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- EMDB-28272: CryoEM structure of the high pH turnover-inactivated nitrogenase ... -

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Basic information

Entry
Database: EMDB / ID: EMD-28272
TitleCryoEM structure of the high pH turnover-inactivated nitrogenase MoFe-protein
Map dataPrimary map sharpened by a B-factor of 50 angstroms.
Sample
  • Complex: Heterotetrameric nitrogenase MoFe-protein
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: UNKNOWN ATOM OR ION
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: CHAPSO
  • Ligand: FE (III) ION
  • Ligand: water
Keywordsnitrogenase / nitrogen fixation / reductase / MoFe / OXIDOREDUCTASE
Function / homology
Function and homology information


molybdenum-iron nitrogenase complex / nitrogenase / : / nitrogenase activity / nitrogen fixation / iron-sulfur cluster binding / ATP binding / metal ion binding
Similarity search - Function
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
Similarity search - Domain/homology
Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase molybdenum-iron protein beta chain
Similarity search - Component
Biological speciesAzotobacter vinelandii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsWarmack RA / Maggiolo AO / Rees DC
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM045162 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143836-01 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural consequences of turnover-induced homocitrate loss in nitrogenase.
Authors: Rebeccah A Warmack / Ailiena O Maggiolo / Andres Orta / Belinda B Wenke / James B Howard / Douglas C Rees /
Abstract: Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor ...Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor contains a [7Fe:1Mo:9S:1C] metallocluster coordinated with an R-homocitrate (HCA) molecule. Here, we establish through single particle cryoEM and chemical analysis of two forms of the Azotobacter vinelandii MoFe-protein - a high pH turnover inactivated species and a ∆NifV variant that cannot synthesize HCA - that loss of HCA is coupled to α-subunit domain and FeMo-cofactor disordering, and formation of a histidine coordination site. We further find a population of the ∆NifV variant complexed to an endogenous protein identified through structural and proteomic approaches as the uncharacterized protein NafT. Recognition by endogenous NafT demonstrates the physiological relevance of the HCA-compromised form, perhaps for cofactor insertion or repair. Our results point towards a dynamic active site in which HCA plays a role in enabling nitrogenase catalysis by facilitating activation of the FeMo-cofactor from a relatively stable form to a state capable of reducing dinitrogen under ambient conditions.
History
DepositionSep 30, 2022-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28272.png

Downloads & links

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Map

FileDownload / File: emd_28272.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map sharpened by a B-factor of 50 angstroms.
Voxel sizeX=Y=Z: 0.65 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.326944 - 2.1949053
Average (Standard dev.)0.00020935347 (±0.038472753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions576576576
Spacing576576576
CellA=B=C: 374.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Secondary blurred map.

Fileemd_28272_additional_1.map
AnnotationSecondary blurred map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28272_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_28272_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 : Heterotetrameric nitrogenase MoFe-protein

EntireName: Heterotetrameric nitrogenase MoFe-protein
Components
  • Complex: Heterotetrameric nitrogenase MoFe-protein
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: UNKNOWN ATOM OR ION
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: CHAPSO
  • Ligand: FE (III) ION
  • Ligand: water

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Supramolecule #1: Heterotetrameric nitrogenase MoFe-protein

SupramoleculeName: Heterotetrameric nitrogenase MoFe-protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Azotobacter vinelandii (bacteria)

