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Yorodumi- EMDB-3505: Substrate specificity in plant nitrilase helical assemblies is de... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3505 | |||||||||
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Title | Substrate specificity in plant nitrilase helical assemblies is determined by their twist. | |||||||||
Map data | Sinapis alba NITRILASE 1C filament | |||||||||
Sample |
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Biological species | Sinapis alba (white mustard) | |||||||||
Method | helical reconstruction / negative staining / Resolution: 20.0 Å | |||||||||
Authors | Woodward JD / Trompetter I / Sewell BT / Piotrowski M | |||||||||
Citation | Journal: Commun Biol / Year: 2018 Title: Substrate specificity of plant nitrilase complexes is affected by their helical twist. Authors: Jeremy D Woodward / Inga Trompetter / B Trevor Sewell / Markus Piotrowski / Abstract: Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important ...Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar nitrilases from the plant , that modifying nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket. | |||||||||
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_3505.map.gz | 297.9 KB | EMDB map data format | |
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Header (meta data) | emd-3505-v30.xml emd-3505.xml | 13.1 KB 13.1 KB | Display Display | EMDB header |
Images | emd_3505.png | 92.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3505 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3505 | HTTPS FTP |
-Validation report
Summary document | emd_3505_validation.pdf.gz | 211.7 KB | Display | EMDB validaton report |
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Full document | emd_3505_full_validation.pdf.gz | 210.8 KB | Display | |
Data in XML | emd_3505_validation.xml.gz | 5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3505 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3505 | HTTPS FTP |
-Related structure data
Related structure data | 3486C 3496C 3497C 3498C 3499C 3500C 3501C 3503C 3504C C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_3505.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Sinapis alba NITRILASE 1C filament | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.22 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 : Sinapis alba NITRILASE 1C filament
Entire | Name: Sinapis alba NITRILASE 1C filament |
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Components |
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-Supramolecule #1: Sinapis alba NITRILASE 1C filament
Supramolecule | Name: Sinapis alba NITRILASE 1C filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Sinapis alba (white mustard) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET21-b(+) |
Molecular weight | Theoretical: 24 kDa/nm |
-Macromolecule #1: NITRILASE 1C
Macromolecule | Name: NITRILASE 1C / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: nitrilase |
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Source (natural) | Organism: Sinapis alba (white mustard) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MSKTTKDTTP VSNDFPSTIV RATIVQASSV YNDTPKTLEK AEKFIAEAAS DGAQLVVFPE AYVGGYPRGF RFGMAVGVQN EEGRDLFRKY HASAIAVPGP EVDKLAEMAR KYKVYLVTGA IEKDGYTLYC IALFFSSEGH FLGKHRKVMP TAMERVIWGY GDGSTLPVYD ...String: MSKTTKDTTP VSNDFPSTIV RATIVQASSV YNDTPKTLEK AEKFIAEAAS DGAQLVVFPE AYVGGYPRGF RFGMAVGVQN EEGRDLFRKY HASAIAVPGP EVDKLAEMAR KYKVYLVTGA IEKDGYTLYC IALFFSSEGH FLGKHRKVMP TAMERVIWGY GDGSTLPVYD TPLGKLGGAI CWENRMPSLR TSLYAKGVEL YCAPTADWSK EWQSSVMHIA MEGGCFVMAA CQFCLRKDYP EDPNYYFTDE SDDHSPEAVV SPGGSVIISP LGKILAGPNF GSEGLVTADL DLGDIAKAKL YFDVVGHYSR PDIFNLTVNE KEKKPVTFVS KPEDDSEPQD K |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 0.3 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Staining | Type: NEGATIVE / Material: Uranyl Acetate Details: The protein was allowed to adhere for 30 s, blotted, washed three-times with distilled water and stained with uranyl acetate, blotted again and allowed to dry at room temperature. | |||||||||
Grid | Model: Grid-tech / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 20.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Image recording | Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 30 / Average exposure time: 1.0 sec. / Average electron dose: 20.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated defocus max: 0.5 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 50200 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: PHILIPS ROTATION HOLDER |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Number classes used: 90 Applied symmetry - Helical parameters - Δz: 14.8 Å Applied symmetry - Helical parameters - Δ&Phi: -70.6 ° Applied symmetry - Helical parameters - Axial symmetry: D2 (2x2 fold dihedral) Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER (ver. 11) / Software - details: IHRSR / Number images used: 1158 |
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Segment selection | Number selected: 1158 / Software - Name: EMAN / Software - details: Boxer / Details: Picked using Eman Boxer in helix mode |
Startup model | Type of model: INSILICO MODEL In silico model: Featureless cylinder approximating the diameter of the filament. |
Final angle assignment | Type: NOT APPLICABLE / Software - Name: SPIDER (ver. 11) / Software - details: IHRSR |