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- EMDB-48381: Azotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using... -

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Basic information

Entry
Database: EMDB / ID: EMD-48381
TitleAzotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using the SPT Labtech chameleon of 5 mM sodium dithionite under Al's oil
Map dataSharpened EM map of MoFeP from Azotobacter vinellandii (C1 symmetry) in the presence of 5 mM sodium dithionite under Al's oil
Sample
  • Complex: Azotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using the SPT Labtech chameleon of 5 mM sodium dithionite under Al's oil
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
  • Ligand: 3-HYDROXY-3-CARBOXY-ADIPIC ACID
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: FE (III) ION
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: water
KeywordsNitrogenase / FeMoCo / nitrogen / P-cluster / METAL BINDING PROTEIN
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: 2.33 Å
AuthorsCook BD / Narehood SM / McGuire KL / Li Y / Tezcan FA / Herzik MA
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35GM138206 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM148607-02 United States
Other privateSearle Scholars
CitationJournal: Nat Commun / Year: 2025
Title: Preparation of oxygen-sensitive proteins for high-resolution cryoEM structure determination using blot-free vitrification.
Authors: Brian D Cook / Sarah M Narehood / Kelly L McGuire / Yizhou Li / F Akif Tezcan / Mark A Herzik /
Abstract: High-quality grid preparation for single-particle cryogenic electron microscopy (cryoEM) remains a bottleneck for routinely obtaining high-resolution structures. The issues that arise from ...High-quality grid preparation for single-particle cryogenic electron microscopy (cryoEM) remains a bottleneck for routinely obtaining high-resolution structures. The issues that arise from traditional grid preparation workflows are particularly exacerbated for oxygen-sensitive proteins, including metalloproteins, whereby oxygen-induced damage and alteration of oxidation states can result in protein inactivation, denaturation, and/or aggregation. Indeed, 99% of the current structures in the EMBD were prepared aerobically and limited successes for anaerobic cryoEM grid preparation exist. Current practices for anaerobic grid preparation involve a vitrification device located in an anoxic chamber, which presents significant challenges including temperature and humidity control, optimization of freezing conditions, costs for purchase and operation, as well as accessibility. Here, we present a streamlined approach that allows for the vitrification of oxygen-sensitive proteins in reduced states using an automated blot-free grid vitrification device - the SPT Labtech chameleon. This robust workflow allows for high-resolution structure determination of dynamic, oxygen-sensitive proteins, of varying complexity and molecular weight.
History
DepositionDec 19, 2024-
Header (metadata) releaseApr 30, 2025-
Map releaseApr 30, 2025-
UpdateApr 30, 2025-
Current statusApr 30, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48381.png

Downloads & links

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Map

FileDownload / File: emd_48381.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened EM map of MoFeP from Azotobacter vinellandii (C1 symmetry) in the presence of 5 mM sodium dithionite under Al's oil
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 384 pix.
= 282.24 Å		0.74 Å/pix.
x 384 pix.
= 282.24 Å		0.74 Å/pix.
x 384 pix.
= 282.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.735 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.32304063 - 0.47621417
Average (Standard dev.)0.00018176304 (±0.010687516)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 282.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48381_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened EM map of MoFeP from Azotobacter vinellandii...

Fileemd_48381_additional_1.map
AnnotationUnsharpened EM map of MoFeP from Azotobacter vinellandii (C1 symmetry) in the presence of 5 mM sodium dithionite under Al's oil
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Resolve sharpened EM map of MoFeP from Azotobacter...

Fileemd_48381_additional_2.map
AnnotationResolve sharpened EM map of MoFeP from Azotobacter vinellandii (C1 symmetry) in the presence of 5 mM sodium dithionite under Al's oil
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B of MoFeP from Azotobacter vinellandii...

Fileemd_48381_half_map_1.map
AnnotationHalf Map B of MoFeP from Azotobacter vinellandii (C1 symmetry) in the presence of 5 mM sodium dithionite under Al's oil
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B of MoFeP from Azotobacter vinellandii...

Fileemd_48381_half_map_2.map
AnnotationHalf Map B of MoFeP from Azotobacter vinellandii (C1 symmetry) in the presence of 5 mM sodium dithionite under Al's oil
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Azotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using...

EntireName: Azotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using the SPT Labtech chameleon of 5 mM sodium dithionite under Al's oil
Components
  • Complex: Azotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using the SPT Labtech chameleon of 5 mM sodium dithionite under Al's oil
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
  • Ligand: 3-HYDROXY-3-CARBOXY-ADIPIC ACID
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: FE (III) ION
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: water

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Supramolecule #1: Azotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using...

SupramoleculeName: Azotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using the SPT Labtech chameleon of 5 mM sodium dithionite under Al's oil
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Azotobacter vinelandii (bacteria)
Molecular weightTheoretical: 230 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 (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: 3-HYDROXY-3-CARBOXY-ADIPIC ACID

MacromoleculeName: 3-HYDROXY-3-CARBOXY-ADIPIC ACID / type: ligand / ID: 3 / 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 #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: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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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: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 1037 / 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

Concentration8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
500.0 mMNaClSodium Chloride
GridModel: Quantifoil Active R1.2/0.8 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 298.15 K / Instrument: SPOTITON
Details: Samples were frozen with the SPT Labtech chameleon.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
SoftwareName: EPU (ver. 2)
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 2501 / Average exposure time: 5.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 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: 881772
Startup modelType of model: EMDB MAP
EMDB ID:

0407

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.33 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 121967
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6nbc


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 51
Output model

PDB-9mly:
Azotobacter vinelandii Reduced MoFeP (C1 symmetry) obtained using the SPT Labtech chameleon of 5 mM sodium dithionite under Al's oil

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