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- EMDB-4418: Cryo-EM informed directed evolution of Nitrilase 4 leads to a cha... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-4418 | |||||||||
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Title | Cryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure. | |||||||||
![]() | Class with an average helical twist of 72.8 degrees. | |||||||||
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![]() | NITRILASE / Cyanide detoxification / Beta-cyano-L-alanine hydrolase / Helical filament / HYDROLASE | |||||||||
Function / homology | ![]() 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 | ![]() ![]() | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||
![]() | Mulelu AE / Woodward JD | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 25.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.5 KB 13.5 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.1 KB | Display | ![]() |
Images | ![]() | 100.7 KB | ||
Filedesc metadata | ![]() | 6.3 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6i5tMC ![]() 4420C ![]() 6i5uC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Class with an average helical twist of 72.8 degrees. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.85 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Active helical nitrilase complex
Entire | Name: Active helical nitrilase complex |
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Components |
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-Supramolecule #1: Active helical nitrilase complex
Supramolecule | Name: Active helical nitrilase complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Bifunctional nitrilase/nitrile hydratase NIT4
Macromolecule | Name: Bifunctional nitrilase/nitrile hydratase NIT4 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: nitrilase |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 39.766113 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MSMQQETSHM TAAPQTNGHQ IFPEIDMSAG DSSSIVRATV VQASTVFYDT PATLDKAERL LSEAAENGSQ LVVFPEAFIG GYPRGSTFE LAIGSRTAKG RDDFRKYHAS AIDVPGPEVE RLALMAKKYK VYLVMGVIER EGYTLYCTVL FFDSQGLFLG K HRKLMPTA ...String: MSMQQETSHM TAAPQTNGHQ IFPEIDMSAG DSSSIVRATV VQASTVFYDT PATLDKAERL LSEAAENGSQ LVVFPEAFIG GYPRGSTFE LAIGSRTAKG RDDFRKYHAS AIDVPGPEVE RLALMAKKYK VYLVMGVIER EGYTLYCTVL FFDSQGLFLG K HRKLMPTA LERCIWGFGD GSTIPVFDTP IGKIGAAICW ENRMPSLRTA MYAKGIEIYC APTADSRETW LASMTHIALE GG CFVLSAN QFCRRKDYPS PPEYMFSGSE ESLTPDSVVC AGGSSIISPL GIVLAGPNYR GEALITADLD LGDIARAKFD FDV VGHYSR PEVFSLNIRE HPRKAVSFKT SKVMEDESVH HHHHH UniProtKB: Bifunctional nitrilase/nitrile hydratase NIT4 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | helical reconstruction |
Aggregation state | filament |
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Sample preparation
Concentration | 0.15 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 50 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK I Details: The sample (2.5 ul) was applied to the grid and incubated for 30 seconds at 50% humidity before blotting and plunging.. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Number real images: 1266 / Average exposure time: 7.0 sec. / Average electron dose: 45.5 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.75 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Details | Ab initio fitting was performed using Buccaneer, cleaned up with Coot and Phenix Real-Space Refine. |
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Refinement | Space: REAL / Protocol: AB INITIO MODEL / Target criteria: Cross-correlation coefficient |
Output model | ![]() PDB-6i5t: |