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- PDB-8pou: Crystal Structure of the C19G/C120G variant of the membrane-bound... -

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Basic information

Entry
Database: PDB / ID: 8pou
TitleCrystal Structure of the C19G/C120G variant of the membrane-bound [NiFe]-Hydrogenase from Cupriavidus necator in the air-oxidized state at 1.65 A Resolution.
Components(Uptake hydrogenase ...) x 2
KeywordsOXIDOREDUCTASE / [NIFE]-HYDROGENASE / HYDROGENASE / OXYGEN-TOLERANCE / HYDROGEN CATALYSIS / KNALLGASBACTERIA / PROTEOBACTERIA / METALLOENZYME / BIMETALLIC / NI-FE ACTIVE SITE / [4FE-3S] / PROXIMAL CLUSTER / AEROBIC HYDROGEN BACTERIA / ELECTRON TRANSFER / METALLOPROTEIN / CATALYTIC CENTER / MEMBRANE / MEMBRANE-BOUND / OXIDOREDUCTASE-OXIDOREDUCTASE COMPLEX / ELECTRON RELAY / CUBANE CLUSTER / LOW POTENTIAL / CLUSTER TUNING / ELECTRON TRANSPORT
Function / homology
Function and homology information


hydrogenase (acceptor) / [Ni-Fe] hydrogenase complex / ferredoxin hydrogenase complex / hydrogenase (acceptor) activity / ferredoxin hydrogenase activity / anaerobic respiration / 3 iron, 4 sulfur cluster binding / nickel cation binding / 4 iron, 4 sulfur cluster binding / electron transfer activity ...hydrogenase (acceptor) / [Ni-Fe] hydrogenase complex / ferredoxin hydrogenase complex / hydrogenase (acceptor) activity / ferredoxin hydrogenase activity / anaerobic respiration / 3 iron, 4 sulfur cluster binding / nickel cation binding / 4 iron, 4 sulfur cluster binding / electron transfer activity / metal ion binding / plasma membrane
Similarity search - Function
: / [NiFe]-hydrogenase, small subunit / Cytochrome-c3 hydrogenase, C-terminal / [NiFe]-hydrogenase, small subunit, C-terminal domain superfamily / NiFe/NiFeSe hydrogenase small subunit C-terminal / Nickel-dependent hydrogenases large subunit signature 2. / Nickel-dependent hydrogenases large subunit signature 1. / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / Nickel-dependent hydrogenase, large subunit, nickel binding site / Nickel-dependent hydrogenase, large subunit ...: / [NiFe]-hydrogenase, small subunit / Cytochrome-c3 hydrogenase, C-terminal / [NiFe]-hydrogenase, small subunit, C-terminal domain superfamily / NiFe/NiFeSe hydrogenase small subunit C-terminal / Nickel-dependent hydrogenases large subunit signature 2. / Nickel-dependent hydrogenases large subunit signature 1. / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / Nickel-dependent hydrogenase, large subunit, nickel binding site / Nickel-dependent hydrogenase, large subunit / Nickel-dependent hydrogenase / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / NADH:ubiquinone oxidoreductase-like, 20kDa subunit / NADH ubiquinone oxidoreductase, 20 Kd subunit / [NiFe]-hydrogenase, large subunit / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence
Similarity search - Domain/homology
FE3-S4 CLUSTER / NI-FE OXIDIZED ACTIVE CENTER / IRON/SULFUR CLUSTER / Uptake hydrogenase large subunit / Uptake hydrogenase small subunit
Similarity search - Component
Biological speciesCupriavidus necator H16 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.65 Å
AuthorsSchmidt, A. / Kalms, J. / Scheerer, P.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)DFG Cluster of Excellence Unifying Concepts in Catalysis - Research Field D3/E3-1 Germany
German Research Foundation (DFG)UniSysCat - Germany Excellence Strategy EXC2008/1-390540038 Germany
CitationJournal: Chem Sci / Year: 2023
Title: Stepwise conversion of the Cys 6 [4Fe-3S] to a Cys 4 [4Fe-4S] cluster and its impact on the oxygen tolerance of [NiFe]-hydrogenase.
Authors: Schmidt, A. / Kalms, J. / Lorent, C. / Katz, S. / Frielingsdorf, S. / Evans, R.M. / Fritsch, J. / Siebert, E. / Teutloff, C. / Armstrong, F.A. / Zebger, I. / Lenz, O. / Scheerer, P.
History
DepositionJul 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2024Group: Refinement description / Category: pdbx_initial_refinement_model / Item: _pdbx_initial_refinement_model.entity_id_list

