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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 / GAF domain / AAA+ ATPase lid 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 / GAF domain / AAA+ ATPase lid 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 / PAS-associated, C-terminal / PAC domain profile. / Histidine kinase/HSP90-like ATPase / 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: FEBS J / Year: 2025
Title: Structural analysis of the NifL-NifA complex reveals the molecular basis of anti-activation of nitrogen fixation gene expression in Azotobacter vinelandii.
Authors: Marcelo Bueno Batista / Jake Richardson / Michael W Webster / Dmitry Ghilarov / John W Peters / David M Lawson / Ray Dixon /
Abstract: Understanding the molecular basis of regulated nitrogen (N) fixation is essential for engineering N-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on ...Understanding the molecular basis of regulated nitrogen (N) fixation is essential for engineering N-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system comprising the anti-activator protein (NifL) and the Nif-specific transcriptional activator (NifA)controls the expression of nif genes, encoding the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. While genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-electron microscopy (cryo-EM) map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.
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
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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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