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- EMDB-26759: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1... -

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Entry
Database: EMDB / ID: EMD-26759
TitleLocally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1 complex (ATP-bound) with MoFeP during catalytic N2 reduction
Map datadeepEMhancer sharpened map
Sample
  • Complex: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1 complex (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: 2.75 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_26759.png

Downloads & links

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Map

FileDownload / File: emd_26759.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMhancer sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 384 pix.
= 320.64 Å		0.84 Å/pix.
x 384 pix.
= 320.64 Å		0.84 Å/pix.
x 384 pix.
= 320.64 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.054129638 - 1.7228051
Average (Standard dev.)0.0005479099 (±0.012617971)
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_26759_msk_1.map
Projections & Slices
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Additional map: unsharpened map

Fileemd_26759_additional_1.map
Annotationunsharpened map
Projections & Slices
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Half map: gold-standard half map A

Fileemd_26759_half_map_1.map
Annotationgold-standard half map A
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: gold-standard half map B

Fileemd_26759_half_map_2.map
Annotationgold-standard half map B
Projections & Slices
AxesZYX

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Density Histograms

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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 (ATP-bound) with MoFeP during catalytic N2 reduction
Components
  • Complex: Locally refined cryoEM map of Azotobacter vinelandii FeP in a 1:1 complex (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 (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 MAAEAGTVE DLELEDVLKA GYGGVKCVES GGPEPGVGCA GRGVITAINF LEEEGAYEDD L DFVFYDVL GDVVCGGFAM PIRENKAQEI YIVCSGEMMA MYAANNISKG ...String:
MAMRQCAIYG KGGIGKSTTT QNLVAALAEM GKKVMIVGCD PKADSTRLIL HSKAQNTIME MAAEAGTVE DLELEDVLKA GYGGVKCVES GGPEPGVGCA GRGVITAINF LEEEGAYEDD L DFVFYDVL GDVVCGGFAM PIRENKAQEI YIVCSGEMMA MYAANNISKG IVKYANSGSV RL GGLICNS RNTDREDELI IALANKLGTQ MIHFVPRDNV VQRAEIRRMT VIEYDPKAKQ ADE YRALAR KVVDNKLLVI PNPITMDELE ELLMEFGIME VEDESIVGKT AEEV

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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 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..
Details3.6 mg/mL FeP Sample also contained 1.44 mg/mL MoFeP

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Electron microscopy

MicroscopeTFS KRIOS
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
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: 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
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
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 38099
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 / Avg.num./class: 42412 / Software - Name: cryoSPARC (ver. 3.3.2)
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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