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- EMDB-71144: Cryo-EM structure of Nitrogenase MoFe protein from Methanosarcina... -

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Basic information

Entry
Database: EMDB / ID: EMD-71144
TitleCryo-EM structure of Nitrogenase MoFe protein from Methanosarcina acetivorans bound to the NifI inhibitor complex
Map data
Sample
  • Complex: Trimeric complex of MoFe protein bound to NifI inhibitor complex
    • Protein or peptide: Nitrogenase protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
    • Protein or peptide: P-II family nitrogen regulatory protein
    • Protein or peptide: P-II family nitrogen regulatory protein
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: 3-HYDROXY-3-CARBOXY-ADIPIC ACID
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: 2-OXOGLUTARIC ACID
KeywordsMetalloproteins / Nitrogenase / Oxidoreductase / Methanogen / cryo-EM
Function / homology
Function and homology information


molybdenum-iron nitrogenase complex / nitrogenase / nitrogenase activity / regulation of nitrogen utilization / iron-sulfur cluster binding / enzyme regulator activity / DNA-templated transcription / ATP binding / metal ion binding / cytosol
Similarity search - Function
Nitrogen regulatory protein PII, conserved site / P-II protein C-terminal region signature. / Nitrogen regulatory protein P-II / P-II protein family profile. / Nitrogen regulatory protein PII / Nitrogen regulatory protein P-II / Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase molybdenum-iron protein beta chain / Nitrogenase component 1, alpha chain / Nitrogenase component 1, conserved site ...Nitrogen regulatory protein PII, conserved site / P-II protein C-terminal region signature. / Nitrogen regulatory protein P-II / P-II protein family profile. / Nitrogen regulatory protein PII / Nitrogen regulatory protein P-II / 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. / Nitrogen regulatory PII-like, alpha/beta / Nitrogen regulatory protein PII/ATP phosphoribosyltransferase, C-terminal / : / Nitrogenase/oxidoreductase, component 1 / Nitrogenase component 1 type Oxidoreductase
Similarity search - Domain/homology
Nitrogenase molybdenum-iron protein beta chain / Nitrogenase protein alpha chain / P-II family nitrogen regulatory protein / P-II family nitrogen regulatory protein
Similarity search - Component
Biological speciesMethanosarcina acetivorans (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsKashyap R / Antony E
Funding support United States, 1 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-SC0020965 United States
CitationJournal: To Be Published
Title: Cryo-EM structure of Nitrogenase MoFe protein from Methanosarcina acetivorans bound to the NifI inhibitor complex
Authors: Kashyap R / Antony E
History
DepositionJun 11, 2025-
Header (metadata) releaseJul 1, 2026-
Map releaseJul 1, 2026-
UpdateJul 1, 2026-
Current statusJul 1, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71144.png

Downloads & links

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Map

FileDownload / File: emd_71144.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 400 pix.
= 371.2 Å		0.93 Å/pix.
x 400 pix.
= 371.2 Å		0.93 Å/pix.
x 400 pix.
= 371.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.928 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0204
Minimum - Maximum-0.0017576038 - 2.7677546
Average (Standard dev.)0.002761585 (±0.039059836)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 371.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_71144_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_71144_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Trimeric complex of MoFe protein bound to NifI inhibitor complex

EntireName: Trimeric complex of MoFe protein bound to NifI inhibitor complex
Components
  • Complex: Trimeric complex of MoFe protein bound to NifI inhibitor complex
    • Protein or peptide: Nitrogenase protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
    • Protein or peptide: P-II family nitrogen regulatory protein
    • Protein or peptide: P-II family nitrogen regulatory protein
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: 3-HYDROXY-3-CARBOXY-ADIPIC ACID
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: 2-OXOGLUTARIC ACID

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Supramolecule #1: Trimeric complex of MoFe protein bound to NifI inhibitor complex

SupramoleculeName: Trimeric complex of MoFe protein bound to NifI inhibitor complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Methanosarcina acetivorans (archaea)

