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- EMDB-3498: Substrate specificity in plant nitrilase helical assemblies is de... -

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Basic information

Entry
Database: EMDB / ID: EMD-3498
TitleSubstrate specificity in plant nitrilase helical assemblies is determined by their twist.
Map dataCaenorhabditis elegans NITRILASE
Sample
  • Complex: Caenorhabditis elegans NITRILASE filament
    • Protein or peptide: NITRILASE
Biological speciesCaenorhabditis elegans (invertebrata)
Methodhelical reconstruction / negative staining / Resolution: 20.0 Å
AuthorsWoodward JD / Trompetter I / Sewell BT / Piotrowski M
CitationJournal: Commun Biol / Year: 2018
Title: Substrate specificity of plant nitrilase complexes is affected by their helical twist.
Authors: Jeremy D Woodward / Inga Trompetter / B Trevor Sewell / Markus Piotrowski /
Abstract: Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important ...Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar nitrilases from the plant , that modifying nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket.
History
DepositionNov 14, 2016-
Header (metadata) releaseDec 14, 2016-
Map releaseJan 17, 2018-
UpdateNov 21, 2018-
Current statusNov 21, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0223
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0223
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3498.png

Downloads & links

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Map

FileDownload / File: emd_3498.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCaenorhabditis elegans NITRILASE
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: 0.0223 / Movie #1: 0.0223
Minimum - Maximum-0.11266248 - 0.14269295
Average (Standard dev.)-0.0011506807 (±0.024447069)
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
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-0.1130.143-0.001

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Supplemental data

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Sample components

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Entire : Caenorhabditis elegans NITRILASE filament

EntireName: Caenorhabditis elegans NITRILASE filament
Components
  • Complex: Caenorhabditis elegans NITRILASE filament
    • Protein or peptide: NITRILASE

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Supramolecule #1: Caenorhabditis elegans NITRILASE filament

SupramoleculeName: Caenorhabditis elegans NITRILASE filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21-b(+)
Molecular weightTheoretical: 22.5 kDa/nm

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Macromolecule #1: NITRILASE

MacromoleculeName: NITRILASE / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: nitrilase
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: mpkiaivqag tplfdkpatl ekvkknveea agngaelvlf peafiggypk wnsfgitmgt rtpegrkefk ryfenaieen geeskliesl aaqnnihivi gvvereastl ycsvffysps gylgkhrkll ptalercvwg qgdgstmpvf stsvgkigsa icwenymply ...String:
mpkiaivqag tplfdkpatl ekvkknveea agngaelvlf peafiggypk wnsfgitmgt rtpegrkefk ryfenaieen geeskliesl aaqnnihivi gvvereastl ycsvffysps gylgkhrkll ptalercvwg qgdgstmpvf stsvgkigsa icwenymply rmtlyskeiq iylaptvddr dvwlstmrti alegrcfvvs acqflkssdy pldhplrkeh gedkvlirgg scavdplgtv lvepdftket irytefdlsd lalgkmdldv vghysrpdvf qlkvnensqs tvvkk

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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.
GridModel: Grid-tech / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 20.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa

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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 reconstructionNumber classes used: 90
Applied symmetry - Helical parameters - Δz: 15.0 Å
Applied symmetry - Helical parameters - Δ&Phi: -74 °
Applied symmetry - Helical parameters - Axial symmetry: D2 (2x2 fold dihedral)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER (ver. 11) / Software - details: IHRSR / Number images used: 957
Segment selectionNumber selected: 969 / 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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