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- PDB-8rq5: Cryo-EM structure of the light-driven sodium-pumping rhodopsin KR2 -

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Basic information

Entry
Database: PDB / ID: 8rq5
TitleCryo-EM structure of the light-driven sodium-pumping rhodopsin KR2
ComponentsSodium pumping rhodopsin
KeywordsMEMBRANE PROTEIN / retinal / ion transport / sodium / rhodopsin / photocycle / pentamer
Function / homologyBacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein / membrane / EICOSANE / OLEIC ACID / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / RETINAL / Sodium pumping rhodopsin
Function and homology information
Biological speciesDokdonia eikasta (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.32 Å
AuthorsKovalev, K. / Linares, R.
Funding support Germany, 1items
OrganizationGrant numberCountry
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND847543 Germany
CitationJournal: Nat Methods / Year: 2026
Title: EasyGrid: a versatile platform for automated cryo-EM sample preparation and quality control.
Authors: Olivier Gemin / Victor Armijo / Léa Lecomte / Michael Hons / Thibault Deckers / Caroline Bissardon / Christopher Rossi / Kévin Lauzier / Robert Janocha / Franck Felisaz / Jérémy Sinoir / ...Authors: Olivier Gemin / Victor Armijo / Léa Lecomte / Michael Hons / Thibault Deckers / Caroline Bissardon / Christopher Rossi / Kévin Lauzier / Robert Janocha / Franck Felisaz / Jérémy Sinoir / Romain Linares / Anastasiia Babenko / Kirill Kovalev / Irina Prokhorova / Iskander Khusainov / Claudia Schreiner / Olga Kolesnikova / Veijo T Salo / Sarah Schneider / Matthew W Bowler / Georg Wolff / Wojciech P Galej / Julia Mahamid / Christoph W Müller / Kristina Djinovic Carugo / Sebastian Eustermann / Simone Mattei / Florent Cipriani / Gergely Papp /
Abstract: Optimized sample preparation is essential for imaging biological macromolecules in their native state using single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography ...Optimized sample preparation is essential for imaging biological macromolecules in their native state using single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET). Here we present EasyGrid, a modular, automated platform designed to streamline and standardize cryo-EM/ET sample preparation. EasyGrid integrates in-line plasma treatment of the sample support, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and grid quality control via light interferometry. We demonstrate its effectiveness by preparing grids for multiple purified macromolecular complexes and resolving their structures with cryo-EM. Additionally, EasyGrid achieves improved vitrification of large mammalian cells compared to conventional plunge-freezing. By enabling systematic and high-throughput optimization, EasyGrid provides a robust and time-saving solution for both structural and cellular cryo-EM applications.
History
DepositionJan 17, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 29, 2025Provider: repository / Type: Initial release
Revision 1.0Jan 29, 2025Data content type: EM metadata / Data content type: EM metadata / 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: Sodium pumping rhodopsin
B: Sodium pumping rhodopsin
C: Sodium pumping rhodopsin
D: Sodium pumping rhodopsin
E: Sodium pumping rhodopsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,80090
Polymers157,7025
Non-polymers23,09885
Water6,341352
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

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Protein , 1 types, 5 molecules ABCDE

#1: Protein
Sodium pumping rhodopsin


Mass: 31540.439 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dokdonia eikasta (bacteria) / Gene: NaR / Production host: Escherichia coli (E. coli) / References: UniProt: N0DKS8

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Non-polymers , 6 types, 437 molecules

#2: Chemical
ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C21H40O4
#3: Chemical...
ChemComp-LFA / EICOSANE / LIPID FRAGMENT


Mass: 282.547 Da / Num. of mol.: 66 / Source method: obtained synthetically / Formula: C20H42
#4: Chemical
ChemComp-RET / RETINAL


Mass: 284.436 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C20H28O / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-OLA / OLEIC ACID


Mass: 282.461 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C18H34O2
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 352 / Source method: isolated from a natural source / Formula: H2O

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Details

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: Sodium-pumping rhodopsin / Type: COMPLEX / Details: KR2 rhodopsin solubilized in DDM / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Dokdonia eikasta (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
Specimen supportDetails: copper grids were first plasma-treated 15 times in EasyGrid,
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 297 K / Details: Jet vitrified with EasyGrid

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.6 sec. / Electron dose: 40.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6671

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2particle selection
2EPUimage acquisition
4cryoSPARC4.2CTF correction
9cryoSPARC4.2initial Euler assignment
10cryoSPARC4.2final Euler assignment
11cryoSPARC4.2classification
12cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1916000
Details: A first round of blob picking was performed and used to obtain an initial model. this initial model was used to generate templates that were then used to performed template picking, ...Details: A first round of blob picking was performed and used to obtain an initial model. this initial model was used to generate templates that were then used to performed template picking, resulting in 1916000 particles
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 333078 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementResolution: 2.32→2.32 Å / Cor.coef. Fo:Fc: 0.646 / SU B: 5.243 / SU ML: 0.11 / ESU R: 0.275
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.36083 --
obs0.36083 158830 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 38.091 Å2
Refinement stepCycle: 1 / Total: 12137
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.01212058
ELECTRON MICROSCOPYr_bond_other_d00.01612090
ELECTRON MICROSCOPYr_angle_refined_deg1.4271.64616052
ELECTRON MICROSCOPYr_angle_other_deg0.4721.54727850
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.02851371
ELECTRON MICROSCOPYr_dihedral_angle_2_deg8.53573
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.413101720
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0830.21710
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.0213306
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022914
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_it3.9423.025444
ELECTRON MICROSCOPYr_mcbond_other3.9373.025444
ELECTRON MICROSCOPYr_mcangle_it5.6865.4436827
ELECTRON MICROSCOPYr_mcangle_other5.6865.4436828
ELECTRON MICROSCOPYr_scbond_it5.6444.1296614
ELECTRON MICROSCOPYr_scbond_other5.6444.1296615
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other8.7157.0689226
ELECTRON MICROSCOPYr_long_range_B_refined10.21130.0213816
ELECTRON MICROSCOPYr_long_range_B_other10.21130.0313817
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.3→2.36 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.952 11797 -
obs--100 %

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