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C12 symmetrised 3D reconstruction of the Shigella flexneri T3SS needle complex from negatively stained sample electron micrographs

by single particle reconstruction, at 25 A resolution

Movie

Orientation:

#1: Surface view with section colored by density value, Surface level: 1.2, Made by UCSF CHIMERA

#2: Surface view colored by cylindrical radius, Surface level: 1.2, Made by UCSF CHIMERA

Entry
Summary
Database / IDEM DATA BANK (EMDB) / 1617
TitleC12 symmetrised 3D reconstruction of the Shigella flexneri T3SS needle complex from negatively stained sample electron micrographs
MapThis is a 3D reconstruction of the Shigella flexneri 'needle complex' from negative stain images, done with C12 symmetry imposed. The resolution is 21-25A.
SampleShigella flexneri T3SS needle complex
KeywordsShigella flexneri Type III secretion system Needle complex Microbial pathogenesis
AuthorsHodgkinson JL, Horsley A, Stabat D, Simon M, Johnson S, da Fonseca PCA, Morris EP, Wall JS, Lea SM, Blocker AJ
DateDeposition: 2009-05-01, Header release: 2009-05-20, Map release: 2009-05-27, Last update: 2010-04-23
EMDB SitesEMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
MoviesMovie Page

#1: Surface view with section colored by density value, Surface level: 1.2, Made by UCSF CHIMERA

#2: Surface view colored by cylindrical radius, Surface level: 1.2, Made by UCSF CHIMERA

Supplemental images
Structure viewersYorodumi, Launch PeppeR (About PeppeR), Volume viewer (RCSB, PDBe)
Related Structure Data
Similar strucutres (beta)
List of similar structure data about Omokage system
Article
Citation - Primary
ArticleNat. Struct. Mol. Biol., Vol. 16, Issue 5, Page 477-85, Year 2009
TitleThree-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout.
AuthorsJulie L Hodgkinson, Ashley Horsley, David Stabat, Martha Simon, Steven Johnson, Paula C A da Fonseca, Edward P Morris, Joseph S Wall, Susan M Lea, Ariel J Blocker
Sir William Dunn School of Pathology, University of Oxford, UK.
KeywordsBacterial Proteins (chemistry), Cell Membrane (chemistry), Crystallography, X-Ray, Membrane Proteins (chemistry), Microscopy, Electron, Scanning Transmission, Models, Molecular, Protein Structure, Secondary, Secretin (chemistry, 1393-25-5), Shigella flexneri (metabolism)
LinksDOI: 10.1038/nsmb.1599, PubMed: 19396171, PMC: PMC2681179
Map
Fileemd_1617.map.gz ( map file in CCP4 format, 23551 KB )
Projections & SlicesSize of images:
AxesZ (Sec.)Y (Row.)X (Col.)
182 pix
2.62 A/pix
= 476.84 A
182 pix
2.62 A/pix
= 476.84 A
182 pix
2.62 A/pix
= 476.84 A

