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- PDB-9hfk: Cryo-EM structure of the freshwater actinorhodopsin, Rhodoluna la... -

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Basic information

Entry
Database: PDB / ID: 9hfk
TitleCryo-EM structure of the freshwater actinorhodopsin, Rhodoluna lacicola (RlActR)
ComponentsBacteriorhodopsin
KeywordsPROTON TRANSPORT / bacterial / rhodopsin / fresh-water / proton transporter
Function / homology
Function and homology information


photoreceptor activity / phototransduction / monoatomic ion channel activity / membrane
Similarity search - Function
Bacterial rhodopsins signature 1. / Rhodopsin, retinal binding site / Bacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein
Similarity search - Domain/homology
: / : / : / RETINAL / Bacteriorhodopsin
Similarity search - Component
Biological speciesRhodoluna lacicola (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsDjabeur, N. / Jeckelmann, J.-M. / Fotiadis, D.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation205608 Switzerland
CitationJournal: J Mol Biol / Year: 2026
Title: Structural, Mechanistic and Phylogenetic Insights Into a Freshwater Actinorhodopsin.
Authors: Nadia Djabeur / Jean-Marc Jeckelmann / Nooraldeen Ayoub / Daniel Harder / Dimitrios Fotiadis /
Abstract: Actinorhodopsins represent a unique subgroup of microbial rhodopsins, predominantly found in non-marine Actinobacteria and proposed to contribute to the global energy cycle. Despite their ecological ...Actinorhodopsins represent a unique subgroup of microbial rhodopsins, predominantly found in non-marine Actinobacteria and proposed to contribute to the global energy cycle. Despite their ecological significance, structural information on this family has remained scarce. Here, we present the high-resolution three-dimensional structure of the pentameric actinorhodopsin RlActR from the actinobacterium Rhodoluna lacicola, as determined by cryo-electron microscopy and single-particle 3D reconstruction. The structure provides molecular insights into key functional amino acid residues involved in retinal cofactor binding and the proton translocation pathway. In addition to describing the organization of the retinal Schiff base region, we present a comparative analysis of this region in RlActR and in prototypical microbial rhodopsins from two distinct phyla, namely, the green-light-absorbing proteorhodopsin from Bacteria and bacteriorhodopsin from Archaea. We also describe the amino acid interactions at the oligomerization interface that stabilize the pentamer. Furthermore, the structure reveals a pentameric architecture with a lipid-filled central cavity and lipid-occupied, membrane-facing interprotomer crevices, further highlighting molecular interactions that stabilize the assembly. Phylogenetic analysis and structural comparisons with selected microbial rhodopsins exhibiting light-driven proton-pumping activity position RlActR within a distinct group of proton-pumping rhodopsins, underscoring its evolutionary and functional relevance.
History
DepositionNov 17, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 18, 2026Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)161,24525
Polymers150,4365
Non-polymers10,81020
Water00
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
Bacteriorhodopsin / Putative actinorhodopsin / Actinorhodopsin


Mass: 30087.166 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Details: Freshwater bacterium / Source: (gene. exp.) Rhodoluna lacicola (bacteria) / Strain: MWH-Ta8 / Gene: Rhola_00012080 / Plasmid: pZUDF21 / Cell (production host): Bacterial cell / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: C0K2L3
#2: Chemical
ChemComp-A1INB / [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoyloxy-propan-2-yl]-tridecnoate


Mass: 593.773 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C30H60NO8P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-A1IVO / [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoyloxy-propan-2-yl] octadecanoate


Mass: 663.906 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C35H70NO8P
#4: Chemical
ChemComp-A1INC / [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-nonanoyloxy-propan-2-yl] (~{E})-octadec-9-enoate


Mass: 619.810 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C32H62NO8P
#5: 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
Has protein modificationY

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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 actinorhodopsin / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.226 MDa / Experimental value: NO
Source (natural)Organism: Rhodoluna lacicola (bacteria) / Strain: MWH-Ta8 / Cellular location: Membrane
Source (recombinant)Organism: Escherichia coli C43(DE3) / Strain: C43(DE3) / Cell: Bacterial cell / Plasmid: pZUDF21
Buffer solutionpH: 7.5
Details: Elution buffer B (20 mM BTP-HCl pH 7.5 adjusted at 4 degree, 150 mM NaCl, 200 mM L-histidine, 0.25% (w/v) Cymal-5)
Buffer component
IDConc.NameFormulaBuffer-ID
120 4.874Bis-Tris Propane HydrochlorideBTP-HCl1
2150 8.766Sodium ChlorideNaCl1
3200 31.032L-histidineL-histidine1
40.25 2.55-Cyclohexyl-1-pentyl-BETA-D-maltosideCYMAL-51
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: In brief, grids were incubated for 30 seconds, and excess liquid blotted off for 5 seconds applying a blotting force of -6 at 4 degrees and a relative humidity of about 100% before plunging ...Details: In brief, grids were incubated for 30 seconds, and excess liquid blotted off for 5 seconds applying a blotting force of -6 at 4 degrees and a relative humidity of about 100% before plunging into liquid ethane (-177 degrees to -171 degrees).

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50.01 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.00313 sec. / Electron dose: 1.01 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9890
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1crYOLO1.5particle selection
2SerialEMimage acquisition
4Gctf1.6CTF correction
7UCSF Chimera1.7.1model fitting
9RELION4initial Euler assignment
10cryoSPARCfinal Euler assignment
11RELION4classification
12cryoSPARC3D reconstruction
13PHENIX1.20-4459model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 431658
Details: a non-template-driven convolutional neural network (CNN) based-particle picking was performed within CrYOLO (version 1.5). A total of 431,658 particles were selected with the pre-trained ...Details: a non-template-driven convolutional neural network (CNN) based-particle picking was performed within CrYOLO (version 1.5). A total of 431,658 particles were selected with the pre-trained general model and extracted from the remaining 8170 dose-weighted micrographs with Relion4
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 184961 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingDetails: Shiff base bond created between K234 and RETINAL (RET)
Atomic model buildingDetails: created by modelAngelo / Source name: Other / Type: in silico model

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