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- EMDB-4804: Cryo-EM informed directed evolution of Nitrilase 4 leads to a cha... -

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Basic information

Entry
Database: EMDB / ID: EMD-4804
TitleCryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure.
Map dataAtNIT4 R95T nitrilase filament
Sample
  • Complex: Arabidopsis thaliana NITRILASE 4 filament
    • Protein or peptide: Arabidopsis thaliana Nitrilase 4 R95T
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 Africa92556 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
DepositionApr 12, 2019-
Header (metadata) releaseAug 7, 2019-
Map releaseAug 7, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4804.png

Downloads & links

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Map

FileDownload / File: emd_4804.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAtNIT4 R95T nitrilase filament
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.22 Å/pix.
x 64 pix.
= 270.08 Å		4.22 Å/pix.
x 64 pix.
= 270.08 Å		4.22 Å/pix.
x 64 pix.
= 270.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.22 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 7.0 / Movie #1: 7
Minimum - Maximum-8.383659 - 16.416553
Average (Standard dev.)0.6341259 (±3.2814233)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 270.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.224.224.22
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z270.080270.080270.080
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-8.38416.4170.634

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Supplemental data

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Sample components

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Entire : Arabidopsis thaliana NITRILASE 4 filament

EntireName: Arabidopsis thaliana NITRILASE 4 filament
Components
  • Complex: Arabidopsis thaliana NITRILASE 4 filament
    • Protein or peptide: Arabidopsis thaliana Nitrilase 4 R95T

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Supramolecule #1: Arabidopsis thaliana NITRILASE 4 filament

SupramoleculeName: Arabidopsis thaliana NITRILASE 4 filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Wild-type Arabidopsis thaliana NIT4 modified by site directed mutagenesis at position 95.
Source (natural)Organism: Arabidopsis thaliana (thale cress) / Strain: Columbia
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET28
Molecular weightTheoretical: 49 kDa/nm

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Macromolecule #1: Arabidopsis thaliana Nitrilase 4 R95T

MacromoleculeName: Arabidopsis thaliana Nitrilase 4 R95T / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: nitrilase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MSMQQETSHM TAAPQTNGHQ IFPEIDMSAG DSSSIVRATV VQASTVFYDT PATLDKAERL LSEAAENGSQ LVVFPEAFIG GYPRGSTFEL AIGSTTAKGR DDFRKYHASA IDVPGPEVER LALMAKKYKV YLVMGVIERE GYTLYCTVLF FDSQGLFLGK HRKLMPTALE ...String:
MSMQQETSHM TAAPQTNGHQ IFPEIDMSAG DSSSIVRATV VQASTVFYDT PATLDKAERL LSEAAENGSQ LVVFPEAFIG GYPRGSTFEL AIGSTTAKGR DDFRKYHASA IDVPGPEVER LALMAKKYKV YLVMGVIERE GYTLYCTVLF FDSQGLFLGK HRKLMPTALE RCIWGFGDGS TIPVFDTPIG KIGAAICWEN RMPSLRTAMY AKGIEIYCAP TADSRETWLA SMTHIALEGG CFVLSANQFC RRKDYPSPPE YMFSGSEESL TPDSVVCAGG SSIISPLGIV LAGPNYRGEA LITADLDLGD IARAKFDFDV VGHYSRPEVF SLNIREHPRK AVSFKTSKVM EDESV

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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
200.0 mMNaClsodium chloride
50.0 mMC4H11NO3TRIS-Hcl
StainingType: 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.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm 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 beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 stageSpecimen holder model: PHILIPS ROTATION HOLDER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 16.0 Å
Applied symmetry - Helical parameters - Δ&Phi: -73 °
Applied symmetry - Helical parameters - Axial symmetry: D2 (2x2 fold dihedral)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 1/2 BIT CUT-OFF / Software - Name: SPIDER (ver. 11) / Software - details: IHRSR / Number images used: 3167
Segment selectionNumber selected: 1015 / Software - Name: EMAN / Software - details: Boxer / Details: Picked using Eman Boxer in helix mode
Startup modelType of model: INSILICO MODEL
In silico model: Featureless cylinder approximating the diameter of the filament.
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER (ver. 11) / Software - details: IHRSR

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