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Yorodumi- PDB-6i5u: Cryo-EM informed directed evolution of Nitrilase 4 leads to a cha... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6i5u | |||||||||
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Title | Cryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure. | |||||||||
Components | Bifunctional nitrilase/nitrile hydratase NIT4 | |||||||||
Keywords | HYDROLASE / NITRILASE / Cyanide detoxification / Beta-cyano-L-alanine hydrolase / Helical filament | |||||||||
Function / homology | Function and homology information cyanoalanine nitrilase / 3-cyanoalanine hydratase / cyanoalanine nitrilase activity / 3-cyanoalanine hydratase activity / cyanide metabolic process / nitrilase / indole-3-acetonitrile nitrilase activity / nitrilase activity / detoxification of nitrogen compound / nitrile hydratase activity ...cyanoalanine nitrilase / 3-cyanoalanine hydratase / cyanoalanine nitrilase activity / 3-cyanoalanine hydratase activity / cyanide metabolic process / nitrilase / indole-3-acetonitrile nitrilase activity / nitrilase activity / detoxification of nitrogen compound / nitrile hydratase activity / plasma membrane / cytosol Similarity search - Function | |||||||||
Biological species | Arabidopsis thaliana (thale cress) | |||||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||
Authors | Mulelu, A.E. / Woodward, J.D. | |||||||||
Funding support | South Africa, United Kingdom, 2items
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Citation | Journal: Commun Biol / Year: 2019 Title: Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure. Authors: Andani E Mulelu / Angela M Kirykowicz / Jeremy D Woodward / Abstract: Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced ...Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana. We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6i5u.cif.gz | 301.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6i5u.ent.gz | 248 KB | Display | PDB format |
PDBx/mmJSON format | 6i5u.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6i5u_validation.pdf.gz | 876 KB | Display | wwPDB validaton report |
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Full document | 6i5u_full_validation.pdf.gz | 885.7 KB | Display | |
Data in XML | 6i5u_validation.xml.gz | 54.1 KB | Display | |
Data in CIF | 6i5u_validation.cif.gz | 74.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/i5/6i5u ftp://data.pdbj.org/pub/pdb/validation_reports/i5/6i5u | HTTPS FTP |
-Related structure data
Related structure data | 4420MC 4418C 6i5tC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 39766.113 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: NIT4, At5g22300, MWD9.8 / Plasmid: pET21B(+) / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: P46011, nitrilase, cyanoalanine nitrilase, 3-cyanoalanine hydratase |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: Active helical nitrilase complex / Type: COMPLEX Details: Arabidopsis thaliana NITRILASE 4 in 73.2 degree conformation Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||
Source (natural) | Organism: Arabidopsis thaliana (thale cress) | |||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET21b(+) | |||||||||||||||
Buffer solution | pH: 8 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3 | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 50 % / Chamber temperature: 293 K Details: The sample (2.5 ul) was applied to the grid and incubated for 30 seconds at 50% humidity before blotting and plunging. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 7 sec. / Electron dose: 45.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1266 |
Image scans | Sampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 45 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: -73.2 ° / Axial rise/subunit: 17.68 Å / Axial symmetry: C1 | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 133106 / Details: 3D class with a 73.2 degree average helical twist | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33812 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient Details: Ab initio fitting was performed using Buccaneer, cleaned up with Coot and Phenix Real-Space Refine. |