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- EMDB-4420: Cryo-EM informed directed evolution of Nitrilase 4 leads to a cha... -

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Basic information

Entry
Database: EMDB / ID: EMD-4420
TitleCryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure.
Map data73.2 degree conformation
Sample
  • Complex: Active helical nitrilase complex
    • Protein or peptide: Bifunctional nitrilase/nitrile hydratase NIT4
KeywordsNITRILASE / Cyanide detoxification / Beta-cyano-L-alanine hydrolase / Helical filament / HYDROLASE
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 / detoxification of nitrogen compound / nitrilase activity / nitrile hydratase activity ...cyanoalanine nitrilase / 3-cyanoalanine hydratase / cyanoalanine nitrilase activity / 3-cyanoalanine hydratase activity / cyanide metabolic process / nitrilase / indole-3-acetonitrile nitrilase activity / detoxification of nitrogen compound / nitrilase activity / nitrile hydratase activity / plasma membrane / cytosol
Similarity search - Function
Nitrilases / cyanide hydratase active site signature. / Nitrilase/Cyanide hydratase / Nitrilases / cyanide hydratase signature 1. / Nitrilase/cyanide hydratase, conserved site / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
Bifunctional nitrilase/nitrile hydratase NIT4
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodhelical reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsMulelu AE / Woodward JD
Funding support South Africa, 1 items
OrganizationGrant numberCountry
National Research Foundation in South AfricaResearch Career Advancement Fellowship South Africa
CitationJournal: 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
DepositionNov 14, 2018-
Header (metadata) releaseNov 20, 2019-
Map releaseNov 20, 2019-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6i5u
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6i5u
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4420.png

Downloads & links

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Map

FileDownload / File: emd_4420.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation73.2 degree conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 260 pix.
= 221. Å		0.85 Å/pix.
x 260 pix.
= 221. Å		0.85 Å/pix.
x 260 pix.
= 221. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.0683879 - 0.109094195
Average (Standard dev.)0.00045632457 (±0.007359177)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 221.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z221.000221.000221.000
α/β/γ90.00090.00090.000
start NX/NY/NZ929262
NX/NY/NZ290290360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.0680.1090.000

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Supplemental data

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Sample components

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Entire : Active helical nitrilase complex

EntireName: Active helical nitrilase complex
Components
  • Complex: Active helical nitrilase complex
    • Protein or peptide: Bifunctional nitrilase/nitrile hydratase NIT4

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Supramolecule #1: Active helical nitrilase complex

SupramoleculeName: Active helical nitrilase complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Arabidopsis thaliana NITRILASE 4 in 73.2 degree conformation
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Bifunctional nitrilase/nitrile hydratase NIT4

MacromoleculeName: Bifunctional nitrilase/nitrile hydratase NIT4 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: nitrilase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 39.766113 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloride
50.0 mMTris-HclTris
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen 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

MicroscopeFEI TITAN KRIOS
Image recordingFilm 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.75 µm
Sample stageSpecimen 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

Final reconstructionApplied symmetry - Helical parameters - Δz: 17.68 Å
Applied symmetry - Helical parameters - Δ&Phi: -73.20 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 33812
Segment selectionNumber selected: 133106 / Details: 3D class with a 73.2 degree average helical twist
Startup modelType of model: EMDB MAP
EMDB ID:

3497


Details: Low-pass filtered to 60 A
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsAb initio fitting was performed using Buccaneer, cleaned up with Coot and Phenix Real-Space Refine.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Cross-correlation coefficient
Output model

PDB-6i5u:
Cryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure.

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