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- PDB-9r21: Cryo-EM structure of the flotillin-associated rhodopsin PsFAR in ... -

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Basic information

Entry
Database: PDB / ID: 9r21
TitleCryo-EM structure of the flotillin-associated rhodopsin PsFAR in detergent micelle
Componentsflotillin-associated rhodopsin
KeywordsMEMBRANE PROTEIN / flotillin / rhodopsin / retinal
Function / homologyEICOSANE
Function and homology information
Biological speciesCandidatus Pseudothioglobus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.56 Å
AuthorsKovalev, K. / Stetsenko, A. / Marin, E. / Guskov, A.
Funding support Netherlands, Germany, 3items
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, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: flotillin-associated rhodopsin
B: flotillin-associated rhodopsin
C: flotillin-associated rhodopsin
D: flotillin-associated rhodopsin
E: flotillin-associated rhodopsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)160,47886
Polymers137,5915
Non-polymers22,88681
Water1,74797
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
flotillin-associated rhodopsin


Mass: 27518.244 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Pseudothioglobus (bacteria) / Production host: Escherichia coli (E. coli)
#2: Chemical...
ChemComp-LFA / EICOSANE / LIPID FRAGMENT


Mass: 282.547 Da / Num. of mol.: 81 / Source method: obtained synthetically / Formula: C20H42
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 97 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationN

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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: flotillin-associated rhodopsin PsFAR / Type: COMPLEX / Details: pentamer of the protein in detergent micelle / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.135 MDa / Experimental value: NO
Source (natural)Organism: Candidatus Pseudothioglobus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288 K / Details: Blotting 4-6 second, force 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212403 / Symmetry type: POINT
RefinementResolution: 2.56→108.68 Å / Cor.coef. Fo:Fc: 0.567 / SU B: 7.248 / SU ML: 0.14 / ESU R: 0.417
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.47041 --
obs0.47041 126248 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 45.843 Å2
Refinement stepCycle: 1 / Total: 10231
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0110.01210273
ELECTRON MICROSCOPYr_bond_other_d00.01610886
ELECTRON MICROSCOPYr_angle_refined_deg2.0941.83813602
ELECTRON MICROSCOPYr_angle_other_deg0.6741.91725044
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.93351175
ELECTRON MICROSCOPYr_dihedral_angle_2_deg15.308545
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.428101525
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0970.21505
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.0211050
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022340
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it5.3823.4224715
ELECTRON MICROSCOPYr_mcbond_other5.3813.4224715
ELECTRON MICROSCOPYr_mcangle_it86.1445885
ELECTRON MICROSCOPYr_mcangle_other86.1475886
ELECTRON MICROSCOPYr_scbond_it7.3024.9895558
ELECTRON MICROSCOPYr_scbond_other7.3024.9895559
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other11.4668.3177718
ELECTRON MICROSCOPYr_long_range_B_refined14.00533.3111765
ELECTRON MICROSCOPYr_long_range_B_other14.00433.3111766
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.56→2.626 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.866 9436 -
obs--100 %

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