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- EMDB-10040: Inner membrane ring and secretin N0 N1 domains of the Shigella ty... -

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Basic information

Entry
Database: EMDB / ID: EMD-10040
TitleInner membrane ring and secretin N0 N1 domains of the Shigella type 3 secretion system
Map dataMasked C8 map of IM ring and connector, post-processed filtered at 3.86 A and sharpened (b=-120)
Sample
  • Complex: Oligomer of the secretin N0 and N1 domains in complex with the inner membrane ring of the Shigella type 3 secretion system
    • Protein or peptide: Outer membrane protein MxiD
    • Protein or peptide: Protein MxiG
Function / homology
Function and homology information


type III protein secretion system complex / protein secretion by the type III secretion system / cell outer membrane / plasma membrane
Similarity search - Function
Type III secretion system, PrgH/EprH-like / Type III secretion system protein PrgH-EprH (PrgH) / : / SPI-1 type 3 secretion system secretin, N0 domain / Type III secretion system outer membrane pore YscC/HrcC / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / NolW-like / Bacterial type II/III secretion system short domain / NolW-like superfamily ...Type III secretion system, PrgH/EprH-like / Type III secretion system protein PrgH-EprH (PrgH) / : / SPI-1 type 3 secretion system secretin, N0 domain / Type III secretion system outer membrane pore YscC/HrcC / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / NolW-like / Bacterial type II/III secretion system short domain / NolW-like superfamily / Type II/III secretion system / Bacterial type II and III secretion system protein
Similarity search - Domain/homology
Protein MxiG / Type 3 secretion system secretin
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.86 Å
AuthorsLunelli M / Kamprad A
Funding support2 items
OrganizationGrant numberCountry
European Research Council311371
European Union653706
CitationJournal: PLoS Pathog / Year: 2020
Title: Cryo-EM structure of the Shigella type III needle complex.
Authors: Michele Lunelli / Antje Kamprad / Jörg Bürger / Thorsten Mielke / Christian M T Spahn / Michael Kolbe /
Abstract: The Type III Secretion Systems (T3SS) needle complex is a conserved syringe-shaped protein translocation nanomachine with a mass of about 3.5 MDa essential for the survival and virulence of many Gram- ...The Type III Secretion Systems (T3SS) needle complex is a conserved syringe-shaped protein translocation nanomachine with a mass of about 3.5 MDa essential for the survival and virulence of many Gram-negative bacterial pathogens. This system is composed of a membrane-embedded basal body and an extracellular needle that deliver effector proteins into host cells. High-resolution structures of the T3SS from different organisms and infection stages are needed to understand the underlying molecular mechanisms of effector translocation. Here, we present the cryo-electron microscopy structure of the isolated Shigella T3SS needle complex. The inner membrane (IM) region of the basal body adopts 24-fold rotational symmetry and forms a channel system that connects the bacterial periplasm with the export apparatus cage. The secretin oligomer adopts a heterogeneous architecture with 16- and 15-fold cyclic symmetry in the periplasmic N-terminal connector and C-terminal outer membrane ring, respectively. Two out of three IM subunits bind the secretin connector via a β-sheet augmentation. The cryo-EM map also reveals the helical architecture of the export apparatus core, the inner rod, the needle and their intervening interfaces.
History
DepositionJun 5, 2019-
Header (metadata) releaseFeb 12, 2020-
Map releaseFeb 12, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rwk
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6rwk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10040.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked C8 map of IM ring and connector, post-processed filtered at 3.86 A and sharpened (b=-120)
Voxel sizeX=Y=Z: 1.38369 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.24032187 - 0.47087005
Average (Standard dev.)0.0005601756 (±0.009396414)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 664.1712 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.38368958333331.38368958333331.3836895833333
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z664.171664.171664.171
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.2400.4710.001

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Supplemental data

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Mask #1

Fileemd_10040_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Masked C8 map of IM ring and connector,...

Fileemd_10040_additional.map
AnnotationMasked C8 map of IM ring and connector, post-processed filtered at 3.5 A and sharpened (b=-120), used for model building and refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Unfiltered half map of C8 reconstruction of IM ring and connector.

Fileemd_10040_half_map_1.map
AnnotationUnfiltered half map of C8 reconstruction of IM ring and connector.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Unfiltered half map of C8 reconstruction of IM ring and connector.

Fileemd_10040_half_map_2.map
AnnotationUnfiltered half map of C8 reconstruction of IM ring and connector.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Oligomer of the secretin N0 and N1 domains in complex with the in...

EntireName: Oligomer of the secretin N0 and N1 domains in complex with the inner membrane ring of the Shigella type 3 secretion system
Components
  • Complex: Oligomer of the secretin N0 and N1 domains in complex with the inner membrane ring of the Shigella type 3 secretion system
    • Protein or peptide: Outer membrane protein MxiD
    • Protein or peptide: Protein MxiG

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Supramolecule #1: Oligomer of the secretin N0 and N1 domains in complex with the in...

