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- PDB-9qq6: Structure of the Azotobacter vinelandii NifL-NifA complex -

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Basic information

Entry
Database: PDB / ID: 9qq6
TitleStructure of the Azotobacter vinelandii NifL-NifA complex
Components
  • Nif-specific regulatory protein
  • histidine kinase
KeywordsGENE REGULATION / biological nitrogen fixation / transcriptional regulation / sustainable agriculture
Function / homology
Function and homology information


protein histidine kinase activity / nitrogen fixation / histidine kinase / phosphorelay signal transduction system / sequence-specific DNA binding / DNA-binding transcription factor activity / regulation of DNA-templated transcription / ATP binding
Similarity search - Function
Nif-specific regulatory protein / Nitrogen fixation negative regulator NifL / AAA+ ATPase lid domain / GAF domain / Sigma-54 interaction domain ATP-binding region A signature. / Sigma-54 interaction domain, conserved site / Sigma-54 interaction domain C-terminal part signature. / Sigma-54 interaction domain, ATP-binding site 1 / Sigma-54 interaction domain, ATP-binding site 2 / Sigma-54 interaction domain ATP-binding region B signature. ...Nif-specific regulatory protein / Nitrogen fixation negative regulator NifL / AAA+ ATPase lid domain / GAF domain / Sigma-54 interaction domain ATP-binding region A signature. / Sigma-54 interaction domain, conserved site / Sigma-54 interaction domain C-terminal part signature. / Sigma-54 interaction domain, ATP-binding site 1 / Sigma-54 interaction domain, ATP-binding site 2 / Sigma-54 interaction domain ATP-binding region B signature. / Sigma-54 interaction domain profile. / Sigma-54 interaction domain / RNA polymerase sigma factor 54 interaction domain / DNA binding HTH domain, Fis-type / Bacterial regulatory protein, Fis family / Histidine kinase/HSP90-like ATPase / PAS-associated, C-terminal / PAC domain profile. / PAC motif / Motif C-terminal to PAS motifs (likely to contribute to PAS structural domain) / Signal transduction histidine kinase-related protein, C-terminal / Histidine kinase domain / Histidine kinase domain profile. / Domain present in phytochromes and cGMP-specific phosphodiesterases. / GAF domain / GAF-like domain superfamily / PAS fold / PAS fold / PAS domain / PAS repeat profile. / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / PAS domain / PAS domain superfamily / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase superfamily / Homeobox-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / FLAVIN-ADENINE DINUCLEOTIDE / histidine kinase / Nif-specific regulatory protein
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.45 Å
AuthorsBueno Batista, M. / Richardson, J. / Webster, M.W. / Ghilarov, D. / Peters, J.W. / Lawson, D.M. / Dixon, R.
Funding support United Kingdom, United States, 9items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/W009986/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X01102X/1 United Kingdom
Wellcome Trust221868/Z/20/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X01097X/1 United Kingdom
Department of Energy (DOE, United States)DE-SC0018143 United States
Biotechnology and Biological Sciences Research Council (BBSRC)BBS/E/J/000PR9797 United Kingdom
Royal SocietyICA/R1/180088 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/CCG2240/1 United Kingdom
Wellcome Trust218785/Z/19/Z United Kingdom
CitationJournal: To Be Published
Title: Structural and functional analysis of the NifL-NifA complex for engineered control of nitrogen fixation in Proteobacteria
Authors: Bueno Batista, M. / Richardson, J. / Webster, M.W. / Ghilarov, D. / Peters, J.W. / Lawson, D.M. / Dixon, R.
History
DepositionMar 31, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 18, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: histidine kinase
B: histidine kinase
C: Nif-specific regulatory protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)181,6268
Polymers178,7743
Non-polymers2,8535
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein histidine kinase


Mass: 59897.203 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The wild-type sequence was pre-pended by a Strep-Tag II in place of the N-terminal methionine with sequence MASWSHPQFEKGADDDDKV
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Gene: nifL, Avin_50990 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C1DMA9, histidine kinase
#2: Protein Nif-specific regulatory protein


Mass: 58979.191 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: A hexahistidine tag was appended directly to the C-terminus of the wild-type sequence
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Gene: nifA, Avin_51000 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C1DMB0
#3: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationN

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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: NifL-NifA complex from Azotobacter vinelandii / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: YES
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Details: 20 mM HEPES pH 7.5, 300 mM NaCl, 2.5 mM MgCl2, 1 mM ADP, 1 mM TCEP
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The particles had a tendency to aggregate, were prone to orientational bias and because they were long and thin, they were difficult to distinguish from the background.
Specimen supportDetails: glow discharged for 60 seconds at 8 mA using an ACE 200 (Leica Microsystems)
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 2.27 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14218
Details: mode: counting; super resolution, x2 binning, magnified pixel size 0.83 Angstrom
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.3particle selection
2EPU3.5.1image acquisition
4cryoSPARC4.5.3CTF correction
7UCSF ChimeraX1.9model fitting
8Coot0.9.8.95 ELmodel fitting
10cryoSPARC4.5.3initial Euler assignment
11cryoSPARC4.5.3final Euler assignment
12cryoSPARC4.5.3classification
13cryoSPARC4.5.33D reconstruction
14PHENIX1.21_5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2186245
Details: Used template-based picking with 2D-classes from blob picker
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209009 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 456 / Protocol: OTHER / Space: REAL / Target criteria: cross-correlation coefficient / Details: Real space refinement in COOT and PHENIX
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210127
ELECTRON MICROSCOPYf_angle_d0.56413739
ELECTRON MICROSCOPYf_dihedral_angle_d7.3131490
ELECTRON MICROSCOPYf_chiral_restr0.041555
ELECTRON MICROSCOPYf_plane_restr0.0051770

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