Database of articles cited by EMDB/PDB/SASBDB data

Keywords
Structure methods	All X-ray diffraction NMR electron microscopy small angle scattering neutron diffraction fiber diffraction powder diffraction infrared spectroscopy EPR fluorescence transfer theoretical model Hybrid
Author
Journal
IF	>30 >20 >10 >5 all

Title	Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins.
Journal, issue, pages	Nat Protoc, Year 2024
Publish date	Apr 4, 2024
Authors	Rebeccah A Warmack / Belinda B Wenke / Thomas Spatzal / Douglas C Rees /
PubMed 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.
External links	Nat Protoc / PubMed:38575747
Methods	EM (single particle)
Resolution	2.12 - 2.57 Å
Structure data	27317 8dby EMDB-27317, PDB-8dby: CryoEM structure of anaerobically prepared nitrogenase MoFe-protein on ultrathin carbon Method: EM (single particle) / Resolution: 2.26 Å 27404 8dfc EMDB-27404, PDB-8dfc: CryoEM structure of the 1:1 ADP-tetrafluoroaluminate stabilized nitrogenase complex from Azotobacter vinelandii Method: EM (single particle) / Resolution: 2.48 Å 27405 8dfd EMDB-27405, PDB-8dfd: CryoEM structure of the 2:1 ADP-tetrafluoroaluminate stabilized nitrogenase complex from Azotobacter vinelandii Method: EM (single particle) / Resolution: 2.12 Å 41151 8tc3 EMDB-41151, PDB-8tc3: CryoEM structure of nucleotide-free form of the nitrogenase iron protein from A. vinelandii Method: EM (single particle) / Resolution: 2.57 Å
Chemicals	ChemComp-ICS: iron-sulfur-molybdenum cluster with interstitial carbon ChemComp-HCA: 3-HYDROXY-3-CARBOXY-ADIPIC ACID ChemComp-FE: Unknown entry / Iron ChemComp-CLF: FE(8)-S(7) CLUSTER ChemComp-HOH: WATER / Water ChemComp-SF4: IRON/SULFUR CLUSTER / Iron–sulfur cluster ChemComp-ADP: ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying moleculeYM / Adenosine diphosphate ChemComp-ALF: TETRAFLUOROALUMINATE ION ChemComp-MG*: Unknown entry
Source	azotobacter vinelandii (bacteria) azotobacter vinelandii dj (bacteria) discosoma sp. lw-2004 (sea anemone)
Keywords	METAL BINDING PROTEIN / OXIDOREDUCTASE / Nitrogenase / metalloenzyme / ATPase / metalloprotein / iron-sulfur