SupramoleculeName: Oligomer of the secretin N0 and N1 domains in complex with the inner membrane ring of the Shigella type 3 secretion system
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Focused reconstruction from isolated needle complex of the Shigella type 3 secretion system
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: M90T / Location in cell: Membrane

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Macromolecule #1: Outer membrane protein MxiD

MacromoleculeName: Outer membrane protein MxiD / type: protein_or_peptide / ID: 1
Details: MxiD N0 and N1 N-terminal domains in complex with MxiG C-terminal domain
Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 63.230414 KDa
SequenceString: MKKFNIKSLT LLIVLLPLIV NANNIDSHLL EQNDIAKYVA QSDTVGSFFE RFSALLNYPI VVSKQAAKKR ISGEFDLSNP EEMLEKLTL LVGLIWYKDG NALYIYDSGE LISKVILLEN ISLNYLIQYL KDANLYDHRY PIRGNISDKT FYISGPPALV E LVANTATL ...String:
MKKFNIKSLT LLIVLLPLIV NANNIDSHLL EQNDIAKYVA QSDTVGSFFE RFSALLNYPI VVSKQAAKKR ISGEFDLSNP EEMLEKLTL LVGLIWYKDG NALYIYDSGE LISKVILLEN ISLNYLIQYL KDANLYDHRY PIRGNISDKT FYISGPPALV E LVANTATL LDKQVSSIGT DKVNFGVIKL KNTFVSDRTY NMRGEDIVIP GVATVVERLL NNGKALSNRQ AQNDPMPPFN IT QKVSEDS NDFSFSSVTN SSILEDVSLI AYPETNSILV KGNDQQIQII RDIITQLDVA KRHIELSLWI IDIDKSELNN LGV NWQGTA SFGDSFGASF NMSSSASIST LDGNKFIASV MALNQKKKAN VVSRPVILTQ ENIPAIFDNN RTFYVSLVGE RNSS LEHVT YGTLINVIPR FSSRGQIEMS LTIEDGTGNS QSNYNYNNEN TSVLPEVGRT KISTIARVPQ GKSLLIGGYT HETNS NEII SIPFLSSIPV IGNVFKYKTS NISNIVRVFL IQPREIKESS YYNTAEYKSL ISEREIQKTT QIIPSETTLL EDEKSL VSY LNY

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Macromolecule #2: Protein MxiG

MacromoleculeName: Protein MxiG / type: protein_or_peptide / ID: 2
Details: MxiG C-terminal domain in complex with secretin MxiD
Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 43.053844 KDa
SequenceString: MSEAKNSNLA PFRLLVKLTN GVGDEFPLYY GNNLIVLGRT IETLEFGNDN FPENIIPVTD SKSDGIIYLT ISKDNICQFS DEKGEQIDI NSQFNSFEYD GISFHLKNMR EDKSRGHILN GMYKNHSVFF FFAVIVVLII IFSLSLKKDE VKEIAEIIDD K RYGIVNTG ...String:
MSEAKNSNLA PFRLLVKLTN GVGDEFPLYY GNNLIVLGRT IETLEFGNDN FPENIIPVTD SKSDGIIYLT ISKDNICQFS DEKGEQIDI NSQFNSFEYD GISFHLKNMR EDKSRGHILN GMYKNHSVFF FFAVIVVLII IFSLSLKKDE VKEIAEIIDD K RYGIVNTG QCNYILAETQ NDAVWASVAL NKTGFTKCRY ILVSNKEINR IQQYINQRFP FINLYVLNLV SDKAELLVFL SK ERNSSKD TELDKLKNAL IVEFPYIKNI KFNYLSDHNA RGDAKGIFTK VNVQYKEICE NNKVTYSVRE ELTDEKLELI NRL ISEHKN IYGDQYIEFS VLLIDDDFKG KSYLNSKDSY VMLNDKHWFF LDKNK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: Sample applied on grid 5 ul, incubation time 5 min on ice, then moved into Vitrobot and 5 ul sample applied again. Blot time: 2 sec Blot force: -2 Drain time: 0 sec.
DetailsIsolated needle complex in detergent solution

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.004 µm / Nominal defocus min: 0.0015 µm / Nominal magnification: 101179
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 5238 / Average exposure time: 1.5 sec. / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 171833
CTF correctionSoftware - Name: CTFFIND (ver. 3.5)
Startup modelType of model: INSILICO MODEL / In silico model: Initial model generated with EMAN2
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C8 (8 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 72298
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A
DetailsModel was built and refined in the map low-pass filtered at 3.5 A
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 120 / Target criteria: Correlation coefficient and geometry
Output model

PDB-6rwk:
MxiD N0 N1 and MxiG C-terminal domains of the Shigella type 3 secretion system

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