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- EMDB-53519: Cryo-EM structure of the flotillin-associated rhodopsin PsFAR in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-53519
TitleCryo-EM structure of the flotillin-associated rhodopsin PsFAR in detergent micelle
Map dataSharpened map used for manual and automatic structure refinement
Sample
  • Complex: flotillin-associated rhodopsin PsFAR
    • Protein or peptide: flotillin-associated rhodopsin
  • Ligand: EICOSANE
  • Ligand: water
Keywordsflotillin / rhodopsin / retinal / MEMBRANE PROTEIN
Biological speciesCandidatus Pseudothioglobus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.56 Å
AuthorsKovalev K / Stetsenko A / Marin E / Guskov A
Funding support Netherlands, Germany, 3 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
Netherlands Organisation for Scientific Research (NWO)OCENW.KLEIN.141 Netherlands
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND847543 Germany
CitationJournal: Structure / Year: 2025
Title: Structural basis for no retinal binding in flotillin-associated rhodopsins.
Authors: Kirill Kovalev / Artem Stetsenko / Florian Trunk / Egor Marin / Jose M Haro-Moreno / Gerrit H U Lamm / Alexey Alekseev / Francisco Rodriguez-Valera / Thomas R Schneider / Josef Wachtveitl / Albert Guskov /
Abstract: Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins lack the conserved lysine and ...Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins lack the conserved lysine and showed no retinal binding. Recently, flotillin-associated rhodopsins (FArhodopsins or FARs) were identified and suggested to lack the retinal-binding pocket despite preserving the lysine residue in many members of the group. Here, we present cryoelectron microscopic (cryo-EM) structures of paralog FArhodopsin and proteorhodopsin from marine bacterium Pseudothioglobus, both forming pentamers similar to those of other microbial rhodopsins. We demonstrate no binding of retinal to the FArhodopsin despite preservation of the lysine residue and overall similarity of the protein fold and internal organization to those of the retinal-binding paralog. Mutational analysis confirmed that two amino acids, H84 and E120, prevent retinal binding within the FArhodopsin. Our work provides insights into the natural retinal loss in microbial rhodopsins and might contribute to the further understanding of FArhodopsins.
History
DepositionApr 29, 2025-
Header (metadata) releaseJul 23, 2025-
Map releaseJul 23, 2025-
UpdateSep 17, 2025-
Current statusSep 17, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53519.png

Downloads & links

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Map

FileDownload / File: emd_53519.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map used for manual and automatic structure refinement
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 384 pix.
= 321.024 Å		0.84 Å/pix.
x 384 pix.
= 321.024 Å		0.84 Å/pix.
x 384 pix.
= 321.024 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.836 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.0834544 - 1.5537978
Average (Standard dev.)-0.000014945021 (±0.02927946)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 321.02402 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Volume map used for validation against coordinates

Fileemd_53519_additional_1.map
AnnotationVolume map used for validation against coordinates
Projections & Slices
AxesZYX

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Half map: Half map A

Fileemd_53519_half_map_1.map
AnnotationHalf map A
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Half map: Half map B

Fileemd_53519_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : flotillin-associated rhodopsin PsFAR

EntireName: flotillin-associated rhodopsin PsFAR
Components
  • Complex: flotillin-associated rhodopsin PsFAR
    • Protein or peptide: flotillin-associated rhodopsin
  • Ligand: EICOSANE
  • Ligand: water

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Supramolecule #1: flotillin-associated rhodopsin PsFAR

SupramoleculeName: flotillin-associated rhodopsin PsFAR / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: pentamer of the protein in detergent micelle
Source (natural)Organism: Candidatus Pseudothioglobus (bacteria)
Molecular weightTheoretical: 135 KDa

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Macromolecule #1: flotillin-associated rhodopsin

MacromoleculeName: flotillin-associated rhodopsin / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Pseudothioglobus (bacteria)
Molecular weightTheoretical: 27.518244 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNSTLLPTDI VGGTFWLLSM ALIGASIFFL LERNRVDGRW HTTMTLLGVT MLISAIFYYY VQGMWVDTGK APIVLRYLDW ILTHSMQVV MFYVILTAVT KVSSALFWRL LIGALVMVIG EFLGAAGYMS ATLGFIIGVV GWLYILGEIY MGEASRCNIE S GNEATHMA ...String:
MNSTLLPTDI VGGTFWLLSM ALIGASIFFL LERNRVDGRW HTTMTLLGVT MLISAIFYYY VQGMWVDTGK APIVLRYLDW ILTHSMQVV MFYVILTAVT KVSSALFWRL LIGALVMVIG EFLGAAGYMS ATLGFIIGVV GWLYILGEIY MGEASRCNIE S GNEATHMA FNGLRLILTI GWAIYPLGYF INNLGGGVDA NSLNIIYNLT DFLNKIIFGF VVYRAAMNDT QARLDEIKKL EH HHHHH

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Macromolecule #2: EICOSANE

MacromoleculeName: EICOSANE / type: ligand / ID: 2 / Number of copies: 81 / Formula: LFA
Molecular weightTheoretical: 282.547 Da
Chemical component information

ChemComp-LFA:
EICOSANE

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 97 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration10 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting 4-6 second, force 0.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.56 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 212403
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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