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- EMDB-15700: Type 3 secretion system needle complex of Shigella flexneri -

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Basic information

Entry
Database: EMDB / ID: EMD-15700
TitleType 3 secretion system needle complex of Shigella flexneri
Map dataMain map (sharpened and masked)
Sample
  • Complex: Needle complex of the type 3 secretion system
    • Complex: Export apparatus core
      • Protein or peptide: Surface presentation of antigens protein SpaP
      • Protein or peptide: Surface presentation of antigens protein SpaR
      • Protein or peptide: Surface presentation of antigens protein SpaQ
      • Protein or peptide: Surface presentation of antigens protein SpaS
    • Complex: Inner rod
      • Protein or peptide: Protein MxiI
    • Complex: Needle filament
      • Protein or peptide: Protein MxiH
    • Complex: Ring formed by the N1 domain of the secretin MxiD
      • Protein or peptide: Outer membrane protein MxiD
    • Complex: MxiJ loop interacting with the export apparatus
      • Protein or peptide: Lipoprotein MxiJ
Function / homology
Function and homology information


type III protein secretion system complex / protein secretion by the type III secretion system / protein secretion / protein targeting / cell outer membrane / protein transport / cell surface / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
: / Type III secretion protein SpaR/YscT / Type III secretion protein HrpO / Yop virulence translocation protein R / Type III exporter system, secretion apparatus protein BsaZ / Type III secretion system substrate exporter / Type III secretion system substrate exporter, C-terminal / FlhB HrpN YscU SpaS Family / Type III secretion system lipoprotein HrcJ/YscJ / : ...: / Type III secretion protein SpaR/YscT / Type III secretion protein HrpO / Yop virulence translocation protein R / Type III exporter system, secretion apparatus protein BsaZ / Type III secretion system substrate exporter / Type III secretion system substrate exporter, C-terminal / FlhB HrpN YscU SpaS Family / Type III secretion system lipoprotein HrcJ/YscJ / : / SPI-1 type 3 secretion system secretin, N0 domain / Type III secretion system outer membrane pore YscC/HrcC / Type III secretion, needle-protein-like / Type III secretion, needle-protein-like superfamily / Type III secretion needle MxiH, YscF, SsaG, EprI, PscF, EscF / Type III secretion system, needle protein / Type III secretion system inner membrane R protein / Bacterial export protein family 3 / Bacterial export proteins, family 1 / Bacterial export proteins, family 3 / Flagella transport protein fliP family signature 1. / Type III secretion system inner membrane P protein / FliP family / Flagella transport protein fliP family signature 2. / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / : / NolW-like / NolW-like superfamily / Bacterial type II/III secretion system short domain / Type II/III secretion system / Bacterial type II and III secretion system protein / 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
Surface presentation of antigens protein SpaP / Surface presentation of antigens protein SpaQ / Surface presentation of antigens protein SpaR / Surface presentation of antigens protein SpaS / Type 3 secretion system needle filament protein / Protein MxiI / Type 3 secretion system secretin / Lipoprotein MxiJ
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.05 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_15700.png

Downloads & links

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Map

FileDownload / File: emd_15700.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map (sharpened and masked)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 480 pix.
= 664.171 Å		1.38 Å/pix.
x 480 pix.
= 664.171 Å		1.38 Å/pix.
x 480 pix.
= 664.171 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel size
XYZ
EMDB info.1.038371.038371.03837
CCP4 map header1.383691.383691.38369
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.20915963 - 0.30591616
Average (Standard dev.)0.0004833863 (±0.0075753983)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 498.41714 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15700_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15700_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_15700_half_map_2.map
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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: Export apparatus core
      • Protein or peptide: Surface presentation of antigens protein SpaP
      • Protein or peptide: Surface presentation of antigens protein SpaR
      • Protein or peptide: Surface presentation of antigens protein SpaQ
      • Protein or peptide: Surface presentation of antigens protein SpaS
    • Complex: Inner rod
      • Protein or peptide: Protein MxiI
    • Complex: Needle filament
      • Protein or peptide: Protein MxiH
    • Complex: Ring formed by the N1 domain of the secretin MxiD
      • Protein or peptide: Outer membrane protein MxiD
    • Complex: MxiJ loop interacting with the export apparatus
      • Protein or peptide: Lipoprotein MxiJ

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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: Export apparatus core

SupramoleculeName: Export apparatus core / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Shigella flexneri (bacteria)

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Supramolecule #3: Inner rod

SupramoleculeName: Inner rod / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Shigella flexneri (bacteria)

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Supramolecule #4: Needle filament

SupramoleculeName: Needle filament / type: complex / ID: 4 / Chimera: Yes / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: Shigella flexneri (bacteria)

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Supramolecule #5: Ring formed by the N1 domain of the secretin MxiD

SupramoleculeName: Ring formed by the N1 domain of the secretin MxiD / type: complex / ID: 5 / Chimera: Yes / Parent: 1 / Macromolecule list: #7
Source (natural)Organism: Shigella flexneri (bacteria)

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Supramolecule #6: MxiJ loop interacting with the export apparatus

