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- EMDB-15703: Inner membrane ring of the type 3 secretion system of Shigella fl... -

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Basic information

Entry
Database: EMDB / ID: EMD-15703
TitleInner membrane ring of the type 3 secretion system of Shigella flexneri
Map dataMain map (sharpened and masked)
Sample
  • Complex: Needle complex of the type 3 secretion system
    • Complex: Inner membrane ring
      • Protein or peptide: Protein MxiG (SctD)
      • Protein or peptide: Lipoprotein MxiJ (SctJ)
Biological speciesShigella flexneri (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsLunelli M
Funding support Germany, 1 items
OrganizationGrant numberCountry
Helmholtz Association Germany
CitationJournal: Protein Sci / Year: 2023
Title: Integrative structural analysis of the type III secretion system needle complex from Shigella flexneri.
Authors: Lara Flacht / Michele Lunelli / Karol Kaszuba / Zhuo Angel Chen / Francis J O' Reilly / Juri Rappsilber / Jan Kosinski / Michael Kolbe /
Abstract: The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram-negative bacteria to transport virulence factors into the host cell during infection. ...The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram-negative bacteria to transport virulence factors into the host cell during infection. Understanding the structure of T3SSs is crucial for future developments of therapeutics that could target this system. However, much of the knowledge about the structure of T3SS is available only for Salmonella, and it is unclear how this large assembly is conserved across species. Here, we combined cryo-electron microscopy, cross-linking mass spectrometry, and integrative modeling to determine the structure of the T3SS needle complex from Shigella flexneri. We show that the Shigella T3SS exhibits unique features distinguishing it from other structurally characterized T3SSs. The secretin pore complex adopts a new fold of its C-terminal S domain and the pilotin MxiM[SctG] locates around the outer surface of the pore. The export apparatus structure exhibits a conserved pseudohelical arrangement but includes the N-terminal domain of the SpaS[SctU] subunit, which was not present in any of the previously published virulence-related T3SS structures. Similar to other T3SSs, however, the apparatus is anchored within the needle complex by a network of flexible linkers that either adjust conformation to connect to equivalent patches on the secretin oligomer or bind distinct surface patches at the same height of the export apparatus. The conserved and unique features delineated by our analysis highlight the necessity to analyze T3SS in a species-specific manner, in order to fully understand the underlying molecular mechanisms of these systems. The structure of the type III secretion system from Shigella flexneri delineates conserved and unique features, which could be used for the development of broad-range therapeutics.
History
DepositionAug 31, 2022-
Header (metadata) releaseMar 1, 2023-
Map releaseMar 1, 2023-
UpdateApr 12, 2023-
Current statusApr 12, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15703.png

Downloads & links

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Map

FileDownload / File: emd_15703.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map (sharpened and masked)
Voxel sizeX=Y=Z: 1.38369 Å
Density
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.25173992 - 0.4521722
Average (Standard dev.)0.00016843258 (±0.00728263)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_15703_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15703_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15703_half_map_2.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 : Needle complex of the type 3 secretion system

EntireName: Needle complex of the type 3 secretion system
Components
  • Complex: Needle complex of the type 3 secretion system
    • Complex: Inner membrane ring
      • Protein or peptide: Protein MxiG (SctD)
      • Protein or peptide: Lipoprotein MxiJ (SctJ)

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Supramolecule #1: Needle complex of the type 3 secretion system

SupramoleculeName: Needle complex of the type 3 secretion system / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Shigella flexneri (bacteria)

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Supramolecule #2: Inner membrane ring

SupramoleculeName: Inner membrane ring / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Shigella flexneri (bacteria)

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Macromolecule #1: Protein MxiG (SctD)

MacromoleculeName: Protein MxiG (SctD) / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
SequenceString: MSEAKNSNLA PFRLLVKLTN GVGDEFPLYY GNNLIVLGRT IETLEFGNDN FPENIIPVTD SKSDGIIYL TISKDNICQF SDEKGEQIDI NSQFNSFEYD GISFHLKNMR EDKSRGHILN G MYKNHSVF FFFAVIVVLI IIFSLSLKKD EVKEIAEIID DKRYGIVNTG ...String:
MSEAKNSNLA PFRLLVKLTN GVGDEFPLYY GNNLIVLGRT IETLEFGNDN FPENIIPVTD SKSDGIIYL TISKDNICQF SDEKGEQIDI NSQFNSFEYD GISFHLKNMR EDKSRGHILN G MYKNHSVF FFFAVIVVLI IIFSLSLKKD EVKEIAEIID DKRYGIVNTG QCNYILAETQ ND AVWASVA LNKTGFTKCR YILVSNKEIN RIQQYINQRF PFINLYVLNL VSDKAELLVF LSK ERNSSK DTELDKLKNA LIVEFPYIKN IKFNYLSDHN ARGDAKGIFT KVNVQYKEIC ENNK VTYSV REELTDEKLE LINRLISEHK NIYGDQYIEF SVLLIDDDFK GKSYLNSKDS YVMLN DKHW FFLDKNK

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Macromolecule #2: Lipoprotein MxiJ (SctJ)

MacromoleculeName: Lipoprotein MxiJ (SctJ) / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
SequenceString: MIRYKGFILF LLLMLIGCEQ REELISNLSQ RQANEIISVL ERHNITARKV DGGKQGISVQ VEKGTFASA VDLMRMYDLP NPERVDISQM FPTDSLVSSP RAEKARLYSA IEQRLEQSLV S IGGVISAK IHVSYDLEEK NISSKPMHIS VIAIYDSPKE SELLVSNIKR ...String:
MIRYKGFILF LLLMLIGCEQ REELISNLSQ RQANEIISVL ERHNITARKV DGGKQGISVQ VEKGTFASA VDLMRMYDLP NPERVDISQM FPTDSLVSSP RAEKARLYSA IEQRLEQSLV S IGGVISAK IHVSYDLEEK NISSKPMHIS VIAIYDSPKE SELLVSNIKR FLKNTFSDVK YE NISVILT PKEEYVYTNV QPVKEVKSEF LTNEVIYLFL GMAVLVVILL VWAFKTGWFK RNK 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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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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: 4.0 µm / Nominal defocus min: 1.5 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) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-6 / 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

Startup modelType of model: EMDB MAP
EMDB ID:

10045

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C24 (24 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Details: Focused reconstruction / Number images used: 90547
FSC plot (resolution estimation)

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