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Density
Contour Level:1 (by author), 1.2 (movie #1):
Minimum - Maximum: -13.1014 - 9.57075
Average (Standard dev.): -1.06342e-09 (1)
Data TypeImage stored as Reals
Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions182182182
Origin-91-91-91
Limit909090
Spacing182182182
Unit CellA= B= C: 476.84 A
Alpha=beta=gamma: 90 degrees
Pixel SpacingX= Y= Z: 2.62 A
CCP4 map header info
modeImage stored as Reals
A/pix X/Y/Z2.622.622.62
M x/y/z182182182
origin x/y/z0.0000.0000.000
length x/y/z476.840476.840476.840
alpha/beta/gamma90.00090.00090.000
start NX/NY/NZ-17-17-200
NX/NY/NZ123123401
MAP C/R/S123
start NC/NR/NS-91-91-91
NC/NR/NS182182182
start NC,NX/NR,NY/NS,NZ
NC,NX/NR,NY/NS,NZ
D min/max/mean-13.1019.571-0.000
Annotation DetailsThis is a 3D reconstruction of the Shigella flexneri 'needle complex' from negative stain images, done with C12 symmetry imposed. The resolution is 21-25A.
Supplement
Images
Images
Sample
NameShigella flexneri T3SS needle complex
Number of Components8
Oligomeric StateLarge macromolecular complex (total list of components not yet known)
DetailsAffinity purified using His6 tag on MxiG N-term Contains detergent (see Zenk et al., 2007)
Mass-estimation MethodSTEM analysis
Experimental Mass3.6MDa
Component #1: protein - Spa24
Scientific nameSpa24
Theoretical Mass0.24 MDa
DetailsThis is the MW of the monomer, which must be cleaved in its cytoplasmic portion during T3SS biogenesis.
Oligomeric DetailsUnknown
Scientific Name of SpeciesShigella flexneri
Common Name of SpeciesDysentery bacillus
NCBI taxonomy623
StrainShigella flexneri M90T
Recombinant expressionNo
Natural SourceOrganelle: Type III secretion system
Cell Location: inner membrane protein
Cell: Gram-negative bacterium
LinksInter Pro: IPR:005773, Gene Ontology: GO:0009306
Component #2: protein - Spa40
Scientific nameSpa40
Theoretical Mass0.4 MDa
DetailsThis is the MW of the monomer, which must be cleaved in its cytoplasmic portion during T3SS biogenesis.
Oligomeric DetailsUnknown
Scientific Name of SpeciesShigella flexneri
Common Name of SpeciesDysentery bacillus
NCBI taxonomy623
StrainShigella flexneri M90T
Recombinant expressionNo
Natural SourceCell: Gram-negative bacterium
Cell Location: polytopic-inner membrane protein
Organelle: Type III secretion system
LinksGene Ontology: GO:0009306, Inter Pro: IPR:006307
Component #3: protein - MxiM
Scientific nameMxiM
Theoretical Mass0.15 MDa
DetailsThis is the MW of the monomer.
Number of Copies12
Scientific Name of SpeciesShigella flexneri
Common Name of SpeciesDysentery bacillus
NCBI taxonomy623
StrainShigella flexneri M90T
Recombinant expressionNo
Natural SourceOrganelle: Type III secretion system
Cell: Gram-negative bacterium
Cell Location: outside face of outer membrane
LinksGene Ontology: GO:0009279
Component #4: protein - MxiI
Scientific nameMxiI
Theoretical Mass0.1 MDa
DetailsThis is the MW of the monomer.
Oligomeric DetailsHelical polymer
Number of Copies10
Scientific Name of SpeciesShigella flexneri
Common Name of SpeciesDysentery bacillus
NCBI taxonomy623
StrainShigella flexneri M90T
Recombinant expressionNo
Natural SourceCell Location: periplasmic space
Organelle: Type III secretion system
Cell: Gram-negative bacterium
LinksGene Ontology: GO:0015031
Component #5: protein - MxiG
Scientific nameMxiG
Theoretical Mass0.43 MDa
DetailsThis is the MW of the monomer.
Oligomeric DetailsProbable homo-24mer
Number of Copies24
Scientific Name of SpeciesShigella flexneri
Common Name of SpeciesDysentery bacillus
NCBI taxonomy623
StrainShigella flexneri M90T
Recombinant expressionNo
Natural SourceOrganelle: Type III secretion system
Cell: Gram-negative bacterium
Cell Location: spans inner membrane
LinksGene Ontology: GO:0009405
