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- EMDB-53294: Structure of the Azotobacter vinelandii NifL-NifA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-53294
TitleStructure of the Azotobacter vinelandii NifL-NifA complex
Map dataSharpened map at 6.45 A resolution used for model building and refinement
Sample
  • Complex: NifL-NifA complex from Azotobacter vinelandii
    • Protein or peptide: histidine kinase
    • Protein or peptide: Nif-specific regulatory protein
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Keywordsbiological nitrogen fixation / transcriptional regulation / sustainable agriculture / GENE REGULATION
Function / homology
Function and homology information


nitrogen fixation / histidine kinase / phosphorelay sensor kinase activity / phosphorelay signal transduction system / sequence-specific DNA binding / DNA-binding transcription factor activity / regulation of DNA-templated transcription / ATP hydrolysis activity / ATP binding
Similarity search - Function
Nif-specific regulatory protein / Nitrogen fixation negative regulator NifL / 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. ...Nif-specific regulatory protein / Nitrogen fixation negative regulator NifL / 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 / 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 / Histidine kinase/HSP90-like ATPase / 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
histidine kinase / Nif-specific regulatory protein
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.45 Å
AuthorsBueno Batista M / Richardson J / Webster MW / Ghilarov D / Peters JW / Lawson DM / Dixon R
Funding support United Kingdom, United States, 9 items
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 MW / Ghilarov D / Peters JW / Lawson DM / Dixon R
History
DepositionMar 31, 2025-
Header (metadata) releaseJun 18, 2025-
Map releaseJun 18, 2025-
UpdateJun 18, 2025-
Current statusJun 18, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53294.png

Downloads & links

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Map

FileDownload / File: emd_53294.map.gz / Format: CCP4 / Size: 18.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map at 6.45 A resolution used for model building and refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.48 Å/pix.
x 168 pix.
= 249.295 Å		1.48 Å/pix.
x 168 pix.
= 249.295 Å		1.48 Å/pix.
x 168 pix.
= 249.295 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4839 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.24982455 - 0.6893031
Average (Standard dev.)-0.0005880738 (±0.024454223)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions168168168
Spacing168168168
CellA=B=C: 249.2952 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_53294_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_53294_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_53294_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_53294_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NifL-NifA complex from Azotobacter vinelandii

EntireName: NifL-NifA complex from Azotobacter vinelandii
Components
  • Complex: NifL-NifA complex from Azotobacter vinelandii
    • Protein or peptide: histidine kinase
    • Protein or peptide: Nif-specific regulatory protein
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: NifL-NifA complex from Azotobacter vinelandii

SupramoleculeName: NifL-NifA complex from Azotobacter vinelandii / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: histidine kinase

MacromoleculeName: histidine kinase / type: protein_or_peptide / ID: 1
Details: The wild-type sequence was pre-pended by a Strep-Tag II in place of the N-terminal methionine with sequence MASWSHPQFEKGADDDDKV
Number of copies: 2 / Enantiomer: LEVO / EC number: histidine kinase
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 59.897203 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MASWSHPQFE KGADDDDKVT PANPTLSNEP QAPHAESDEL LPEIFRQTVE HAPIAISITD LKANILYANR AFRTITGYGS EEVLGKNES ILSNGTTPRL VYQALWGRLA QKKPWSGVLV NRRKDKTLYL AELTVAPVLN EAGETIYYLG MHRDTSELHE L EQRVNNQR ...String:
MASWSHPQFE KGADDDDKVT PANPTLSNEP QAPHAESDEL LPEIFRQTVE HAPIAISITD LKANILYANR AFRTITGYGS EEVLGKNES ILSNGTTPRL VYQALWGRLA QKKPWSGVLV NRRKDKTLYL AELTVAPVLN EAGETIYYLG MHRDTSELHE L EQRVNNQR LMIEAVVNAA PAAMVVLDRQ HRVMLSNPSF CRLARDLVED GSSESLVALL RENLAAPFET LENQGSAFSG KE ISFDLGG RSPRWLSCHG RAIHIENEQA HVFFAPTEER YLLLTINDIS ELRQKQQDSR LNALKALMAE EELLEGMRET FNA AIHRLQ GPVNLISAAM RMLERRLGDK AGNDPVLSAM REASTAGMEA LENLSGSIPV RMAESKMPVN LNQLIREVIT LCTD QLLAQ GIVVDWQPAL RLPWVMGGES SLRSMIKHLV DNAIESMSQN QVSRRELFIS TRVENHLVRM EITDSGPGIP PDLVL KVFE PFFSTKPPHR VGRGMGLPVV QEIVAKHAGM VHVDTDYREG CRIVVELPFS AST

UniProtKB: histidine kinase

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Macromolecule #2: Nif-specific regulatory protein

MacromoleculeName: Nif-specific regulatory protein / type: protein_or_peptide / ID: 2
Details: A hexahistidine tag was appended directly to the C-terminus of the wild-type sequence
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 58.979191 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNATIPQRSA KQNPVELYDL QLQALASIAR TLSREQQIDE LLEQVLAVLH NDLGLLHGLV TISDPEHGAL QIGAIHTDSE AVAQACEGV RYRSGEGVIG NVLKHGNSVV LGRISADPRF LDRLALYDLE MPFIAVPIKN PEGNTIGVLA AQPDCRADEH M PARTRLLE ...String:
MNATIPQRSA KQNPVELYDL QLQALASIAR TLSREQQIDE LLEQVLAVLH NDLGLLHGLV TISDPEHGAL QIGAIHTDSE AVAQACEGV RYRSGEGVIG NVLKHGNSVV LGRISADPRF LDRLALYDLE MPFIAVPIKN PEGNTIGVLA AQPDCRADEH M PARTRLLE IVANLLAQTV RLVVNIEDGR EAADERDELR REVRGKYGFE NMVVGHTPTM RRVFDQIRRV AKWNSTVLVL GE SGTGKEL IASAIHYNSP RAHRPFVRLN CAALPETLLE SELFGHEKGA FTGAVKQRKG RFEQADGGTL FLDEIGEISP MFQ AKLLRV LQEGEFERVG GNQTVRVNVR IVAATNRDLE SEVEKGKFRE DLYYRLNVMA IRIPPLRERT ADIPELAEFL LGKI GRQQG RPLTVTDSAI RLLMSHRWPG NVRELENCLE RSAIMSEDGT ITRDVVSLTG VDNESPPLAA PLPEVNLADE TLDDR ERVI AALEQAGWVQ AKAARLLGMT PRQIAYRIQT LNIHMRKIHH HHHH

UniProtKB: Nif-specific regulatory protein

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Macromolecule #3: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 2 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES pH 7.5, 300 mM NaCl, 2.5 mM MgCl2, 1 mM ADP, 1 mM TCEP
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
Details: glow discharged for 60 seconds at 8 mA using an ACE 200 (Leica Microsystems)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThe 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.

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 14218 / Average exposure time: 2.27 sec. / Average electron dose: 50.0 e/Å2
Details: mode: counting; super resolution, x2 binning, magnified pixel size 0.83 Angstrom
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2186245
Details: Used template-based picking with 2D-classes from blob picker
CTF correctionSoftware - Name: cryoSPARC (ver. 4.5.3) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold3 model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.45 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Number images used: 209009
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final 3D classificationNumber classes: 4 / Avg.num./class: 52252 / Software - Name: cryoSPARC (ver. 4.5.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsReal space refinement in COOT and PHENIX
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 456 / Target criteria: cross-correlation coefficient
Output model

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

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