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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 (bacteria)
Molecular weightTheoretical: 54.000559 KDa
SequenceString: MSREEVESLI QEVLEVYPEK ARKDRNKHLA VNDPAVTQSK KCIISNKKSQ PGLMTIRGCA YAGSKGVVWG PIKDMIHISH GPVGCGQYS RAGRRNYYIG TTGVNAFVTM NFTSDFQEKD IVFGGDKKLA KLIDEVETLF PLNKGISVQS ECPIGLIGDD I ESVSKVKG ...String:
MSREEVESLI QEVLEVYPEK ARKDRNKHLA VNDPAVTQSK KCIISNKKSQ PGLMTIRGCA YAGSKGVVWG PIKDMIHISH GPVGCGQYS RAGRRNYYIG TTGVNAFVTM NFTSDFQEKD IVFGGDKKLA KLIDEVETLF PLNKGISVQS ECPIGLIGDD I ESVSKVKG AELSKTIVPV RCEGFRGVSQ SLGHHIANDA VRDWVLGKRD EDTTFASTPY DVAIIGDYNI GGDAWSSRIL LE EMGLRCV AQWSGDGSIS EIELTPKVKL NLVHCYRSMN YISRHMEEKY GIPWMEYNFF GPTKTIESLR AIAAKFDESI QKK CEEVIA KYKPEWEAVV AKYRPRLEGK RVMLYIGGLR PRHVIGAYED LGMEVVGTGY EFAHNDDYDR TMKEMGDSTL LYDD VTGYE FEEFVKRIKP DLIGSGIKEK FIFQKMGIPF REMHSWDYSG PYHGFDGFAI FARDMDMTLN NPCWKKLQAP WE

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 (bacteria)
Molecular weightTheoretical: 59.404684 KDa
SequenceString: SQQVDKIKAS YPLFLDQDYK DMLAKKRDGF EEKYPQDKID EVFQWTTTKE YQELNFQREA LTVNPAKACQ PLGAVLCALG FEKTMPYVH GSQGCVAYFR SYFNRHFREP VSCVSDSMTE DAAVFGGQQN MKDGLQNCKA TYKPDMIAVS TTCMAEVIGD D LNAFINNS ...String:
SQQVDKIKAS YPLFLDQDYK DMLAKKRDGF EEKYPQDKID EVFQWTTTKE YQELNFQREA LTVNPAKACQ PLGAVLCALG FEKTMPYVH GSQGCVAYFR SYFNRHFREP VSCVSDSMTE DAAVFGGQQN MKDGLQNCKA TYKPDMIAVS TTCMAEVIGD D LNAFINNS KKEGFIPDEF PVPFAHTPSF VGSHVTGWDN MFEGIARYFT LKSMDDKVVG SNKKINIVPG FETYLGNFRV IK RMLSEMG VGYSLLSDPE EVLDTPADGQ FRMYAGGTTQ EEMKDAPNAL NTVLLQPWHL EKTKKFVEGT WKHEVPKLNI PMG LDWTDE FLMKVSEISG QPIPASLTKE RGRLVDMMTD SHTWLHGKRF ALWGDPDFVM GLVKFLLELG CEPVHILCHN GNKR WKKAV DAILAASPYG KNATVYIGKD LWHLRSLVFT DKPDFMIGNS YGKFIQRDTL HKGKEFEVPL IRIGFPIFDR HHLHR STTL GYEGAMQILT TLVNSILERL DEETRGMQAT DYNHDLVR

UniProtKB: Nitrogenase molybdenum-iron protein beta chain

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Macromolecule #3: iron-sulfur-molybdenum cluster with interstitial carbon

MacromoleculeName: iron-sulfur-molybdenum cluster with interstitial carbon
type: ligand / ID: 3 / 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 #4: UNKNOWN ATOM OR ION

MacromoleculeName: UNKNOWN ATOM OR ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: UNX
Molecular weightTheoretical: 671.215 Da

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

MacromoleculeName: FE(8)-S(7) CLUSTER / type: ligand / ID: 5 / 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 #6: CHAPSO

MacromoleculeName: CHAPSO / type: ligand / ID: 6 / Number of copies: 2 / Formula: 1N7
Molecular weightTheoretical: 631.884 Da
Chemical component information

ChemComp-1N7:
CHAPSO / detergent*YM

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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: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 926 / 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

Concentration2 mg/mL
BufferpH: 7.8
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: -3.0 µm / Nominal defocus min: -0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 156311
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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