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- EMDB-1205: Three-dimensional structure of a type III glutamine synthetase by... -

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Basic information

Entry
Database: EMDB / ID: EMD-1205
TitleThree-dimensional structure of a type III glutamine synthetase by single-particle reconstruction.
Map dataGSIII from Bacteroides fragilis (negative stain reconstruction)
Sample
  • Sample: B.fragilis GlnN purified from E.coli
  • Protein or peptide: GlnN
Function / homologyGlutamine synthetase, N-terminal domain
Function and homology information
Biological speciesBacteroides fragilis (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 21.0 Å
Authorsvan Rooyen JM / Abratt VR / Sewell BT
CitationJournal: J Mol Biol / Year: 2006
Title: Three-dimensional structure of a type III glutamine synthetase by single-particle reconstruction.
Authors: Jason M van Rooyen / Valerie R Abratt / B Trevor Sewell /
Abstract: GlnN, the type III glutamine synthetase (GSIII) from the medically important, anaerobic, opportunistic pathogen Bacteroides fragilis, has 82.8 kDa subunits that share only 9% sequence identity with ...GlnN, the type III glutamine synthetase (GSIII) from the medically important, anaerobic, opportunistic pathogen Bacteroides fragilis, has 82.8 kDa subunits that share only 9% sequence identity with the type I glutamine synthetases (GSI), the only family for which a structure is known. Active GlnN was found predominantly in a single peak that eluted from a calibrated gel-filtration chromatography column at a position equaivalent to 0.86(+/-0.08) MDa. Negative-stain electron microscopy enabled the identification of double-ringed particles and single hexameric rings ("pinwheels") resulting from partial staining. A 2D average of these pinwheels showed marked similarity to the corresponding structures found in preparations of GSI, except that the arms of the subunits were 40% longer. Reconstructions from particles embedded in vitreous ice showed that GlnN has a double-ringed, dodecameric structure with a 6-fold dihedral space group (D6) symmetry and dimensions of 17.0 nm parallel with the 6-fold axis and 18.3 nm parallel with the 2-fold axes. The structures, combined with a sequence alignment based on structural principles, showed how many aspects of the structure of GSI, and most notably the alpha/beta barrel fold active site were preserved. There was evidence for the presence of this structure in the reconstructed volume, thus, identifying the indentations between the pinwheel spokes as putative active sites and suggesting conservation of the overall molecular geometry found in GSI despite their low level of global homology. Furthermore, docking of GSI into the reconstruction left sufficient plausibly located unoccupied density to account for the additional residues in GSIII, thus validating the structure.
History
DepositionMar 23, 2006-
Header (metadata) releaseMar 23, 2006-
Map releaseAug 9, 2006-
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: 0.07634
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.07634
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1205.gif

Downloads & links

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Map

FileDownload / File: emd_1205.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGSIII from Bacteroides fragilis (negative stain reconstruction)
Voxel sizeX=Y=Z: 4.25 Å
Density
Contour Level1: 0.0128 / Movie #1: 0.07634
Minimum - Maximum0.0 - 0.191964
Average (Standard dev.)0.00442323 (±0.0211771)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 340 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.254.254.25
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z340.000340.000340.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-96-96-96
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean0.0000.1920.004

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Supplemental data

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Sample components

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Entire : B.fragilis GlnN purified from E.coli

EntireName: B.fragilis GlnN purified from E.coli
Components
  • Sample: B.fragilis GlnN purified from E.coli
  • Protein or peptide: GlnN

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Supramolecule #1000: B.fragilis GlnN purified from E.coli

SupramoleculeName: B.fragilis GlnN purified from E.coli / type: sample / ID: 1000 / Oligomeric state: One dodecamer of GlnN / Number unique components: 1
Molecular weightExperimental: 1.3 MDa / Theoretical: 990 KDa / Method: Calibrated gel-filtration

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Macromolecule #1: GlnN

MacromoleculeName: GlnN / type: protein_or_peptide / ID: 1 / Name.synonym: GSIII
Details: Recombinant GlnN purified from GlnA deficient E.coli mutant; SwissProt P15623
Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes
Source (natural)Organism: Bacteroides fragilis (bacteria) / Strain: B. fragilis BF-1 / Tissue: E.coli cytoplasm / Cell: E.coli
Molecular weightExperimental: 990 KDa / Theoretical: 1.3 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pJS139
SequenceInterPro: Glutamine synthetase, N-terminal domain

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7.15 / Details: 10 mM Imidazole-HCl, 10 mM MnCl2
StainingType: NEGATIVE
Details: Aliquots (10ul) were applied to 300 mesh copper grids, which had been coated with thin carbon support film and previously glow discharged in air for 20 seconds, before being stained with 2% ...Details: Aliquots (10ul) were applied to 300 mesh copper grids, which had been coated with thin carbon support film and previously glow discharged in air for 20 seconds, before being stained with 2% uranyl acetate solution using the droplet method.
GridDetails: 300 mesh copper grid
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 65882 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: LEO OMEGA
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
DetailsLeo 912 TEM
DateMay 13, 2004
Image recordingCategory: CCD / Film or detector model: PROSCAN TEM-PIV (2k x 2k) / Digitization - Sampling interval: 14 µm / Number real images: 160
Details: Images were digitized using an Ilford Leafscan and downsized by a factor of 2
Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 166
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 12587

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Atomic model buiding 1

SoftwareName: Situs
DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: CC score

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