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- EMDB-1050: The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is... -

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Basic information

Entry
Database: EMDB / ID: EMD-1050
TitleThe cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral.
Map data
Sample
  • Sample: Cyanide dihydratase from Pseudomonas stutzeri
  • Protein or peptide: cyanide dihydratase
Function / homologyNitrilase/cyanide hydratase, conserved site / nitrilase activity
Function and homology information
Biological speciesPseudomonas stutzeri (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsSewell BT / Berman MN / Meyers PR / Jandhyala D / Benedik MJ
CitationJournal: Structure / Year: 2003
Title: The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral.
Authors: B T Sewell / M N Berman / P R Meyers / D Jandhyala / M J Benedik /
Abstract: The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle ...The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle technique. The structure is a spiral comprising 14 subunits with 2-fold symmetry. Interactions across the groove cause a decrease in the radius of the spiral at the ends and the resulting steric hindrance prevents the addition of further subunits. Similarity to two members of the nitrilase superfamily, the Nit domain of NitFhit and N-carbamyl-D-amino acid amidohydrolase, enabled the construction of a partial atomic model that could be unambiguously fitted to the stain envelope. The model suggests that interactions involving two significant insertions in the sequence relative to these structures leads to the left-handed spiral assembly.
History
DepositionFeb 4, 2003-
Header (metadata) releaseJul 9, 2003-
Map releaseJul 9, 2003-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.206044
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.206044
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1050.gif

Downloads & links

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Map

FileDownload / File: emd_1050.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4 Å/pix.
x 80 pix.
= 320. Å		4 Å/pix.
x 80 pix.
= 320. Å		4 Å/pix.
x 80 pix.
= 320. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.153 / Movie #1: 0.206044
Minimum - Maximum-0.329862 - 0.6125
Average (Standard dev.)-0.00721576 (±0.0641706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 320 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z444
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ0052
NX/NY/NZ12812855
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.3300.612-0.007

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Supplemental data

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Sample components

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Entire : Cyanide dihydratase from Pseudomonas stutzeri

EntireName: Cyanide dihydratase from Pseudomonas stutzeri
Components
  • Sample: Cyanide dihydratase from Pseudomonas stutzeri
  • Protein or peptide: cyanide dihydratase

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Supramolecule #1000: Cyanide dihydratase from Pseudomonas stutzeri

SupramoleculeName: Cyanide dihydratase from Pseudomonas stutzeri / type: sample / ID: 1000
Details: The sample eluted as a single symmetric peak on gel filtration chromatography. Three bands were seen on SDS-PAGE presumably indicating some proteolysis
Oligomeric state: 14 identical subunits / Number unique components: 1
Molecular weightExperimental: 500 KDa / Theoretical: 532 KDa
Method: gel filtration chromatography, sequencing and counting subunits.

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Macromolecule #1: cyanide dihydratase

MacromoleculeName: cyanide dihydratase / type: protein_or_peptide / ID: 1 / Name.synonym: nitrilase
Details: The protein elutes as a sharp symmetric peak on Sephacryl 300 HR chromatography and particles appear homogeneously sized by negative stain EM
Number of copies: 14 / Oligomeric state: 14mer / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas stutzeri (bacteria) / Strain: AK61 / Cell: bacteria / Organelle: bacteria / Location in cell: cytoplasm
Molecular weightExperimental: 38 KDa / Theoretical: 38 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET26b
SequenceGO: nitrilase activity / InterPro: Nitrilase/cyanide hydratase, conserved site

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 8 / Details: 10 mM triethanolamine, 50 mM NaCl
StainingType: NEGATIVE
Details: A carbon-coated copper grid was placed on a droplet of this enzyme preparation for 10 minutes, blotted, and then stained with 2% uranyl acetate for 3 minutes. The grid was then blotted and air-dried.
GridDetails: carbon film on 200 mesh copper grid
VitrificationCryogen name: NONE

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Electron microscopy

MicroscopeJEOL 2000EXII
DetailsAdditional details about microscope model:JEOL 1200EXII
DateMar 1, 2001
Image recordingCategory: CCD / Film or detector model: KODAK SO-163 FILM / Digitization - Sampling interval: 20 µm / Number real images: 15 / Bits/pixel: 16
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 5.6 mm / Nominal defocus max: 0.7 µm / Nominal defocus min: 0.1 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER

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Image processing

Detailsparticles were picked manually
CTF correctionDetails: none
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Initially no symmetry was assumed but a two fold axis became apparent after 47 cycles. The orientation of the twofold axis was determined and the model was rotated so that the twofold axis ...Details: Initially no symmetry was assumed but a two fold axis became apparent after 47 cycles. The orientation of the twofold axis was determined and the model was rotated so that the twofold axis was parallel to the x-axis. In 17 subsequent cycles of refinement the twofold symmetry was imposed by rotating about the x-axis and adding the structure thus formed to the original.
Number images used: 7008
Final angle assignmentDetails: generated at 15 degree intervals by the SPIDER VO EA function.
Final two d classificationNumber classes: 84

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Atomic model buiding 1

SoftwareName: CoLoRes, SITUS
DetailsThe map was fitted using a model generated from the Nit structure (1ems in PDB). Magification was refined by dtermining the CoLoRes correlation coefficient as a function of scale factor. Locations of the 14 subunits were verified by vizualization with O. Fitted co-ordinates are available on request.Initially no symmetry was assumed but a two fold axis became apparent after 47 cycles. The orientation of the twofold axis was determined and the model was rotated so that the twofold axis was parallel to the x-axis. In 17 subsequent cycles of refinement the twofold symmetry was imposed by rotating about the x-axis and adding the structure thus formed to the original.
RefinementProtocol: RIGID BODY FIT

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