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- PDB-8qr0: Cryo-EM structure of the light-driven sodium pump ErNaR in the pe... -

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Basic information

Entry
Database: PDB / ID: 8qr0
TitleCryo-EM structure of the light-driven sodium pump ErNaR in the pentameric form at pH 4.3
ComponentsBacteriorhodopsin-like protein
KeywordsMEMBRANE PROTEIN / retinal / ion transport / rhodopsin / photocycle / sodium transport
Function / homologyBacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein / membrane / EICOSANE / Bacteriorhodopsin-like protein
Function and homology information
Biological speciesErythrobacter (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsKovalev, K. / Podoliak, E. / Lamm, G.H.U. / Marin, E. / Stetsenko, A. / Guskov, A.
Funding support Germany, 1items
OrganizationGrant numberCountry
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND847543 Germany
CitationJournal: Nat Commun / Year: 2024
Title: A subgroup of light-driven sodium pumps with an additional Schiff base counterion.
Authors: E Podoliak / G H U Lamm / E Marin / A V Schellbach / D A Fedotov / A Stetsenko / M Asido / N Maliar / G Bourenkov / T Balandin / C Baeken / R Astashkin / T R Schneider / A Bateman / J ...Authors: E Podoliak / G H U Lamm / E Marin / A V Schellbach / D A Fedotov / A Stetsenko / M Asido / N Maliar / G Bourenkov / T Balandin / C Baeken / R Astashkin / T R Schneider / A Bateman / J Wachtveitl / I Schapiro / V Busskamp / A Guskov / V Gordeliy / A Alekseev / K Kovalev /
Abstract: Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region ...Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.
History
DepositionOct 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2024Provider: 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-like protein
B: Bacteriorhodopsin-like protein
C: Bacteriorhodopsin-like protein
D: Bacteriorhodopsin-like protein
E: Bacteriorhodopsin-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,81546
Polymers160,0905
Non-polymers12,72541
Water2,612145
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37990 Å2
ΔGint-33 kcal/mol
Surface area39250 Å2
MethodPISA

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Components

#1: Protein
Bacteriorhodopsin-like protein / light-driven sodium-pumping rhodopsin


Mass: 32018.041 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Erythrobacter (bacteria) / Gene: SAMN04515621_2824 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1H1XA63
#2: Sugar
ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM
#3: Chemical...
ChemComp-LFA / EICOSANE / LIPID FRAGMENT / Icosane


Mass: 282.547 Da / Num. of mol.: 36 / Source method: obtained synthetically / Formula: C20H42
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 145 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Light-driven sodium pump ErNaR / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.036 MDa / Experimental value: NO
Source (natural)Organism: Erythrobacter (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 4.3
Buffer component
IDConc.NameBuffer-ID
10.1 sodium actetate1
20.1 sodium chloride1
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: OTHER / Nominal magnification: 105000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5688

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.0.2CTF correction
12cryoSPARC4.0.2classification
13cryoSPARC4.0.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 555297 / Symmetry type: POINT
RefinementResolution: 2.5→101.99 Å / Cor.coef. Fo:Fc: 0.641 / SU B: 6.041 / SU ML: 0.119 / ESU R: 0.318
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.32842 --
obs0.32842 123770 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 34.824 Å2
Refinement stepCycle: 1 / Total: 11598
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0120.01211762
ELECTRON MICROSCOPYr_bond_other_d00.01611486
ELECTRON MICROSCOPYr_angle_refined_deg1.7561.64415896
ELECTRON MICROSCOPYr_angle_other_deg0.5931.55226343
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.97951355
ELECTRON MICROSCOPYr_dihedral_angle_2_deg8.1945110
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.565101590
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0890.21740
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.0213465
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022945
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_it4.0822.8775435
ELECTRON MICROSCOPYr_mcbond_other4.0782.8775435
ELECTRON MICROSCOPYr_mcangle_it5.785.1766785
ELECTRON MICROSCOPYr_mcangle_other5.7815.1776786
ELECTRON MICROSCOPYr_scbond_it6.7053.826327
ELECTRON MICROSCOPYr_scbond_other6.7043.826328
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other10.6326.579112
ELECTRON MICROSCOPYr_long_range_B_refined12.18429.113987
ELECTRON MICROSCOPYr_long_range_B_other12.18229.113978
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.5→2.565 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.018 9097 -
obs--100 %

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