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- EMDB-10045: Inner membrane ring of the Shigella type 3 secretion system -

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Basic information

Entry
Database: EMDB / ID: EMD-10045
TitleInner membrane ring of the Shigella type 3 secretion system
Map dataMasked C24 map of inner membrane ring of Shigella type 3 secretion system, post-processed, sharpened (b=-128) and filtered at 3.55 A.
Sample
  • Complex: The periplasmic inner membrane ring of the Shigella type 3 secretion system
    • Protein or peptide: Protein MxiG
    • Protein or peptide: Lipoprotein MxiJ
Function / homology
Function and homology information


protein secretion / cell outer membrane / plasma membrane
Similarity search - Function
Type III secretion system lipoprotein HrcJ/YscJ / Type III secretion system, PrgH/EprH-like / Type III secretion system protein PrgH-EprH (PrgH) / Lipoprotein YscJ/Flagellar M-ring protein / Secretory protein of YscJ/FliF family / Flagellar M-ring , N-terminal / AMP-binding enzyme, C-terminal domain superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Protein MxiG / Lipoprotein MxiJ
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.55 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 6, 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: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.07
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  • Surface view with fitted model
  • Atomic models: PDB-6rwx
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6rwx
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10045.png

Downloads & links

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Map

FileDownload / File: emd_10045.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked C24 map of inner membrane ring of Shigella type 3 secretion system, post-processed, sharpened (b=-128) and filtered at 3.55 A.
Voxel sizeX=Y=Z: 1.38369 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.3866547 - 0.6354907
Average (Standard dev.)0.0004545977 (±0.011630482)
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-0.3870.6350.000

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Supplemental data

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Mask #1

Fileemd_10045_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Masked C24 map of inner membrane ring of...

Fileemd_10045_additional.map
AnnotationMasked C24 map of inner membrane ring of Shigella type 3 secretion system, post-processed, sharpened (b=-128) and filtered at 3.2 A. Used for model building and refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map

Fileemd_10045_half_map_1.map
AnnotationUnfiltered half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map

Fileemd_10045_half_map_2.map
AnnotationUnfiltered half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The periplasmic inner membrane ring of the Shigella type 3 secret...

EntireName: The periplasmic inner membrane ring of the Shigella type 3 secretion system
Components
  • Complex: The periplasmic inner membrane ring of the Shigella type 3 secretion system
    • Protein or peptide: Protein MxiG
    • Protein or peptide: Lipoprotein MxiJ

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Supramolecule #1: The periplasmic inner membrane ring of the Shigella type 3 secret...

SupramoleculeName: The periplasmic 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 complexes 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: Protein MxiG

MacromoleculeName: Protein MxiG / type: protein_or_peptide / ID: 1 / Number of copies: 24 / 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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Macromolecule #2: Lipoprotein MxiJ

MacromoleculeName: Lipoprotein MxiJ / type: protein_or_peptide / ID: 2 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 27.542055 KDa
SequenceString: MIRYKGFILF LLLMLIGCEQ REELISNLSQ RQANEIISVL ERHNITARKV DGGKQGISVQ VEKGTFASAV DLMRMYDLPN PERVDISQM FPTDSLVSSP RAEKARLYSA IEQRLEQSLV SIGGVISAKI HVSYDLEEKN ISSKPMHISV IAIYDSPKES E LLVSNIKR ...String:
MIRYKGFILF LLLMLIGCEQ REELISNLSQ RQANEIISVL ERHNITARKV DGGKQGISVQ VEKGTFASAV DLMRMYDLPN PERVDISQM FPTDSLVSSP RAEKARLYSA IEQRLEQSLV SIGGVISAKI HVSYDLEEKN ISSKPMHISV IAIYDSPKES E LLVSNIKR FLKNTFSDVK YENISVILTP KEEYVYTNVQ PVKEVKSEFL TNEVIYLFLG MAVLVVILLV WAFKTGWFKR NK I

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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 reconstructionApplied symmetry - Point group: C24 (24 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.55 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 model
PDB IDChain

3gr0

chain_id: D, residue_range: 183-362

2y9j

chain_id: t, residue_range: 21-190
DetailsModel was built and refined in the map low-pass filtered at 3.2 A
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 140 / Target criteria: Correlation coefficient and geometry
Output model

PDB-6rwx:
Periplasmic inner membrane ring of the Shigella type 3 secretion system

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