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- EMDB-4406: Cryo-EM informed directed evolution of Nitrilase 4 leads to a cha... -

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Basic information

Entry
Database: EMDB / ID: EMD-4406
TitleCryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure.
Map dataAtNIT4 R95T L169A S224Q filament
Sample
  • Complex: Arabidopsis thaliana NITRILASE 4 mutant
    • Protein or peptide: NITRILASE 4 mutant
Biological speciesArabidopsis thaliana (thale cress)
Methodhelical reconstruction / negative staining / Resolution: 20.0 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 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 . 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 8, 2018-
Header (metadata) releaseAug 7, 2019-
Map releaseAug 7, 2019-
UpdateAug 7, 2019-
Current statusAug 7, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4406.png

Downloads & links

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Map

FileDownload / File: emd_4406.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAtNIT4 R95T L169A S224Q filament
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.11 Å/pix.
x 80 pix.
= 328.8 Å		4.11 Å/pix.
x 80 pix.
= 328.8 Å		4.11 Å/pix.
x 80 pix.
= 328.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.11 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-2.5709467 - 4.9610124
Average (Standard dev.)0.000000004074297 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 328.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.114.114.11
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z328.800328.800328.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-15-9-23
NX/NY/NZ635180
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-2.5714.9610.000

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Supplemental data

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Sample components

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Entire : Arabidopsis thaliana NITRILASE 4 mutant

EntireName: Arabidopsis thaliana NITRILASE 4 mutant
Components
  • Complex: Arabidopsis thaliana NITRILASE 4 mutant
    • Protein or peptide: NITRILASE 4 mutant

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Supramolecule #1: Arabidopsis thaliana NITRILASE 4 mutant

SupramoleculeName: Arabidopsis thaliana NITRILASE 4 mutant / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Generated by directed evolution
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET-21b(+)
Molecular weightExperimental: 400 KDa

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Macromolecule #1: NITRILASE 4 mutant

MacromoleculeName: NITRILASE 4 mutant / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: nitrilase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSMQQETSHM TAAPQTNGHQ IFPEIDMSAG DSSSIVRATV VQASTVFYDT PATLDKAERL LSEAAENGSQ LVVFPEAFIG GYPRGSTFEL AIGSTTAKGR DDFRKYHASA IDVPGPEVER LALMAKKYKV YLVMGVIERE GYTLYCTVLF FDSQGLFLGK HRKLMPTAAE ...String:
MSMQQETSHM TAAPQTNGHQ IFPEIDMSAG DSSSIVRATV VQASTVFYDT PATLDKAERL LSEAAENGSQ LVVFPEAFIG GYPRGSTFEL AIGSTTAKGR DDFRKYHASA IDVPGPEVER LALMAKKYKV YLVMGVIERE GYTLYCTVLF FDSQGLFLGK HRKLMPTAAE RCIWGFGDGS TIPVFDTPIG KIGAAICWEN RMPSLRTAMY AKGIEIYCAP TADQRETWLA SMTHIALEGG CFVLSANQFC RRKDYPSPPE YMFSGSEESL TPDSVVCAGG SSIISPLGIV LAGPNYRGEA LITADLDLGD IARAKFDFDV VGHYSRPEVF SLNIREHPRK AVSFKTSKVM EDESVHHHHH H

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTris-HclTris
200.0 mMNaClsodium chloride
StainingType: NEGATIVE / Material: Uranyl acetate
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 15.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI TECNAI 20
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 1 / Number real images: 50 / Average exposure time: 1.0 sec. / Average electron dose: 10.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: PHILIPS ROTATION HOLDER

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Image processing

DetailsThe images were low-pass filtered to 20 A and normalised.
Final reconstructionNumber classes used: 90
Applied symmetry - Helical parameters - Δz: 14.52459 Å
Applied symmetry - Helical parameters - Δ&Phi: -65.59083 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER (ver. 21.10) / Software - details: IHRSR / Number images used: 10000
Segment selectionNumber selected: 10000 / Software - Name: EMAN
Software - details: Boxer was used in helical mode with 95% overlap
Startup modelType of model: INSILICO MODEL / In silico model: Featureless cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER (ver. 21.10) / Software - details: IHRSR

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