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- PDB-8bts: Nitrogenase MoFe protein from A. vinelandii alpha double mutant C... -

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Basic information

Entry
Database: PDB / ID: 8bts
TitleNitrogenase MoFe protein from A. vinelandii alpha double mutant C45A/L158C
Components
  • Nitrogenase molybdenum-iron protein beta chain
  • Nitrogenase protein alpha chain
KeywordsOXIDOREDUCTASE / N2-fixation / Nitrogenase / cooperativity / ammonia / hydrogen / metalloenzyme / FeMo-cofactor / P cluster / O2-sensitivity
Function / homology
Function and homology information


molybdenum-iron nitrogenase complex / nitrogenase / : / nitrogenase activity / nitrogen fixation / iron-sulfur cluster binding / ATP binding / metal ion binding
Similarity search - Function
Nitrogenase molybdenum-iron protein beta chain, N-terminal / Domain of unknown function (DUF3364) / Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase molybdenum-iron protein beta chain / Nitrogenase component 1, alpha chain / Nitrogenase component 1, conserved site / Nitrogenases component 1 alpha and beta subunits signature 2. / Nitrogenases component 1 alpha and beta subunits signature 1. / Nitrogenase/oxidoreductase, component 1 / Nitrogenase component 1 type Oxidoreductase
Similarity search - Domain/homology
FE(8)-S(7) CLUSTER, OXIDIZED / : / 3-HYDROXY-3-CARBOXY-ADIPIC ACID / Chem-ICS / Nitrogenase protein alpha chain / Nitrogenase molybdenum-iron protein beta chain
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.03 Å
AuthorsWagner, T. / Maslac, N.
Funding support Switzerland, Germany, 4items
OrganizationGrant numberCountry
Swiss National Science Foundation180544 Switzerland
Swiss National Science FoundationSNSF 310030_189246 Switzerland
Max Planck Society Germany
German Research Foundation (DFG)WA 4053/1-1 Germany
CitationJournal: Jacs Au / Year: 2023
Title: Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase.
Authors: Cadoux, C. / Ratcliff, D. / Maslac, N. / Gu, W. / Tsakoumagkos, I. / Hoogendoorn, S. / Wagner, T. / Milton, R.D.
History
DepositionNov 29, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nitrogenase protein alpha chain
B: Nitrogenase molybdenum-iron protein beta chain
C: Nitrogenase protein alpha chain
D: Nitrogenase molybdenum-iron protein beta chain
H: Nitrogenase protein alpha chain
I: Nitrogenase molybdenum-iron protein beta chain
J: Nitrogenase protein alpha chain
L: Nitrogenase molybdenum-iron protein beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)472,79346
Polymers463,8448
Non-polymers8,94838
Water00
1
A: Nitrogenase protein alpha chain
B: Nitrogenase molybdenum-iron protein beta chain
C: Nitrogenase protein alpha chain
D: Nitrogenase molybdenum-iron protein beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)236,76527
Polymers231,9224
Non-polymers4,84323
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area33820 Å2
ΔGint-243 kcal/mol
Surface area58840 Å2
MethodPISA
2
H: Nitrogenase protein alpha chain
I: Nitrogenase molybdenum-iron protein beta chain
J: Nitrogenase protein alpha chain
L: Nitrogenase molybdenum-iron protein beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)236,02819
Polymers231,9224
Non-polymers4,10615
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31980 Å2
ΔGint-240 kcal/mol
Surface area58700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)148.392, 73.444, 211.253
Angle α, β, γ (deg.)90.00, 104.76, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 2 types, 8 molecules ACHJBDIL

#1: Protein
Nitrogenase protein alpha chain


Mass: 56425.152 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: NifD containing two substitutions: C45A and L158C. / Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Strain: RS1 / Tissue: / / Cell: / / Cell line: / / Gene: nifD / Organ: / / Variant: A. vinelandii nifD: C45A/L158C / Details (production host): / / Cell (production host): / / Cell line (production host): / / Organ (production host): / / Production host: Azotobacter vinelandii DJ (bacteria) / Strain (production host): RS1 / Tissue (production host): / / Variant (production host): A. vinelandii nifD: C45A/L158C / References: UniProt: C1DGZ7
#2: Protein
Nitrogenase molybdenum-iron protein beta chain / Dinitrogenase / Nitrogenase component I


Mass: 59535.879 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Strain: RS1 / Tissue: / / Cell: / / Cell line: / / Gene: nifK / Organ: / / Variant: A. vinelandii nifD: C45A/L158C / Details (production host): / / Cell (production host): / / Cell line (production host): / / Organ (production host): / / Production host: Azotobacter vinelandii DJ (bacteria) / Strain (production host): RS1 / Tissue (production host): / / Variant (production host): A. vinelandii nifD: C45A/L158C / References: UniProt: P07329, nitrogenase

