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Yorodumi- PDB-8dfc: CryoEM structure of the 1:1 ADP-tetrafluoroaluminate stabilized n... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8dfc | ||||||||||||
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Title | CryoEM structure of the 1:1 ADP-tetrafluoroaluminate stabilized nitrogenase complex from Azotobacter vinelandii | ||||||||||||
Components |
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Keywords | METAL BINDING PROTEIN / OXIDOREDUCTASE / Nitrogenase / metalloenzyme | ||||||||||||
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 | ||||||||||||
Biological species | Azotobacter vinelandii (bacteria) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.48 Å | ||||||||||||
Authors | Warmack, R.A. / Rees, D.C. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8dfc.cif.gz | 514.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8dfc.ent.gz | 414.7 KB | Display | PDB format |
PDBx/mmJSON format | 8dfc.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/df/8dfc ftp://data.pdbj.org/pub/pdb/validation_reports/df/8dfc | HTTPS FTP |
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-Related structure data
Related structure data | 27404MC 8dbyC 8dfdC 8tc3C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Nitrogenase molybdenum-iron protein ... , 2 types, 4 molecules ACBD
#1: Protein | Mass: 55363.043 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii (bacteria) / References: UniProt: P07328, nitrogenase #2: Protein | Mass: 59535.879 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii (bacteria) / References: UniProt: P07329, nitrogenase |
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-Protein , 1 types, 2 molecules EF
#3: Protein | Mass: 31548.240 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii (bacteria) / References: UniProt: P00459, nitrogenase |
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-Non-polymers , 9 types, 498 molecules
#4: Chemical | #5: Chemical | #6: Chemical | #7: Chemical | #8: Chemical | ChemComp-SF4 / | #9: Chemical | #10: Chemical | #11: Chemical | #12: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Heterohexameric nitrogenase MoFe-protein - Fe-protein 1:1 complex stabilized by aluminum tetrafluoride Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL |
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Source (natural) | Organism: Azotobacter vinelandii (bacteria) |
Buffer solution | pH: 7.8 |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Cryogen name: ETHANE-PROPANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: -3000 nm / Nominal defocus min: -800 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68443 / Symmetry type: POINT | ||||||||||||||||||||||||
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