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Macromolecule #1: Nitrogenase protein alpha chain

MacromoleculeName: Nitrogenase protein alpha chain / type: protein_or_peptide / ID: 1
Details: A Methanosarcina acetivorans strain (DJL70) expressing an N-terminal twin-Strep-tagged NifD was engineered using CRISPR-Cas9. A gRNA targeting nifI2 was designed, and synthetic homology ...Details: A Methanosarcina acetivorans strain (DJL70) expressing an N-terminal twin-Strep-tagged NifD was engineered using CRISPR-Cas9. A gRNA targeting nifI2 was designed, and synthetic homology repair templates introduced the Strep-tag sequence upstream of nifD, along with a silent PAM mutation. The repair construct (pDL707) was assembled through Gibson assembly and Gateway cloning, transformed into M. acetivorans WWM73, and selected with puromycin and 8-ADP to remove the plasmid. PCR and sequencing confirmed successful integration of the Strep-tag at the nifD locus, yielding strain DJL70 which is a natural strain.
Number of copies: 6 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Methanosarcina acetivorans (archaea)
Molecular weightTheoretical: 62.884285 KDa
SequenceString: MWSHPQFEKG GGSGGGSGGG SWSHPQFEKS GMGAEIEDKQ LIVDNILKVL PEKAARDRRK HIVVRDCAAE QHIEADDKVI PGILTNRGC AFAGTKGVVF GPIKDMVHLV HGPIGCAYFT WGTRRNLAKA EEGEDNYMNY SVCTDMKETD IVFGGEKKLK K AIDEVVKI ...String:
MWSHPQFEKG GGSGGGSGGG SWSHPQFEKS GMGAEIEDKQ LIVDNILKVL PEKAARDRRK HIVVRDCAAE QHIEADDKVI PGILTNRGC AFAGTKGVVF GPIKDMVHLV HGPIGCAYFT WGTRRNLAKA EEGEDNYMNY SVCTDMKETD IVFGGEKKLK K AIDEVVKI FNPGAITICA TCPVGLIGDD IESVAREAEE EHGIKVIPAR CEGYRGVSQS AGHHIASNSL MEHLIGTEDI KD PTPFDIN IFGEYNIGGD LWEIKPILEK IGYRIISSLT GDGSFHKISQ AHQAKLSILL CHRSINYTNR MMEEKYGVPW LKV NYIGTK GTEKSLRKMA EFFDDPELTR KTEEVIAEEK AKYADEIERY REKLQGKTAF IYAGGSRSHH YINLFEELGM KVVV AGYQF AHRDDYEGRQ IIPHIKEKAL GSILEDVHYE REENFKPSIN AERITELKEK IGLMDYEGLF PEMKDGTIVV DDLNH HETE FLVKALKPDI FCSGIKDKYW AQKIGVPSRQ IHSYDYSGRY TGFSGVLNFA RDIDMAIHSP TWRFINPPWK GEGAE

UniProtKB: Nitrogenase protein alpha chain

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Macromolecule #2: Nitrogenase molybdenum-iron protein beta chain

