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- PDB-8qlf: Crystal structure of the light-driven sodium pump ErNaR in the mo... -

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Basic information

Entry
Database: PDB / ID: 8qlf
TitleCrystal structure of the light-driven sodium pump ErNaR in the monomeric form at pH 8.8
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 / OLEIC ACID / Bacteriorhodopsin-like protein
Function and homology information
Biological speciesErythrobacter (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.71 Å
AuthorsKovalev, K. / Podoliak, E. / Lamm, G.H.U. / Astashkin, R. / Bourenkov, G.
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
DepositionSep 19, 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

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Assembly

Deposited unit
A: Bacteriorhodopsin-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)37,10419
Polymers32,0181
Non-polymers5,08618
Water2,018112
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6740 Å2
ΔGint32 kcal/mol
Surface area12760 Å2
Unit cell
Length a, b, c (Å)53.382, 53.382, 364.842
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number178
Space group name H-MP6122
Components on special symmetry positions
IDModelComponents
11A-414-

LFA

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Components

#1: Protein Bacteriorhodopsin-like protein


Mass: 32018.041 Da / Num. of mol.: 1
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: Chemical
ChemComp-LFA / EICOSANE / LIPID FRAGMENT


Mass: 282.547 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C20H42
#3: Chemical ChemComp-OLA / OLEIC ACID


Mass: 282.461 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H34O2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 112 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.34 Å3/Da / Density % sol: 47.51 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 8.8
Details: 0.1M sodium acetate, 10% PEG550MME, soaked in 0.1M Tris-HCl pH 8.8, 10% PEG550MME prior to crystal harvesting.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.976 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Feb 21, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 1.707→46.23 Å / Num. obs: 26414 / % possible obs: 94.66 % / Redundancy: 37.6 % / CC1/2: 0.9991 / Rpim(I) all: 0.038 / Net I/σ(I): 13.769
Reflection shellResolution: 1.707→1.875 Å / Mean I/σ(I) obs: 1.59 / Num. unique obs: 1321 / CC1/2: 0.7444 / Rpim(I) all: 0.518

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
MOLREPphasing
XDSdata reduction
STARANISOdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.71→20 Å / Cor.coef. Fo:Fc: 0.962 / Cor.coef. Fo:Fc free: 0.948 / SU B: 2.793 / SU ML: 0.087 / Cross valid method: THROUGHOUT / ESU R: 0.144 / ESU R Free: 0.133 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.21819 1304 4.9 %RANDOM
Rwork0.18271 ---
obs0.18443 25066 75.49 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 29.838 Å2
Baniso -1Baniso -2Baniso -3
1-0.27 Å20.14 Å20 Å2
2--0.27 Å2-0 Å2
3----0.89 Å2
Refinement stepCycle: 1 / Resolution: 1.71→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2168 0 214 112 2494
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0030.0132473
X-RAY DIFFRACTIONr_bond_other_d00.0152542
X-RAY DIFFRACTIONr_angle_refined_deg1.0461.6453301
X-RAY DIFFRACTIONr_angle_other_deg1.1131.5495823
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.1355279
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.6820.42119
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.51515317
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.0131513
X-RAY DIFFRACTIONr_chiral_restr0.0490.2283
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.022643
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02607
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.8282.7161107
X-RAY DIFFRACTIONr_mcbond_other2.8122.7121106
X-RAY DIFFRACTIONr_mcangle_it3.8044.0611389
X-RAY DIFFRACTIONr_mcangle_other3.8084.0661390
X-RAY DIFFRACTIONr_scbond_it3.6763.3951366
X-RAY DIFFRACTIONr_scbond_other3.6813.3961367
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other5.4494.8051913
X-RAY DIFFRACTIONr_long_range_B_refined6.72233.1812828
X-RAY DIFFRACTIONr_long_range_B_other6.72833.1922829
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.712→1.756 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.348 4 -
Rwork0.384 80 -
obs--3.42 %

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