Component #6: protein - MxiJ
Scientific nameMxiJ
Theoretical Mass0.25 MDa
DetailsThis is the MW of the monomer with its N-terminal lipid modification site processed (but MW of lipid moiety unknown).
Oligomeric DetailsProbable homo-dodecamer
Number of Copies12
Scientific Name of SpeciesShigella flexneri
Common Name of SpeciesDysentery bacillus
NCBI taxonomy623
StrainShigella flexneri M90T
Recombinant expressionNo
Natural SourceOrganelle: Type III secretion system
Cell: Gram-negative bacterium
Cell Location: mostly periplasmic face of inner membrane
LinksGene Ontology: GO:0009306, Inter Pro: IPR:006182
Component #7: protein - MxiH
Scientific nameMxiH
Theoretical Mass0.09 MDa
DetailsThis is the MW of the monomer.
Oligomeric Detailshelical polymer
Number of Copies120
Scientific Name of SpeciesShigella flexneri
Common Name of SpeciesDysentery bacillus
NCBI taxonomy623
StrainShigella flexneri M90T
Recombinant expressionNo
Natural SourceCell Location: Extracellular
Organelle: Type III secretion system
Cell: Gram-negative bacterium
LinksInter Pro: IPR:011841, Gene Ontology: GO:0009405
Component #8: protein - MxiD
Scientific nameMxiD
Theoretical Mass0.62 MDa
DetailsThis is the MW of the monomer without it's signal sequence included
Oligomeric Detailshomo-dodecamer
Number of Copies12
Scientific Name of SpeciesShigella flexneri
Common Name of SpeciesDysentery bacillus
NCBI taxonomy623
StrainShigella flexneri M90T
Recombinant expressionNo
Natural SourceCell: Gram-negative bacterium
Organelle: Type III secretion system
Cell Location: Outer membrane
LinksInter Pro: IPR:003522, Gene Ontology: GO:0009306
Experiment
Sample Preparation
Stainingnegatively stained in unbuffered 2% aqueous uranyl acetate
Specimen Conc0.05 mg/ml
Specimen Support Details400 mesh copper grids (Athene) covered with holey carbon film over which thin plain carbon was laid were used
Specimen Stateparticle
BufferDetails: 10mM Tris pH 8, 0.1% Triton X-100 and 1mM EDTA buffer
pH: 8
Vitrification
Cryogen NameNONE
InstrumentNONE
Imaging
MicroscopeFEI/PHILIPS CM200FEG
DetailsLow-dose mode was used
Electron Gun
Electron SourceFIELD EMISSION GUN
Accelerating Voltage200 kV
Electron Dose20 e/A**2
Illumination ModeSPOT SCAN
Lens
MagnificationNominal: 50000, Calibrated: 48600
Astigmatismobjective lens astigmatism was corrected at 100,000 times magnification
Nominal Cs2.2 mm
Imaging ModeBRIGHT FIELD
Defocus700 nm - 900 nm
Specimen Holder
Holdereucentric
ModelOTHER
Camera
DetectorKODAK SO-163 FILM
Image Acquisition
Od Range1
ScannerNIKON SUPER COOLSCAN 9000
Number of Digital Images126
Sampling Size6.35
Quant Bit Number16
Processing
Methodsingle particle reconstruction
3D reconstruction
Algorithmweighted back projection
Euler Angles DetailsImagic beta 87-93 degrees gamma 0-30 degrees
SoftwareSPIDER IMAGIC5
DetailsMaps were generated from 41 individual images (selected through image processing out of an initial pool of 3000)
Resolution By Author25 A
Resolution MethodFSC at 3-sigma and 0.5 cut-offs
Single Particle
Number of Class Averages4
Number of Projections41
DetailsNumbers of particles given are for final reconstruction. We started with over 3000. Particles were selected using Ximdisp software, cut out to an initial box size of 400 by 400 pixel and coarsened to 2.62A per pixel using Label prior to further processing.
Download
Data from EMDB
Header (meta data in XML format)emd-1617.xml (17.4 KB)
Map dataemd_1617.map.gz (13.3 MB)
Images1617.gif (32.1 KB)
1617.png (327.4 KB)
FTP directoryftp://ftp.pdbj.org/pub/emdb/structures/EMD-1617
Movie files
movie #1
.mp4 (H.264/MPEG-4 AVC format), 3.5 MB
.webm (WebM/VP8 format), 5.4 MB
Session file for UCSF-Chimera, 26.8 KB
movie #2
.mp4 (H.264/MPEG-4 AVC format), 3.4 MB
.webm (WebM/VP8 format), 5.1 MB
Session file for UCSF-Chimera, 26.8 KB