MacromoleculeName: Nitrogenase molybdenum-iron protein beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Methanosarcina acetivorans (archaea)
Molecular weightTheoretical: 49.478605 KDa
SequenceString: MLDYTPCEEV ERSAITINPA KICQPIGAVY AALGVHNCMP HSHGSQGCLS YLRMCLSRHY REAALATTSS FSEGTAVFGG AANLKEALV NLTTVYQPEV IAIHTTCVAE TIGDDVGVIL EDVSAEELID PSIKICAAST PSYVGTHITG YDNMVKSFVT T FASKSKPN ...String:
MLDYTPCEEV ERSAITINPA KICQPIGAVY AALGVHNCMP HSHGSQGCLS YLRMCLSRHY REAALATTSS FSEGTAVFGG AANLKEALV NLTTVYQPEV IAIHTTCVAE TIGDDVGVIL EDVSAEELID PSIKICAAST PSYVGTHITG YDNMVKSFVT T FASKSKPN GKLNIIPGFV EPADIREIKR ILSIMGISSI VFPDTTDVFD APLTKEHTFY PKGGTPIGDI EDSANSLGTI AL CRMAGGA AAKILESRYK VPAKIGPTPI GIRNTDRFIM NAAKLANVAI PPELEDERGR LVDMMTDAHP HYHGKKVAIY GDP DILSGL TSLVLEMGME PSVVLTGTQN SEFEKEIEAL IGSEYPDADI ISGGDLFMLH QIIKRKPVDL LIGNTYGKFI SRAE DVPLV RVGFPIMDRA NLHYFPIMGY AGAARLVERI GNTLLDRKDR DAPDWLLETI Q

UniProtKB: Nitrogenase molybdenum-iron protein beta chain

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Macromolecule #3: P-II family nitrogen regulatory protein

MacromoleculeName: P-II family nitrogen regulatory protein / type: protein_or_peptide / ID: 3 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Methanosarcina acetivorans (archaea)
Molecular weightTheoretical: 11.873998 KDa
SequenceString:
MQMIRAVIRP GMESKVVECL EKEGCISLTK MEVFGRGKQK GIHIADIHYD ELQKTMLLMV VEDEHKDKVI KTIMEAARTG KYGDGRIFV NPVEEAYTIR TGKAGL

UniProtKB: P-II family nitrogen regulatory protein

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Macromolecule #4: P-II family nitrogen regulatory protein

MacromoleculeName: P-II family nitrogen regulatory protein / type: protein_or_peptide / ID: 4 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Methanosarcina acetivorans (archaea)
Molecular weightTheoretical: 13.980253 KDa
Recombinant expressionOrganism: Methanosarcina acetivorans (archaea)
SequenceString:
MKEITAIIRM NKVQKTKNAL LGCGFPSFTV RRVMGRGKQR GLCFEFNPPL PDPEKEAETC IRFIPKRMFT VVVDDENVSE VVQKIIEVN QTGNAGDGKI FVSNITEAIR IRTGESGEAT ISKELM

UniProtKB: P-II family nitrogen regulatory protein

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Macromolecule #5: FE(8)-S(7) CLUSTER

MacromoleculeName: FE(8)-S(7) CLUSTER / type: ligand / ID: 5 / Number of copies: 6 / Formula: CLF
Molecular weightTheoretical: 671.215 Da
Chemical component information

ChemComp-CLF:
FE(8)-S(7) CLUSTER

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Macromolecule #6: iron-sulfur-molybdenum cluster with interstitial carbon

MacromoleculeName: iron-sulfur-molybdenum cluster with interstitial carbon
type: ligand / ID: 6 / Number of copies: 6 / Formula: ICS
Molecular weightTheoretical: 787.451 Da
Chemical component information

ChemComp-ICE:
iron-sulfur-molybdenum cluster with interstitial carbon

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Macromolecule #7: 3-HYDROXY-3-CARBOXY-ADIPIC ACID

MacromoleculeName: 3-HYDROXY-3-CARBOXY-ADIPIC ACID / type: ligand / ID: 7 / Number of copies: 6 / Formula: HCA
Molecular weightTheoretical: 206.15 Da
Chemical component information

ChemComp-HCA:
3-HYDROXY-3-CARBOXY-ADIPIC ACID

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Macromolecule #8: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 7 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Macromolecule #9: 2-OXOGLUTARIC ACID

MacromoleculeName: 2-OXOGLUTARIC ACID / type: ligand / ID: 9 / Number of copies: 3 / Formula: AKG
Molecular weightTheoretical: 146.098 Da
Chemical component information

ChemComp-AKG:
2-OXOGLUTARIC ACID

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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

BufferpH: 7.2
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 200 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: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 150000

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Image processing

CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Number images used: 74083
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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