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- EMDB-26764: CryoEM structure of Azotobacter vinelandii nitrogenase complex (2... -

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Entry
Database: EMDB / ID: EMD-26764
TitleCryoEM structure of Azotobacter vinelandii nitrogenase complex (2:1 FeP:MoFeP) inhibited by BeFx during catalytic N2 reduction
Map datadeepEMhanced sharpened map
Sample
  • Complex: BeFx inhibited Azotobacter vinelandii nitrogenase complex. Inhibition occurred during catalytic N2 reduction
    • Protein or peptide: x 3 types
  • Ligand: x 9 types
Keywordsnitrogenase / MoFeP / nitrogen fixation / OXIDOREDUCTASE
Function / homology
Function and homology information


molybdenum-iron nitrogenase complex / nitrogenase / : / nitrogenase activity / nitrogen fixation / iron-sulfur cluster binding / 4 iron, 4 sulfur cluster binding / ATP binding / metal ion binding
Similarity search - Function
Nitrogenase iron protein NifH / NifH/frxC family / NifH/chlL conserved site / 4Fe-4S iron sulfur cluster binding proteins, NifH/frxC family / NifH/frxC family signature 2. / NifH/frxC family signature 1. / NIFH_FRXC family profile. / Nitrogenase molybdenum-iron protein beta chain, N-terminal / Domain of unknown function (DUF3364) / Nitrogenase molybdenum-iron protein alpha chain ...Nitrogenase iron protein NifH / NifH/frxC family / NifH/chlL conserved site / 4Fe-4S iron sulfur cluster binding proteins, NifH/frxC family / NifH/frxC family signature 2. / NifH/frxC family signature 1. / NIFH_FRXC family profile. / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nitrogenase iron protein / Nitrogenase molybdenum-iron protein beta chain / Nitrogenase molybdenum-iron protein alpha chain
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsRutledge HL / Cook BD / Nguyen HPM / Tezcan FA / Herzik MA
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM099813 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138206 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM008326 United States
National Aeronautic Space Administration (NASA, United States)80NSSC18M0093 United States
CitationJournal: Science / Year: 2022
Title: Structures of the nitrogenase complex prepared under catalytic turnover conditions.
Authors: Hannah L Rutledge / Brian D Cook / Hoang P M Nguyen / Mark A Herzik / F Akif Tezcan /
Abstract: The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ...The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo-electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.
History
DepositionApr 26, 2022-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26764.png

Downloads & links

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Map

FileDownload / File: emd_26764.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMhanced sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 384 pix.
= 312.96 Å		0.82 Å/pix.
x 384 pix.
= 312.96 Å		0.82 Å/pix.
x 384 pix.
= 312.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.815 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.065
Minimum - Maximum-0.109596916 - 1.8871024
Average (Standard dev.)0.0010434651 (±0.020868808)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 312.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26764_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_26764_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: gold-standard half map A

Fileemd_26764_half_map_1.map
Annotationgold-standard half map A
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: gold-standard half map B

Fileemd_26764_half_map_2.map
Annotationgold-standard half map B
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : BeFx inhibited Azotobacter vinelandii nitrogenase complex. Inhibi...

EntireName: BeFx inhibited Azotobacter vinelandii nitrogenase complex. Inhibition occurred during catalytic N2 reduction
Components
  • Complex: BeFx inhibited Azotobacter vinelandii nitrogenase complex. Inhibition occurred during catalytic N2 reduction
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
    • Protein or peptide: Nitrogenase iron protein gamma chain
  • Ligand: 3-HYDROXY-3-CARBOXY-ADIPIC ACID
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: FE (III) ION
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: BERYLLIUM
  • Ligand: water

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Supramolecule #1: BeFx inhibited Azotobacter vinelandii nitrogenase complex. Inhibi...

SupramoleculeName: BeFx inhibited Azotobacter vinelandii nitrogenase complex. Inhibition occurred during catalytic N2 reduction
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Wild-type MoFeP and FeP were purified from the native organism, Azotobacter vinelandii
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 359.21 KDa

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Macromolecule #1: Nitrogenase molybdenum-iron protein alpha chain

MacromoleculeName: Nitrogenase molybdenum-iron protein alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 55.363043 KDa
SequenceString: MTGMSREEVE SLIQEVLEVY PEKARKDRNK HLAVNDPAVT QSKKCIISNK KSQPGLMTIR GCAYAGSKGV VWGPIKDMIH ISHGPVGCG QYSRAGRRNY YIGTTGVNAF VTMNFTSDFQ EKDIVFGGDK KLAKLIDEVE TLFPLNKGIS VQSECPIGLI G DDIESVSK ...String:
MTGMSREEVE SLIQEVLEVY PEKARKDRNK HLAVNDPAVT QSKKCIISNK KSQPGLMTIR GCAYAGSKGV VWGPIKDMIH ISHGPVGCG QYSRAGRRNY YIGTTGVNAF VTMNFTSDFQ EKDIVFGGDK KLAKLIDEVE TLFPLNKGIS VQSECPIGLI G DDIESVSK VKGAELSKTI VPVRCEGFRG VSQSLGHHIA NDAVRDWVLG KRDEDTTFAS TPYDVAIIGD YNIGGDAWSS RI LLEEMGL RCVAQWSGDG SISEIELTPK VKLNLVHCYR SMNYISRHME EKYGIPWMEY NFFGPTKTIE SLRAIAAKFD ESI QKKCEE VIAKYKPEWE AVVAKYRPRL EGKRVMLYIG GLRPRHVIGA YEDLGMEVVG TGYEFAHNDD YDRTMKEMGD STLL YDDVT GYEFEEFVKR IKPDLIGSGI KEKFIFQKMG IPFREMHSWD YSGPYHGFDG FAIFARDMDM TLNNPCWKKL QAPWE ASEG AEKVAASA

