18L / 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.: 4.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 49.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: cryoSPARC / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 237013 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211114
ELECTRON MICROSCOPYf_angle_d0.39215174
ELECTRON MICROSCOPYf_dihedral_angle_d9.233652
ELECTRON MICROSCOPYf_chiral_restr0.0351716
ELECTRON MICROSCOPYf_plane_restr0.0031788

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