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- EMDB-26762: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1... -

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Entry
Database: EMDB / ID: EMD-26762
TitleLocally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1 complex (ADP/ATP-bound) with MoFeP during catalytic N2 reduction
Map datadeepEMhanced sharpened map
Sample
  • Complex: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1 complex (ADP/ATP-bound) with MoFeP during catalytic N2 reduction
    • Protein or peptide: Nitrogenase iron protein gamma chain
Keywordsnitrogenase / FeP / nitrogen fixation / nitrogenase complex / OXIDOREDUCTASE
Biological speciesAzotobacter vinelandii DJ (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsRutledge HL / Cook B / 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-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26762.png

Downloads & links

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Map

FileDownload / File: emd_26762.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMhanced sharpened map
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.21
Minimum - Maximum-0.054769065 - 2.0882769
Average (Standard dev.)0.00064766675 (±0.013469655)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 320.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26762_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_26762_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: gold-standard half map A

Fileemd_26762_half_map_1.map
Annotationgold-standard half map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: gold-standard half map B

Fileemd_26762_half_map_2.map
Annotationgold-standard half map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

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Sample components

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Entire : Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1...

EntireName: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1 complex (ADP/ATP-bound) with MoFeP during catalytic N2 reduction
Components
  • Complex: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1 complex (ADP/ATP-bound) with MoFeP during catalytic N2 reduction
    • Protein or peptide: Nitrogenase iron protein gamma chain

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Supramolecule #1: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1...

SupramoleculeName: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1 complex (ADP/ATP-bound) with MoFeP during catalytic N2 reduction
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Wild-type MoFeP and FeP were purified from the native organism, Azotobacter vinelandii. This map is the locally refined map for the FeP portion of the 1:1 complex.
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 63 KDa

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Macromolecule #1: Nitrogenase iron protein gamma chain

MacromoleculeName: Nitrogenase iron protein gamma chain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
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

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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

Concentration3.6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2TRIS
25.0 mMNaClSodium chloridesodium chloride

Details: Solutions were prepared and filtered immediately prior to the experiment.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / 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..
Details3.6 mg/mL FeP Sample also contained 1.44 mg/mL MoFeP

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 135000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 93.0 K / Max: 123.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 14903 / Average electron dose: 65.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 19711170
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4wza


Details: Used chains E,F for FeP
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0/beta2)
Final 3D classificationNumber classes: 4 / Avg.num./class: 42412 / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0/beta2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 48293
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4wza


Chain - Chain ID: EF / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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