UniProtKB: Nitrogenase molybdenum-iron 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: 2 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 59.535879 KDa
SequenceString: MSQQVDKIKA SYPLFLDQDY KDMLAKKRDG FEEKYPQDKI DEVFQWTTTK EYQELNFQRE ALTVNPAKAC QPLGAVLCAL GFEKTMPYV HGSQGCVAYF RSYFNRHFRE PVSCVSDSMT EDAAVFGGQQ NMKDGLQNCK ATYKPDMIAV STTCMAEVIG D DLNAFINN ...String:
MSQQVDKIKA SYPLFLDQDY KDMLAKKRDG FEEKYPQDKI DEVFQWTTTK EYQELNFQRE ALTVNPAKAC QPLGAVLCAL GFEKTMPYV HGSQGCVAYF RSYFNRHFRE PVSCVSDSMT EDAAVFGGQQ NMKDGLQNCK ATYKPDMIAV STTCMAEVIG D DLNAFINN SKKEGFIPDE FPVPFAHTPS FVGSHVTGWD NMFEGIARYF TLKSMDDKVV GSNKKINIVP GFETYLGNFR VI KRMLSEM GVGYSLLSDP EEVLDTPADG QFRMYAGGTT QEEMKDAPNA LNTVLLQPWH LEKTKKFVEG TWKHEVPKLN IPM GLDWTD EFLMKVSEIS GQPIPASLTK ERGRLVDMMT DSHTWLHGKR FALWGDPDFV MGLVKFLLEL GCEPVHILCH NGNK RWKKA VDAILAASPY GKNATVYIGK DLWHLRSLVF TDKPDFMIGN SYGKFIQRDT LHKGKEFEVP LIRIGFPIFD RHHLH RSTT LGYEGAMQIL TTLVNSILER LDEETRGMQA TDYNHDLVR

UniProtKB: Nitrogenase molybdenum-iron protein beta chain

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Macromolecule #3: Nitrogenase iron protein gamma chain

MacromoleculeName: Nitrogenase iron protein gamma chain / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria) / Strain: DJ / ATCC BAA-1303
Molecular weightTheoretical: 31.54824 KDa
SequenceString: MAMRQCAIYG KGGIGKSTTT QNLVAALAEM GKKVMIVGCD PKADSTRLIL HSKAQNTIME MAAEAGTVED LELEDVLKAG YGGVKCVES GGPEPGVGCA GRGVITAINF LEEEGAYEDD LDFVFYDVLG DVVCGGFAMP IRENKAQEIY IVCSGEMMAM Y AANNISKG ...String:
MAMRQCAIYG KGGIGKSTTT QNLVAALAEM GKKVMIVGCD PKADSTRLIL HSKAQNTIME MAAEAGTVED LELEDVLKAG YGGVKCVES GGPEPGVGCA GRGVITAINF LEEEGAYEDD LDFVFYDVLG DVVCGGFAMP IRENKAQEIY IVCSGEMMAM Y AANNISKG IVKYANSGSV RLGGLICNSR NTDREDELII ALANKLGTQM IHFVPRDNVV QRAEIRRMTV IEYDPKAKQA DE YRALARK VVDNKLLVIP NPITMDELEE LLMEFGIMEV EDESIVGKTA EEV

UniProtKB: Nitrogenase iron protein

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Macromolecule #4: 3-HYDROXY-3-CARBOXY-ADIPIC ACID

MacromoleculeName: 3-HYDROXY-3-CARBOXY-ADIPIC ACID / type: ligand / ID: 4 / Number of copies: 2 / Formula: HCA
Molecular weightTheoretical: 206.15 Da
Chemical component information

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

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Macromolecule #5: iron-sulfur-molybdenum cluster with interstitial carbon

MacromoleculeName: iron-sulfur-molybdenum cluster with interstitial carbon
type: ligand / ID: 5 / Number of copies: 2 / Formula: ICS
Molecular weightTheoretical: 787.451 Da
Chemical component information

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

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Macromolecule #6: FE(8)-S(7) CLUSTER

MacromoleculeName: FE(8)-S(7) CLUSTER / type: ligand / ID: 6 / Number of copies: 2 / Formula: CLF
Molecular weightTheoretical: 671.215 Da
Chemical component information

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

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Macromolecule #7: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #8: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 8 / Number of copies: 2 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 4 / 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 #11: BERYLLIUM

MacromoleculeName: BERYLLIUM / type: ligand / ID: 11 / Number of copies: 4 / Formula: 0BE
Molecular weightTheoretical: 9.012 Da
Chemical component information

ChemComp-0BE:
BERYLLIUM

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Macromolecule #12: water

MacromoleculeName: water / type: ligand / ID: 12 / Number of copies: 55 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration5.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2TRIS
25.0 mMNaClsodium chloride

Details: Solutions were prepared and filtered immediately prior to the experiment.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: Custom manual plunger. Greater than 95% humidity..
Details1.44 mg/mL MoFeP 3.6 mg/mL FeP

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 93.0 K / Max: 123.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 8083 / Average electron dose: 65.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
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: 2628629
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4wza


Details: Used chains A,B,C,D,E,F,G,H.
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 72125
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0/beta2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0/beta2)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4wza


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7uta:
CryoEM structure of Azotobacter vinelandii nitrogenase complex (2:1 FeP:MoFeP) inhibited by BeFx during catalytic N2 reduction

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