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Yorodumi- EMDB-1313: Post-translational cleavage of recombinantly expressed nitrilase ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1313 | |||||||||
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Title | Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form. | |||||||||
Map data | Map of the carboxy-terminal truncated nitrilase helix from Rhodococcus rhodochrous J1 | |||||||||
Sample |
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Function / homology | nitrilase activity / Carbon-nitrogen hydrolase Function and homology information | |||||||||
Biological species | Rhodococcus rhodochrous (bacteria) | |||||||||
Method | helical reconstruction / negative staining / Resolution: 18.0 Å | |||||||||
Authors | Thuku RN / Weber BW / Varsani A / Sewell BT | |||||||||
Citation | Journal: FEBS J / Year: 2007 Title: Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form. Authors: R Ndoria Thuku / Brandon W Weber / Arvind Varsani / B Trevor Sewell / Abstract: Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. ...Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy, followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily, enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1-327. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1313.map.gz | 609.9 KB | EMDB map data format | |
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Header (meta data) | emd-1313-v30.xml emd-1313.xml | 10.3 KB 10.3 KB | Display Display | EMDB header |
Images | 1313.gif | 60.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1313 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1313 | HTTPS FTP |
-Validation report
Summary document | emd_1313_validation.pdf.gz | 200.1 KB | Display | EMDB validaton report |
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Full document | emd_1313_full_validation.pdf.gz | 199.2 KB | Display | |
Data in XML | emd_1313_validation.xml.gz | 4.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1313 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1313 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1313.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Map of the carboxy-terminal truncated nitrilase helix from Rhodococcus rhodochrous J1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Carboxy-terminal truncated nitrilase from Rhodococcus rhodochrous J1
Entire | Name: Carboxy-terminal truncated nitrilase from Rhodococcus rhodochrous J1 |
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Components |
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-Supramolecule #1000: Carboxy-terminal truncated nitrilase from Rhodococcus rhodochrous J1
Supramolecule | Name: Carboxy-terminal truncated nitrilase from Rhodococcus rhodochrous J1 type: sample / ID: 1000 / Number unique components: 1 |
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Molecular weight | Experimental: 36 KDa / Theoretical: 36 KDa / Method: SDS-PAGE MALDI-TOF mass spectrometry |
-Macromolecule #1: J1 nitrilase 1-327
Macromolecule | Name: J1 nitrilase 1-327 / type: protein_or_peptide / ID: 1 / Oligomeric state: helix / Recombinant expression: Yes |
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Source (natural) | Organism: Rhodococcus rhodochrous (bacteria) / Strain: J1 |
Molecular weight | Experimental: 36 KDa / Theoretical: 36 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET30a |
Sequence | GO: nitrilase activity / InterPro: Carbon-nitrogen hydrolase |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 7.8 / Details: 100mM KH2PO4, 400mM KCl, 10% (v/v) EtOH |
Staining | Type: NEGATIVE Details: Sample was applied to glow discharged carbon films which were then washed twice with distilled water and then stained with 2% w/v uranyl acetate |
Grid | Details: 300 mesh |
Vitrification | Cryogen name: NONE |
-Electron microscopy
Microscope | JEOL 2000EX |
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Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 50 / Average electron dose: 30 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN |
Electron optics | Calibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 50000 |
Sample stage | Specimen holder: standard / Specimen holder model: OTHER |
-Image processing
Details | Helices were formed by autolysis of the wild-type protein after storage for one month at 4 degrees C |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 15.8 Å Applied symmetry - Helical parameters - Δ&Phi: 73.65 ° Applied symmetry - Helical parameters - Axial symmetry: D1 (2x1 fold dihedral) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Details: D1 symmetry was imposed on map after convergence |
-Atomic model buiding 1
Software | Name: Situs |
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Details | The helical symmetry was applied to a homology model to generate a helix containing 9 dimers. This was fitted to the map using CoLoRes. A two dimensional search of radial distance and azimuthal angle was conducted to find the best fit. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |