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- EMDB-16796: Cryo-EM structure of hexameric proteorhodopsin A18L mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-16796
TitleCryo-EM structure of hexameric proteorhodopsin A18L mutant
Map data
Sample
  • Complex: Hexameric proteorhodopsin A18L
    • Protein or peptide: Green-light absorbing proteorhodopsin
  • Ligand: RETINAL
KeywordsMembrane protein / Light-driven proton pump / Proteorhodopsin / PROTON TRANSPORT
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
Green-light absorbing proteorhodopsin
Similarity search - Component
Biological speciesuncultured Gammaproteobacteria bacterium (environmental samples)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsHirschi S / Lemmin T / Fotiadis D
Funding support Switzerland, 1 items
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, 2023-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16796.png

Downloads & links

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Map

FileDownload / File: emd_16796.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.26 Å/pix.
x 144 pix.
= 180.84 Å		1.26 Å/pix.
x 144 pix.
= 180.84 Å		1.26 Å/pix.
x 144 pix.
= 180.84 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.25583 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.55
Minimum - Maximum-3.4203637 - 4.4730344
Average (Standard dev.)0.030217573 (±0.1684466)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 180.83995 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_16796_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_16796_half_map_2.map
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Sample components

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Entire : Hexameric proteorhodopsin A18L

EntireName: Hexameric proteorhodopsin A18L
Components
  • Complex: Hexameric proteorhodopsin A18L
    • Protein or peptide: Green-light absorbing proteorhodopsin
  • Ligand: RETINAL

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Supramolecule #1: Hexameric proteorhodopsin A18L

SupramoleculeName: Hexameric proteorhodopsin A18L / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: uncultured Gammaproteobacteria bacterium (environmental samples)

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Macromolecule #1: Green-light absorbing proteorhodopsin

MacromoleculeName: Green-light absorbing proteorhodopsin / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: uncultured Gammaproteobacteria bacterium (environmental samples)
Molecular weightTheoretical: 28.110854 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGKLLLILGS VIALPTFLAG GGDLDASDYT GVSFWLVTAA LLASTVFFFV ERDRVSAKWK TSLTVSGLVT GIAFWHYMYM RGVWIETGD SPTVFRYIDW LLTVPLLICE FYLILAAATN VAGSLFKKLL VGSLVMLVFG YMGEAGIMAA WPAFIIGCLA W VYMIYELW ...String:
MGKLLLILGS VIALPTFLAG GGDLDASDYT GVSFWLVTAA LLASTVFFFV ERDRVSAKWK TSLTVSGLVT GIAFWHYMYM RGVWIETGD SPTVFRYIDW LLTVPLLICE FYLILAAATN VAGSLFKKLL VGSLVMLVFG YMGEAGIMAA WPAFIIGCLA W VYMIYELW AGEGKSACNT ASPAVQSAYN TMMYIIIFGW AIYPVGYFTG YLMGDGGSAL NLNLIYNLAD FVNKILFGLI IW NVAVKES SNAGHHHHH

UniProtKB: Green-light absorbing proteorhodopsin

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Macromolecule #2: RETINAL

MacromoleculeName: RETINAL / type: ligand / ID: 2 / Number of copies: 6 / Formula: RET
Molecular weightTheoretical: 284.436 Da
Chemical component information

ChemComp-RET:
RETINAL

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.2 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 49.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 237013
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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