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- EMDB-41151: CryoEM structure of nucleotide-free form of the nitrogenase iron ... -

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Basic information

Entry
Database: EMDB / ID: EMD-41151
TitleCryoEM structure of nucleotide-free form of the nitrogenase iron protein from A. vinelandii
Map data
Sample
  • Complex: Homodimeric nitrogenase Fe-protein
    • Protein or peptide: Nitrogenase iron protein,Fluorescent protein plum
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
  • Ligand: water
Keywordsnitrogenase / ATPase / metalloprotein / iron-sulfur / OXIDOREDUCTASE
Function / homology
Function and homology information


nitrogenase / carbonyl sulfide nitrogenase activity / nitrogenase activity / nitrogen fixation / bioluminescence / generation of precursor metabolites and energy / 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. / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein ...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. / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nitrogenase iron protein / Fluorescent protein plum
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria) / Discosoma sp. LW-2004 (sea anemone)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.57 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 29, 2023-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41151.png

Downloads & links

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Map

FileDownload / File: emd_41151.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.65 Å
Density
Contour LevelBy AUTHOR: 0.431
Minimum - Maximum-1.5160478 - 2.3003716
Average (Standard dev.)-0.00005301384 (±0.039252773)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 260.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41151_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41151_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: #1

Fileemd_41151_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Homodimeric nitrogenase Fe-protein

EntireName: Homodimeric nitrogenase Fe-protein
Components
  • Complex: Homodimeric nitrogenase Fe-protein
    • Protein or peptide: Nitrogenase iron protein,Fluorescent protein plum
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
  • Ligand: water

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Supramolecule #1: Homodimeric nitrogenase Fe-protein

SupramoleculeName: Homodimeric nitrogenase Fe-protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 114 KDa

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Macromolecule #1: Nitrogenase iron protein,Fluorescent protein plum

MacromoleculeName: Nitrogenase iron protein,Fluorescent protein plum / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Discosoma sp. LW-2004 (sea anemone)
Molecular weightTheoretical: 57.197016 KDa
Recombinant expressionOrganism: unidentified (others)
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 EEVGGGVSKG EEVIKEFMRF KEHMEGSVNG HEF EIEGEG EGRPYEGTQT ARLKVTKGGP LPFAWDILSP QIMYGSKAYV KHPADIPDYL KLSFPEGFKW ERVMNFEDGG VVTV TQDSS LQDGEFIYKV KVRGTNFPSD GPVMQKKTMG WEASSERMYP EDGALKGEMK MRLRLKDGGH YDAEVKTTYM AKKPV QLPG AYKTDIKLDI TSHNEDYTIV EQYERAEGRH STGA

UniProtKB: Nitrogenase iron protein, Fluorescent protein plum

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Macromolecule #2: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 2 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 17 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

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

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 552324
FSC plot (resolution estimation)

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