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Non-polymers , 6 types, 38 molecules

#3: Chemical
ChemComp-HCA / 3-HYDROXY-3-CARBOXY-ADIPIC ACID


Mass: 206.150 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C7H10O7 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-ICS / iron-sulfur-molybdenum cluster with interstitial carbon


Mass: 787.451 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: CFe7MoS9 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: SO4
#7: Chemical
ChemComp-1CL / FE(8)-S(7) CLUSTER, OXIDIZED


Mass: 671.215 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe8S7 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe

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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: 2.4 Å3/Da / Density % sol: 48.75 % / Description: Thin brown plate
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: The purified enzyme in 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 2 mM dithionite was crystallized at 8 mg.ml-1. Crystallization was performed anaerobically in a tent containing 100 % N2 atmosphere. ...Details: The purified enzyme in 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 2 mM dithionite was crystallized at 8 mg.ml-1. Crystallization was performed anaerobically in a tent containing 100 % N2 atmosphere. Crystals were obtained by initial screening at 20 degree Celsius on 96-Well MRC 2-drop crystallization plates in polystyrene (SWISSCI) containing 90 ul of crystallization solution in the reservoir. The protein sample (0.5 ul) was mixed with 0.5 ul reservoir solution. The reservoir solution contained 25 % w/v Polyethylene glycol 3,350, 100 mM BIS-TRIS at pH 5.5, and 200 mM Lithium sulfate. The plate was then incubated in a Coy tent (N2/H2, 97:3) at 20 degree Celsius. Crystals appeared after a few weeks and were soaked in the crystallization solution supplemented with 15 % v/v glycerol as a cryo-protectant before being frozen in liquid nitrogen.
PH range: /
Temp details: Crystallized inside a Coy tent, a fluctuation of 1 degree might have occurred

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: / / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1.00003 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Nov 27, 2021 / Details: /
RadiationMonochromator: / / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.00003 Å / Relative weight: 1
ReflectionResolution: 3.029→143.493 Å / Num. obs: 39936 / % possible obs: 86.6 % / Redundancy: 3.7 % / CC1/2: 0.957 / Rmerge(I) obs: 0.297 / Rpim(I) all: 0.175 / Rrim(I) all: 0.346 / Net I/σ(I): 3.4
Reflection shellResolution: 3.029→3.486 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.514 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 1998 / CC1/2: 0.806 / Rpim(I) all: 0.334 / Rrim(I) all: 0.615 / % possible all: 69.4

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Processing

Software
NameVersionClassification
PHENIX1.20.1-4487refinement
BUSTER2.10.4refinement
autoPROC1.0.5data reduction
autoPROC1.0.5data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.03→95.5 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 16.55 / Stereochemistry target values: ML
Details: No Translation-Libration-Screw-rotation, no non-crystallography symmetry, and no hydrogens were applied for the last cycle in Phenix.
RfactorNum. reflection% reflectionSelection details
Rfree0.2227 1945 4.87 %Random selection
Rwork0.2037 ---
obs0.2046 39924 46.19 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 35.11 Å2
Refinement stepCycle: LAST / Resolution: 3.03→95.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms31844 0 314 0 32158
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00432980
X-RAY DIFFRACTIONf_angle_d0.66444732
X-RAY DIFFRACTIONf_dihedral_angle_d15.20712204
X-RAY DIFFRACTIONf_chiral_restr0.0434660
X-RAY DIFFRACTIONf_plane_restr0.0055712
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.03-3.10.3661110.2926158X-RAY DIFFRACTION3
3.11-3.190.3122170.2798249X-RAY DIFFRACTION4
3.19-3.280.2173130.2844356X-RAY DIFFRACTION6
3.28-3.390.262250.2756531X-RAY DIFFRACTION9
3.39-3.510.2926390.2719751X-RAY DIFFRACTION13
3.51-3.640.22550.2558988X-RAY DIFFRACTION18
3.69-3.820.2267800.2381239X-RAY DIFFRACTION29
3.82-4.020.2972710.22421602X-RAY DIFFRACTION27
4.02-4.270.23381960.21763845X-RAY DIFFRACTION66
4.27-4.60.21712680.20535143X-RAY DIFFRACTION87
4.6-5.060.20352840.19665444X-RAY DIFFRACTION93
5.06-5.790.24632790.21295630X-RAY DIFFRACTION95
5.79-7.30.23032980.20985942X-RAY DIFFRACTION100
7.3-95.50.18413090.15446101X-RAY DIFFRACTION100

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