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: Uptake hydrogenase large subunit
S: Uptake hydrogenase small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,52210
Polymers103,1822
Non-polymers1,3408
Water11,998666
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9440 Å2
ΔGint-138 kcal/mol
Surface area26670 Å2
MethodPISA
Unit cell
Length a, b, c (Å)73.231, 95.740, 120.005
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Uptake hydrogenase ... , 2 types, 2 molecules LS

#1: Protein Uptake hydrogenase large subunit / Hydrogenlyase / Membrane-bound hydrogenase large subunit / HoxG


Mass: 67247.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cupriavidus necator H16 (bacteria) / Gene: hoxG, PHG002 / Production host: Cupriavidus necator H16 (bacteria) / References: UniProt: P31891, hydrogenase (acceptor)
#2: Protein Uptake hydrogenase small subunit / Hydrogenlyase / Membrane-bound hydrogenase small subunit / HoxK


Mass: 35934.844 Da / Num. of mol.: 1 / Mutation: C19G, C120G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cupriavidus necator H16 (bacteria) / Gene: hoxK, PHG001 / Production host: Cupriavidus necator H16 (bacteria) / References: UniProt: P31892, hydrogenase (acceptor)

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Non-polymers , 6 types, 674 molecules

#3: Chemical ChemComp-NFV / NI-FE OXIDIZED ACTIVE CENTER


Mass: 210.583 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3FeN2NiO2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-F3S / FE3-S4 CLUSTER


Mass: 295.795 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe3S4
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 666 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.11 Å3/Da / Density % sol: 42 %
Crystal growTemperature: 277 K / Method: vapor diffusion / pH: 5.5 / Details: 20% - 30% PEG3350, 0.1M BIS-TRIS PH 5.5 / PH range: 5.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.97625 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jul 27, 2013 / Details: MIRROR
RadiationMonochromator: SI(311) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97625 Å / Relative weight: 1
ReflectionResolution: 1.65→47.92 Å / Num. obs: 101451 / % possible obs: 99.7 % / Redundancy: 4.8 % / CC1/2: 0.99 / Net I/σ(I): 11.7
Reflection shellResolution: 1.65→1.74 Å / Num. unique obs: 16026 / CC1/2: 0.74

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Processing

Software
NameVersionClassification
REFMAC5.7.0032refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.65→47.92 Å / Cor.coef. Fo:Fc: 0.98 / Cor.coef. Fo:Fc free: 0.973 / SU B: 4.045 / SU ML: 0.058 / Cross valid method: THROUGHOUT / ESU R: 0.117 / ESU R Free: 0.076 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.16127 5059 5 %RANDOM
Rwork0.13175 ---
obs0.13323 96315 99.63 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 28.302 Å2
Baniso -1Baniso -2Baniso -3
1--0.49 Å2-0 Å20 Å2
2---0.96 Å2-0 Å2
3---1.45 Å2
Refinement stepCycle: 1 / Resolution: 1.65→47.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6801 0 36 666 7503
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0197138
X-RAY DIFFRACTIONr_bond_other_d0.0020.026709
X-RAY DIFFRACTIONr_angle_refined_deg1.1781.9469731
X-RAY DIFFRACTIONr_angle_other_deg0.853315464
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.475908
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.623.67327
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.855151171
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.6951549
X-RAY DIFFRACTIONr_chiral_restr0.0650.21041
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0218197
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021684
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_it1.4751.9933525
X-RAY DIFFRACTIONr_mcbond_other1.4731.9913524
X-RAY DIFFRACTIONr_mcangle_it1.8872.9914417
X-RAY DIFFRACTIONr_mcangle_other1.8872.9934418
X-RAY DIFFRACTIONr_scbond_it1.792.2153613
X-RAY DIFFRACTIONr_scbond_other1.792.2153612
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.1633.2365297
X-RAY DIFFRACTIONr_long_range_B_refined3.47917.6448988
X-RAY DIFFRACTIONr_long_range_B_other3.4817.6438986
X-RAY DIFFRACTIONr_rigid_bond_restr2.245313847
X-RAY DIFFRACTIONr_sphericity_free29.6915191
X-RAY DIFFRACTIONr_sphericity_bonded7.487514176
LS refinement shellResolution: 1.651→1.694 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.286 366 -
Rwork0.239 6863 -
obs--97.58 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.7913-0.54270.02180.91760.00530.3627-0.0394-0.00230.02740.01030.02640.0786-0.0122-0.05140.0130.0075-0.0028-0.00160.01160.00070.0205-2.237398.5671126.9961
20.5302-0.04860.04770.33290.02570.5877-0.0578-0.1480.00740.12730.0343-0.04930.0260.05430.02350.05260.0271-0.01830.0536-0.00340.009113.639889.965144.6543
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1L3 - 603
2X-RAY DIFFRACTION2S5 - 273

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