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Yorodumi- EMDB-27317: CryoEM structure of anaerobically prepared nitrogenase MoFe-prote... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27317 | ||||||||||||
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Title | CryoEM structure of anaerobically prepared nitrogenase MoFe-protein on ultrathin carbon | ||||||||||||
Map data | |||||||||||||
Sample |
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Keywords | Nitrogenase / metalloenzyme / METAL BINDING PROTEIN / OXIDOREDUCTASE | ||||||||||||
Function / homology | Function and homology information molybdenum-iron nitrogenase complex / nitrogenase / : / nitrogenase activity / nitrogen fixation / iron-sulfur cluster binding / ATP binding / metal ion binding Similarity search - Function | ||||||||||||
Biological species | Azotobacter vinelandii (bacteria) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.26 Å | ||||||||||||
Authors | Warmack RA / Rees DC | ||||||||||||
Funding support | United States, 3 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27317.map.gz | 382.9 MB | EMDB map data format | |
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Header (meta data) | emd-27317-v30.xml emd-27317.xml | 16.9 KB 16.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27317_fsc.xml | 19.1 KB | Display | FSC data file |
Images | emd_27317.png | 62.9 KB | ||
Filedesc metadata | emd-27317.cif.gz | 5.8 KB | ||
Others | emd_27317_half_map_1.map.gz emd_27317_half_map_2.map.gz | 691.2 MB 691.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27317 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27317 | HTTPS FTP |
-Related structure data
Related structure data | 8dbyMC 8dfcC 8dfdC 8tc3C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_27317.map.gz / Format: CCP4 / Size: 744.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 0.65 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_27317_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27317_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Heterotetrameric nitrogenase MoFe-protein
Entire | Name: Heterotetrameric nitrogenase MoFe-protein |
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Components |
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-Supramolecule #1: Heterotetrameric nitrogenase MoFe-protein
Supramolecule | Name: Heterotetrameric nitrogenase MoFe-protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Azotobacter vinelandii (bacteria) |
-Macromolecule #1: Nitrogenase molybdenum-iron protein alpha chain
Macromolecule | Name: Nitrogenase molybdenum-iron protein alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitrogenase |
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Source (natural) | Organism: Azotobacter vinelandii (bacteria) |
Molecular weight | Theoretical: 55.363043 KDa |
Sequence | String: MTGMSREEVE SLIQEVLEVY PEKARKDRNK HLAVNDPAVT QSKKCIISNK KSQPGLMTIR GCAYAGSKGV VWGPIKDMIH ISHGPVGCG QYSRAGRRNY YIGTTGVNAF VTMNFTSDFQ EKDIVFGGDK KLAKLIDEVE TLFPLNKGIS VQSECPIGLI G DDIESVSK ...String: MTGMSREEVE SLIQEVLEVY PEKARKDRNK HLAVNDPAVT QSKKCIISNK KSQPGLMTIR GCAYAGSKGV VWGPIKDMIH ISHGPVGCG QYSRAGRRNY YIGTTGVNAF VTMNFTSDFQ EKDIVFGGDK KLAKLIDEVE TLFPLNKGIS VQSECPIGLI G DDIESVSK VKGAELSKTI VPVRCEGFRG VSQSLGHHIA NDAVRDWVLG KRDEDTTFAS TPYDVAIIGD YNIGGDAWSS RI LLEEMGL RCVAQWSGDG SISEIELTPK VKLNLVHCYR SMNYISRHME EKYGIPWMEY NFFGPTKTIE SLRAIAAKFD ESI QKKCEE VIAKYKPEWE AVVAKYRPRL EGKRVMLYIG GLRPRHVIGA YEDLGMEVVG TGYEFAHNDD YDRTMKEMGD STLL YDDVT GYEFEEFVKR IKPDLIGSGI KEKFIFQKMG IPFREMHSWD YSGPYHGFDG FAIFARDMDM TLNNPCWKKL QAPWE ASEG AEKVAASA UniProtKB: Nitrogenase molybdenum-iron protein alpha chain |
-Macromolecule #2: Nitrogenase molybdenum-iron protein beta chain
Macromolecule | Name: Nitrogenase molybdenum-iron protein beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitrogenase |
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Source (natural) | Organism: Azotobacter vinelandii (bacteria) |
Molecular weight | Theoretical: 59.535879 KDa |
Sequence | String: MSQQVDKIKA SYPLFLDQDY KDMLAKKRDG FEEKYPQDKI DEVFQWTTTK EYQELNFQRE ALTVNPAKAC QPLGAVLCAL GFEKTMPYV HGSQGCVAYF RSYFNRHFRE PVSCVSDSMT EDAAVFGGQQ NMKDGLQNCK ATYKPDMIAV STTCMAEVIG D DLNAFINN ...String: MSQQVDKIKA SYPLFLDQDY KDMLAKKRDG FEEKYPQDKI DEVFQWTTTK EYQELNFQRE ALTVNPAKAC QPLGAVLCAL GFEKTMPYV HGSQGCVAYF RSYFNRHFRE PVSCVSDSMT EDAAVFGGQQ NMKDGLQNCK ATYKPDMIAV STTCMAEVIG D DLNAFINN SKKEGFIPDE FPVPFAHTPS FVGSHVTGWD NMFEGIARYF TLKSMDDKVV GSNKKINIVP GFETYLGNFR VI KRMLSEM GVGYSLLSDP EEVLDTPADG QFRMYAGGTT QEEMKDAPNA LNTVLLQPWH LEKTKKFVEG TWKHEVPKLN IPM GLDWTD EFLMKVSEIS GQPIPASLTK ERGRLVDMMT DSHTWLHGKR FALWGDPDFV MGLVKFLLEL GCEPVHILCH NGNK RWKKA VDAILAASPY GKNATVYIGK DLWHLRSLVF TDKPDFMIGN SYGKFIQRDT LHKGKEFEVP LIRIGFPIFD RHHLH RSTT LGYEGAMQIL TTLVNSILER LDEETRGMQA TDYNHDLVR UniProtKB: Nitrogenase molybdenum-iron protein beta chain |
-Macromolecule #3: iron-sulfur-molybdenum cluster with interstitial carbon
Macromolecule | Name: iron-sulfur-molybdenum cluster with interstitial carbon type: ligand / ID: 3 / Number of copies: 2 / Formula: ICS |
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Molecular weight | Theoretical: 787.451 Da |
Chemical component information | ChemComp-ICE: |
-Macromolecule #4: 3-HYDROXY-3-CARBOXY-ADIPIC ACID
Macromolecule | Name: 3-HYDROXY-3-CARBOXY-ADIPIC ACID / type: ligand / ID: 4 / Number of copies: 2 / Formula: HCA |
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Molecular weight | Theoretical: 206.15 Da |
Chemical component information | ChemComp-HCA: |
-Macromolecule #5: FE (III) ION
Macromolecule | Name: FE (III) ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: FE |
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Molecular weight | Theoretical: 55.845 Da |
-Macromolecule #6: FE(8)-S(7) CLUSTER
Macromolecule | Name: FE(8)-S(7) CLUSTER / type: ligand / ID: 6 / Number of copies: 2 / Formula: CLF |
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Molecular weight | Theoretical: 671.215 Da |
Chemical component information | ChemComp-CLF: |
-Macromolecule #7: water
Macromolecule | Name: water / type: ligand / ID: 7 / Number of copies: 770 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.0 mg/mL |
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Buffer | pH: 7.8 |
Vitrification | Cryogen name: ETHANE-PROPANE |
-Electron microscopy
Microscope | TFS KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: -3.0 µm / Nominal defocus min: -0.8 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |