EMDB-1617: C12 symmetrised 3D reconstruction of the Shigella flex...

Entry
Summary
Database / ID	EM DATA BANK (EMDB) / 1617
Title	C12 symmetrised 3D reconstruction of the Shigella flexneri T3SS needle complex from negatively stained sample electron micrographs
Map	This is a 3D reconstruction of the Shigella flexneri 'needle complex' from negative stain images, done with C12 symmetry imposed. The resolution is 21-25A.
Sample	Shigella flexneri T3SS needle complex
Keywords	Shigella flexneri Type III secretion system Needle complex Microbial pathogenesis
Authors	Hodgkinson JL, Horsley A, Stabat D, Simon M, Johnson S, da Fonseca PCA, Morris EP, Wall JS, Lea SM, Blocker AJ
Date	Deposition: 2009-05-01, Header release: 2009-05-20, Map release: 2009-05-27, Last update: 2010-04-23
EMDB Sites	EMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
Movies	Movie 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	1617.gif 1617.png
Structure viewers	Yorodumi, Launch PeppeR (About PeppeR), Volume viewer (RCSB, PDBe)
Related Structure Data
Similar strucutres (beta)	Diagram EMDB-2210 PDB-2ymg PDB-2ymf List of similar structure data about Omokage system

Article
Citation - Primary
Article	Nat. Struct. Mol. Biol., Vol. 16, Issue 5, Page 477-85, Year 2009
Title	Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout.
Authors	Julie 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.
Keywords	Bacterial 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)
Links	DOI: 10.1038/nsmb.1599, PubMed: 19396171, PMC: PMC2681179

Map

File

EMD-1617.map ( map file in CCP4 format, 24116 KB )

Projections & Slices

Size of images:

Axes	Z (Sec.)	Y (Row.)	X (Col.)
Surface
Projections
Slices (1/3)
Slices (1/2)
Slices (2/3)

Images are generated by Spider package.

Density

Histogram

Histogram (log scale)

Contour Level:	1 (by author), 1.2 (movie #1):
Minimum - Maximum:	-13.1014 - 9.57075
Average (Standard dev.):	-1.06342e-09 (1)

Data Type

Image stored as Reals

Space Group Number

Map Geometry

Axis order	X	Y	Z
Dimensions	182	182	182
Origin	-91	-91	-91
Limit	90	90	90
Spacing	182	182	182

Unit Cell

A= B= C: 476.84 A
Alpha=beta=gamma: 90 degrees

Pixel Spacing

X= Y= Z: 2.62 A

CCP4 map header info

mode	Image stored as Reals
A/pix X/Y/Z	2.62	2.62	2.62
M x/y/z	182	182	182
origin x/y/z	0.000	0.000	0.000
length x/y/z	476.840	476.840	476.840
alpha/beta/gamma	90.000	90.000	90.000
start NX/NY/NZ	-17	-17	-200
NX/NY/NZ	123	123	401
MAP C/R/S	1	2	3
start NC/NR/NS	-91	-91	-91
NC/NR/NS	182	182	182
start NC,NX/NR,NY/NS,NZ
NC,NX/NR,NY/NS,NZ
D min/max/mean	-13.101	9.571	-0.000

Annotation Details

This 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	1617.gif 1617.png

Sample
Name	Shigella flexneri T3SS needle complex
Number of Components	8
Oligomeric State	Large macromolecular complex (total list of components not yet known)
Details	Affinity purified using His6 tag on MxiG N-term Contains detergent (see Zenk et al., 2007)
Mass-estimation Method	STEM analysis
Experimental Mass	3.6MDa
Component #1: protein - Spa24
Scientific name	Spa24
Theoretical Mass	0.24 MDa
Details	This is the MW of the monomer, which must be cleaved in its cytoplasmic portion during T3SS biogenesis.
Oligomeric Details	Unknown
Scientific Name of Species	Shigella flexneri
Common Name of Species	Dysentery bacillus
NCBI taxonomy	623
Strain	Shigella flexneri M90T
Recombinant expression	No
Natural Source	Organelle: Type III secretion system Cell Location: inner membrane protein Cell: Gram-negative bacterium
Links	Inter Pro: IPR:005773, Gene Ontology: GO:0009306
Component #2: protein - Spa40
Scientific name	Spa40
Theoretical Mass	0.4 MDa
Details	This is the MW of the monomer, which must be cleaved in its cytoplasmic portion during T3SS biogenesis.
Oligomeric Details	Unknown
Scientific Name of Species	Shigella flexneri
Common Name of Species	Dysentery bacillus
NCBI taxonomy	623
Strain	Shigella flexneri M90T
Recombinant expression	No
Natural Source	Cell: Gram-negative bacterium Cell Location: polytopic-inner membrane protein Organelle: Type III secretion system
Links	Gene Ontology: GO:0009306, Inter Pro: IPR:006307
Component #3: protein - MxiM
Scientific name	MxiM
Theoretical Mass	0.15 MDa
Details	This is the MW of the monomer.
Number of Copies	12
Scientific Name of Species	Shigella flexneri
Common Name of Species	Dysentery bacillus
NCBI taxonomy	623
Strain	Shigella flexneri M90T
Recombinant expression	No
Natural Source	Organelle: Type III secretion system Cell: Gram-negative bacterium Cell Location: outside face of outer membrane
Links	Gene Ontology: GO:0009279
Component #4: protein - MxiI
Scientific name	MxiI
Theoretical Mass	0.1 MDa
Details	This is the MW of the monomer.
Oligomeric Details	Helical polymer
Number of Copies	10
Scientific Name of Species	Shigella flexneri
Common Name of Species	Dysentery bacillus
NCBI taxonomy	623
Strain	Shigella flexneri M90T
Recombinant expression	No
Natural Source	Cell Location: periplasmic space Organelle: Type III secretion system Cell: Gram-negative bacterium
Links	Gene Ontology: GO:0015031
Component #5: protein - MxiG
Scientific name	MxiG
Theoretical Mass	0.43 MDa
Details	This is the MW of the monomer.
Oligomeric Details	Probable homo-24mer
Number of Copies	24
Scientific Name of Species	Shigella flexneri
Common Name of Species	Dysentery bacillus
NCBI taxonomy	623
Strain	Shigella flexneri M90T
Recombinant expression	No
Natural Source	Organelle: Type III secretion system Cell: Gram-negative bacterium Cell Location: spans inner membrane
Links	Gene Ontology: GO:0009405
Component #6: protein - MxiJ
Scientific name	MxiJ
Theoretical Mass	0.25 MDa
Details	This is the MW of the monomer with its N-terminal lipid modification site processed (but MW of lipid moiety unknown).
Oligomeric Details	Probable homo-dodecamer
Number of Copies	12
Scientific Name of Species	Shigella flexneri
Common Name of Species	Dysentery bacillus
NCBI taxonomy	623
Strain	Shigella flexneri M90T
Recombinant expression	No
Natural Source	Organelle: Type III secretion system Cell: Gram-negative bacterium Cell Location: mostly periplasmic face of inner membrane
Links	Gene Ontology: GO:0009306, Inter Pro: IPR:006182
Component #7: protein - MxiH
Scientific name	MxiH
Theoretical Mass	0.09 MDa
Details	This is the MW of the monomer.
Oligomeric Details	helical polymer
Number of Copies	120
Scientific Name of Species	Shigella flexneri
Common Name of Species	Dysentery bacillus
NCBI taxonomy	623
Strain	Shigella flexneri M90T
Recombinant expression	No
Natural Source	Cell Location: Extracellular Organelle: Type III secretion system Cell: Gram-negative bacterium
Links	Inter Pro: IPR:011841, Gene Ontology: GO:0009405
Component #8: protein - MxiD
Scientific name	MxiD
Theoretical Mass	0.62 MDa
Details	This is the MW of the monomer without it's signal sequence included
Oligomeric Details	homo-dodecamer
Number of Copies	12
Scientific Name of Species	Shigella flexneri
Common Name of Species	Dysentery bacillus
NCBI taxonomy	623
Strain	Shigella flexneri M90T
Recombinant expression	No
Natural Source	Cell: Gram-negative bacterium Organelle: Type III secretion system Cell Location: Outer membrane
Links	Inter Pro: IPR:003522, Gene Ontology: GO:0009306

Experiment
Sample Preparation
Staining	negatively stained in unbuffered 2% aqueous uranyl acetate
Specimen Conc	0.05 mg/ml
Specimen Support Details	400 mesh copper grids (Athene) covered with holey carbon film over which thin plain carbon was laid were used
Specimen State	particle
Buffer	Details: 10mM Tris pH 8, 0.1% Triton X-100 and 1mM EDTA buffer pH: 8
Vitrification
Cryogen Name	NONE
Instrument	NONE
Imaging
Microscope	FEI/PHILIPS CM200FEG
Details	Low-dose mode was used
Electron Gun
Electron Source	FIELD EMISSION GUN
Accelerating Voltage	200 kV
Electron Dose	20 e/A**2
Illumination Mode	SPOT SCAN
Lens
Magnification	Nominal: 50000, Calibrated: 48600
Astigmatism	objective lens astigmatism was corrected at 100,000 times magnification
Nominal Cs	2.2 mm
Imaging Mode	BRIGHT FIELD
Defocus	700 nm - 900 nm
Specimen Holder
Holder	eucentric
Model	OTHER
Camera
Detector	Kodak SO163 film
Image Acquisition
Od Range	1
Scanner	NIKON SUPER COOLSCAN 9000
Number of Digital Images	126
Sampling Size	6.35
Quant Bit Number	16

Processing
Method	single particle reconstruction
3D reconstruction
Algorithm	weighted back projection
Euler Angles Details	Imagic beta 87-93 degrees gamma 0-30 degrees
Software	SPIDER IMAGIC5
Details	Maps were generated from 41 individual images (selected through image processing out of an initial pool of 3000)
Resolution By Author	25 A
Resolution Method	FSC at 3-sigma and 0.5 cut-offs
Single Particle
Number of Class Averages	4
Number of Projections	41
Details	Numbers 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 data

emd_1617.map.gz (13.3 MB)

Images

1617.gif (32.1 KB)

1617.png (327.4 KB)

FTP directory

ftp://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