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- EMDB-27404: CryoEM structure of the 1:1 ADP-tetrafluoroaluminate stabilized n... -

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Basic information

Entry
Database: EMDB / ID: EMD-27404
TitleCryoEM structure of the 1:1 ADP-tetrafluoroaluminate stabilized nitrogenase complex from Azotobacter vinelandii
Map data
Sample
  • Complex: Heterohexameric nitrogenase MoFe-protein - Fe-protein 1:1 complex stabilized by aluminum tetrafluoride
    • Protein or peptide: x 3 types
  • Ligand: x 9 types
KeywordsNitrogenase / metalloenzyme / metal binding protein / 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 1 / 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: 2.48 Å
AuthorsWarmack RA / 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 Protoc / Year: 2024
Title: Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins.
Authors: Rebeccah A Warmack / Belinda B Wenke / Thomas Spatzal / Douglas C Rees /
Abstract: Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast ...Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments.
History
DepositionJun 21, 2022-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27404.png

Downloads & links

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Map

FileDownload / File: emd_27404.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 576 pix.
= 479.232 Å		0.83 Å/pix.
x 576 pix.
= 479.232 Å		0.83 Å/pix.
x 576 pix.
= 479.232 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.47
Minimum - Maximum-1.3693515 - 3.2089832
Average (Standard dev.)0.00009449175 (±0.0495603)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions576576576
Spacing576576576
CellA=B=C: 479.232 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27404_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27404_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Heterohexameric nitrogenase MoFe-protein - Fe-protein 1:1 complex...

EntireName: Heterohexameric nitrogenase MoFe-protein - Fe-protein 1:1 complex stabilized by aluminum tetrafluoride
Components
  • Complex: Heterohexameric nitrogenase MoFe-protein - Fe-protein 1:1 complex stabilized by aluminum tetrafluoride
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
    • Protein or peptide: Nitrogenase iron protein 1
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: 3-HYDROXY-3-CARBOXY-ADIPIC ACID
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: FE (III) ION
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Heterohexameric nitrogenase MoFe-protein - Fe-protein 1:1 complex...

SupramoleculeName: Heterohexameric nitrogenase MoFe-protein - Fe-protein 1:1 complex stabilized by aluminum tetrafluoride
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
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: 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 (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 1

MacromoleculeName: Nitrogenase iron protein 1 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Azotobacter vinelandii (bacteria)
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 1

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Macromolecule #4: iron-sulfur-molybdenum cluster with interstitial carbon

MacromoleculeName: iron-sulfur-molybdenum cluster with interstitial carbon
type: ligand / ID: 4 / 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 #5: 3-HYDROXY-3-CARBOXY-ADIPIC ACID

MacromoleculeName: 3-HYDROXY-3-CARBOXY-ADIPIC ACID / type: ligand / ID: 5 / 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 #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: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #9: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 9 / Number of copies: 2 / 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 #10: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #11: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 11 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #12: water

MacromoleculeName: water / type: ligand / ID: 12 / Number of copies: 483 / 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

Concentration1 mg/mL
BufferpH: 7.8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
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: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 68443
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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