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- PDB-7nem: Hydrogenase-2 variant R479K - anaerobically oxidised form -

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Basic information

Entry
Database: PDB / ID: 7nem
TitleHydrogenase-2 variant R479K - anaerobically oxidised form
Components(Hydrogenase-2 ...) x 2
KeywordsOXIDOREDUCTASE / Hydrogenase / hydrogen oxidation / proton reduction
Function / homology
Function and homology information


[Ni-Fe] hydrogenase complex / hydrogenase (acceptor) / ferredoxin hydrogenase complex / hydrogenase (acceptor) activity / anaerobic respiration / ferredoxin hydrogenase activity / 3 iron, 4 sulfur cluster binding / iron-sulfur cluster binding / nickel cation binding / 4 iron, 4 sulfur cluster binding ...[Ni-Fe] hydrogenase complex / hydrogenase (acceptor) / ferredoxin hydrogenase complex / hydrogenase (acceptor) activity / anaerobic respiration / ferredoxin hydrogenase activity / 3 iron, 4 sulfur cluster binding / iron-sulfur cluster binding / nickel cation binding / 4 iron, 4 sulfur cluster binding / electron transfer activity / periplasmic space / membrane / 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 / Nickel-dependent hydrogenase ...[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 / CARBONMONOXIDE-(DICYANO) IRON / NICKEL (II) ION / OXYGEN MOLECULE / IRON/SULFUR CLUSTER / Hydrogenase-2 large chain / Hydrogenase-2 small chain
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.35 Å
AuthorsCarr, S.B.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
CitationJournal: To Be Published
Title: A comprehensive structural and kinetic investigation of the role of the active-site argininein bidirectional hydrogen activation by the [NiFe]-hydrogenase "Hyd-2) from Escherichia coli
Authors: Evans, R.M. / Beaton, S.E. / Kertiss, L. / Myers, W.K. / Carr, S.B. / Armstrong, F.A.
History
DepositionFeb 4, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 2, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
S: Hydrogenase-2 small chain
L: Hydrogenase-2 large chain
T: Hydrogenase-2 small chain
M: Hydrogenase-2 large chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)192,38721
Polymers189,8034
Non-polymers2,58417
Water23,7981321
1
T: Hydrogenase-2 small chain
M: Hydrogenase-2 large chain
hetero molecules

S: Hydrogenase-2 small chain
L: Hydrogenase-2 large chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)192,38721
Polymers189,8034
Non-polymers2,58417
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_575x+1/2,-y+5/2,-z1
Buried area20030 Å2
ΔGint-267 kcal/mol
Surface area49130 Å2
MethodPISA
Unit cell
Length a, b, c (Å)99.402, 100.252, 168.536
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Hydrogenase-2 ... , 2 types, 4 molecules STLM

#1: Protein Hydrogenase-2 small chain / HYD2 / Membrane-bound hydrogenase 2 small subunit / NiFe hydrogenase


Mass: 32371.498 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: hybO, yghV, b2997, JW2965 / Plasmid: pOc / Details (production host): Plasmid expresses small subunit / Production host: Escherichia coli (E. coli) / References: UniProt: P69741, hydrogenase (acceptor)
#2: Protein Hydrogenase-2 large chain / HYD2 / Membrane-bound hydrogenase 2 large subunit / NiFe hydrogenase


Mass: 62529.891 Da / Num. of mol.: 2 / Mutation: R479K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: hybC, b2994, JW2962 / Plasmid: pOc / Production host: Escherichia coli (E. coli) / References: UniProt: P0ACE0, hydrogenase (acceptor)

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Non-polymers , 8 types, 1338 molecules

#3: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe4S4
#4: Chemical ChemComp-F3S / FE3-S4 CLUSTER / Iron–sulfur cluster


Mass: 295.795 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe3S4
#5: Chemical ChemComp-FCO / CARBONMONOXIDE-(DICYANO) IRON


Mass: 135.890 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3FeN2O
#6: Chemical ChemComp-NI / NICKEL (II) ION / Nickel


Mass: 58.693 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ni
#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-OXY / OXYGEN MOLECULE / Oxygen


Mass: 31.999 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: O2 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#10: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1321 / 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: 2.1 Å3/Da / Density % sol: 41.5 % / Description: Long rods
Crystal growTemperature: 296 K / Method: vapor diffusion, sitting drop / pH: 5.9
Details: 100 mM Bis tris pH 5.9, 200 mM MgCl2 19-21 % PEG 3350
PH range: 5.7-5.9 / Temp details: Ambient temperature inside anaerobic chamber

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: cryostream - nitrogen vapour / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.7749 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Dec 12, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.7749 Å / Relative weight: 1
ReflectionResolution: 1.35→44.8 Å / Num. obs: 366877 / % possible obs: 100 % / Redundancy: 13.4 % / CC1/2: 0.999 / Rmerge(I) obs: 0.143 / Rpim(I) all: 0.059 / Rrim(I) all: 0.154 / Χ2: 0.9 / Net I/σ(I): 10.3
Reflection shellResolution: 1.35→1.37 Å / Redundancy: 12.8 % / Rmerge(I) obs: 1.8 / Mean I/σ(I) obs: 1.3 / Num. unique obs: 18056 / CC1/2: 0.62 / Rpim(I) all: 0.77 / Rrim(I) all: 1.97 / Χ2: 0.8 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
Aimlessdata scaling
PHASERphasing
REFMAC5.8.0267refinement
PDB_EXTRACT3.27data extraction
DIALSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6syo

6syo


Resolution: 1.35→44.8 Å / Cor.coef. Fo:Fc: 0.977 / Cor.coef. Fo:Fc free: 0.972 / SU B: 0.915 / SU ML: 0.035 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.045 / ESU R Free: 0.046 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1673 18274 5 %RANDOM
Rwork0.1497 ---
obs0.1505 348420 99.94 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 68.29 Å2 / Biso mean: 14.875 Å2 / Biso min: 5.66 Å2
Baniso -1Baniso -2Baniso -3
1-0.29 Å20 Å20 Å2
2--0.44 Å2-0 Å2
3----0.73 Å2
Refinement stepCycle: final / Resolution: 1.35→44.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12650 0 71 1321 14042
Biso mean--11.6 23.62 -
Num. residues----1638
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.01313136
X-RAY DIFFRACTIONr_bond_other_d0.0010.01712137
X-RAY DIFFRACTIONr_angle_refined_deg1.7951.63617948
X-RAY DIFFRACTIONr_angle_other_deg1.5621.57428026
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.6251658
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.43223.218634
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.101152072
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.1261557
X-RAY DIFFRACTIONr_chiral_restr0.10.21710
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0214991
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022908
LS refinement shellResolution: 1.35→1.385 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.278 1394 -
Rwork0.271 25497 -
all-26891 -
obs--99.74 %

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