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- EMDB-52114: Cryo-EM structure of the freshwater actinorhodopsin, Rhodoluna la... -

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Basic information

Entry
Database: EMDB / ID: EMD-52114
TitleCryo-EM structure of the freshwater actinorhodopsin, Rhodoluna lacicola (RlActR)
Map dataCryo-electron microscopy (cryo-EM) density map of actinorhodin from Rhodoluna lacicola (RlActR), refined to a resolution of 2.8 Angstrom following local refinement procedures.
Sample
  • Complex: Pentamer of the actinorhodopsin
    • Protein or peptide: Bacteriorhodopsin
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoyloxy-propan-2-yl]-tridecnoate
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoyloxy-propan-2-yl] octadecanoate
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-nonanoyloxy-propan-2-yl] (~{E})-octadec-9-enoate
  • Ligand: RETINAL
Keywordsbacterial / rhodopsin / fresh-water / proton transporter / PROTON TRANSPORT
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
Biological speciesRhodoluna lacicola (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsDjabeur N / Jeckelmann J-M / Fotiadis D
Funding support Switzerland, 1 items
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, 2024-
Header (metadata) releaseMar 18, 2026-
Map releaseMar 18, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52114.png

Downloads & links

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Map

FileDownload / File: emd_52114.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron microscopy (cryo-EM) density map of actinorhodin from Rhodoluna lacicola (RlActR), refined to a resolution of 2.8 Angstrom following local refinement procedures.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 240 pix.
= 175.44 Å		0.73 Å/pix.
x 240 pix.
= 175.44 Å		0.73 Å/pix.
x 240 pix.
= 175.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.731 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.298
Minimum - Maximum-1.3495682 - 2.0827518
Average (Standard dev.)0.014204646 (±0.087980464)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 175.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-electron microscopy (cryo-EM) Half-map A of actinorhodopsin from...

Fileemd_52114_half_map_1.map
AnnotationCryo-electron microscopy (cryo-EM) Half-map A of actinorhodopsin from Rhodoluna lacicola (RlActR)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-electron microscopy (cryo-EM) Half-map B of actinorhodopsin from...

Fileemd_52114_half_map_2.map
AnnotationCryo-electron microscopy (cryo-EM) Half-map B of actinorhodopsin from Rhodoluna lacicola (RlActR)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pentamer of the actinorhodopsin

EntireName: Pentamer of the actinorhodopsin
Components
  • Complex: Pentamer of the actinorhodopsin
    • Protein or peptide: Bacteriorhodopsin
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoyloxy-propan-2-yl]-tridecnoate
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoyloxy-propan-2-yl] octadecanoate
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-nonanoyloxy-propan-2-yl] (~{E})-octadec-9-enoate
  • Ligand: RETINAL

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Supramolecule #1: Pentamer of the actinorhodopsin

SupramoleculeName: Pentamer of the actinorhodopsin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rhodoluna lacicola (bacteria) / Strain: MWH-Ta8 / Location in cell: Membrane
Molecular weightTheoretical: 226 KDa

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Macromolecule #1: Bacteriorhodopsin

MacromoleculeName: Bacteriorhodopsin / type: protein_or_peptide / ID: 1
Details: A covalently bond, i.e. a Schiff base between Lys234 and retinal is present, ensuring accurate representation of the retinal-lysine linkage in the protein structure. Actinorhodopsin
Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Rhodoluna lacicola (bacteria) / Strain: MWH-Ta8
Molecular weightTheoretical: 30.087166 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNTLSNALDN GQFNLVYNIL SLGIASMLFT AIFLFVARER VLPRYRIAVM VSATVTAIAA YHYFRMFDNF SHAFAGAENN PDAYNVGYR YVDWLLTVPL LLVELVAVLA LAKAAQSSIL NRLVPAAAAM IVLGYPGDAK LDIWGIAPSV WGLLSTIPFL Y ILYVLFIE ...String:
MNTLSNALDN GQFNLVYNIL SLGIASMLFT AIFLFVARER VLPRYRIAVM VSATVTAIAA YHYFRMFDNF SHAFAGAENN PDAYNVGYR YVDWLLTVPL LLVELVAVLA LAKAAQSSIL NRLVPAAAAM IVLGYPGDAK LDIWGIAPSV WGLLSTIPFL Y ILYVLFIE LGKSLSRQSE AVQKKVKILR LLLIATWGVY PITFILAMGT PPGAPFNASE FVAREVGYSI ADILAKCLFG LI IYSIARI KSAEDDKEFA KAEFKDALEG HHHHH

UniProtKB: Bacteriorhodopsin

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Macromolecule #2: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoylox...

MacromoleculeName: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoyloxy-propan-2-yl]-tridecnoate
type: ligand / ID: 2 / Number of copies: 5 / Formula: A1INB
Molecular weightTheoretical: 593.773 Da

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Macromolecule #3: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoylox...

MacromoleculeName: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-dodecanoyloxy-propan-2-yl] octadecanoate
type: ligand / ID: 3 / Number of copies: 5 / Formula: A1IVO
Molecular weightTheoretical: 663.906 Da

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Macromolecule #4: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-nonanoyloxy-...

MacromoleculeName: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-nonanoyloxy-propan-2-yl] (~{E})-octadec-9-enoate
type: ligand / ID: 4 / Number of copies: 5 / Formula: A1INC
Molecular weightTheoretical: 619.81 Da

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Macromolecule #5: RETINAL

MacromoleculeName: RETINAL / type: ligand / ID: 5 / Number of copies: 5 / 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

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 4.874BTP-HClBis-Tris Propane Hydrochloride
150.0 8.766NaClSodium Chloride
200.0 31.032L-histidineL-histidine
0.25 2.5CYMAL-55-Cyclohexyl-1-pentyl-BETA-D-maltoside

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)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 9890 / Average exposure time: 0.00313 sec. / Average electron dose: 1.01 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.01 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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
CTF correctionSoftware - Name: Gctf (ver. 1.6) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: The structural model of actinorhodopsin from Rhodoluna lacicola (RlActR) was generated in silico using Model Angelo. This model was constructed based on cryo-electron microscopy ...In silico model: The structural model of actinorhodopsin from Rhodoluna lacicola (RlActR) was generated in silico using Model Angelo. This model was constructed based on cryo-electron microscopy (cryo-EM) density maps obtained at a resolution of 2.8 Angstrom and its amino-acid sequence
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 184961
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 7 / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: in silico model / Details: created by modelAngelo
DetailsShiff base bond created between K234 and RETINAL (RET)
Output model

PDB-9hfk:
Cryo-EM structure of the freshwater actinorhodopsin, Rhodoluna lacicola (RlActR)

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