SupramoleculeName: MxiJ loop interacting with the export apparatus / type: complex / ID: 6 / Chimera: Yes / Parent: 1 / Macromolecule list: #8
Source (natural)Organism: Shigella flexneri (bacteria)

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Macromolecule #1: Surface presentation of antigens protein SpaP

MacromoleculeName: Surface presentation of antigens protein SpaP / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 24.215562 KDa
SequenceString: MLSDMSLIAT LSFFTLLPFL VAAGTCYIKF SIVFVMVRNA LGLQQVPSNM TLNGIALIMA LFVMKPIIEA GYENYLNGPQ KFDTISDIV RFSDSGLMEY KQYLKKHTDL ELARFFQRSE EENADLKSAE NNDYSLFSLL PAYALSEIKD AFKIGFYLYL P FVVVDLVI ...String:
MLSDMSLIAT LSFFTLLPFL VAAGTCYIKF SIVFVMVRNA LGLQQVPSNM TLNGIALIMA LFVMKPIIEA GYENYLNGPQ KFDTISDIV RFSDSGLMEY KQYLKKHTDL ELARFFQRSE EENADLKSAE NNDYSLFSLL PAYALSEIKD AFKIGFYLYL P FVVVDLVI SSILLALGMM MMSPITISVP IKLVLFVALD GWGILSKALI EQYINIPA

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Macromolecule #2: Surface presentation of antigens protein SpaR

MacromoleculeName: Surface presentation of antigens protein SpaR / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 28.513773 KDa
SequenceString: MDISSWFESI HVFLILLNGV FFRLAPLFFF LPFLNNGIIS PSIRIPVIFL VASGLITSGK VDIGSSVFEH VYFLMFKEII VGLLLSFCL SLPFWIFHAV GSIIDNQRGA TLSSSIDPAN GVDTSELAKF FNLFSAVVFL YSGGMVFILE SIQLSYNICP L FSQCSFRI ...String:
MDISSWFESI HVFLILLNGV FFRLAPLFFF LPFLNNGIIS PSIRIPVIFL VASGLITSGK VDIGSSVFEH VYFLMFKEII VGLLLSFCL SLPFWIFHAV GSIIDNQRGA TLSSSIDPAN GVDTSELAKF FNLFSAVVFL YSGGMVFILE SIQLSYNICP L FSQCSFRI SNILTFLTLL ASQAVILASP VMIVLLLSEV LLGVLSRFAP QMNAFSVSLT IKSLLAIFII FICSSTIYFS KV QFFLGEH KFFTNLFVR

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Macromolecule #3: Surface presentation of antigens protein SpaQ

MacromoleculeName: Surface presentation of antigens protein SpaQ / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 9.433338 KDa
SequenceString:
MSDIVYMGNK ALYLILIFSL WPVGIATVIG LSIGLLQTVT QLQEQTLPFG IKLIGVSISL LLLSGWYGEV LLSFCHEIMF LIKSGV

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Macromolecule #4: Surface presentation of antigens protein SpaS

MacromoleculeName: Surface presentation of antigens protein SpaS / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 39.903348 KDa
SequenceString: MANKTEKPTP KKLKDAAKKG QSFKFKDLTT VVIILVGTFT IISFFSLSDV MLLYRYVIIN DFEINEGKYF FAVVIVFFKI IGFPLFFCV LSAVLPTLVQ TKFVLATKAI KIDFSVLNPV KGLKKIFSIK TIKEFFKSIL LLIILALTTY FFWINDRKII F SQVFSSVD ...String:
MANKTEKPTP KKLKDAAKKG QSFKFKDLTT VVIILVGTFT IISFFSLSDV MLLYRYVIIN DFEINEGKYF FAVVIVFFKI IGFPLFFCV LSAVLPTLVQ TKFVLATKAI KIDFSVLNPV KGLKKIFSIK TIKEFFKSIL LLIILALTTY FFWINDRKII F SQVFSSVD GLYLIWGRLF KDIILFFLAF SILVIILDFV IEFILYMKDM MMDKQEIKRE YIEQEGHFET KSRRRELHIE IL SEQTKSD IRNSKLVVMN PTHIAIGIYF NPEIAPAPFI SLIETNQCAL AVRKYANEVG IPTVRDVKLA RKLYKTHTKY SFV DFEHLD EVLRLIVWLE QVENTH

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Macromolecule #5: Protein MxiI

MacromoleculeName: Protein MxiI / type: protein_or_peptide / ID: 5 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 10.64 KDa
SequenceString:
MNYIYPVNQV DIIKASDFQS QEISSLEDVV SAKYSDIKMD TDIQVSQIME MVSNPESLNP ESLAKLQTTL SNYSIGVSLA GTLARKTVS AVETLLKS

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Macromolecule #6: Protein MxiH

MacromoleculeName: Protein MxiH / type: protein_or_peptide / ID: 6 / Number of copies: 28 / Enantiomer: LEVO
Source (natural)Organism: Shigella flexneri (bacteria)
Molecular weightTheoretical: 11.060279 KDa
Recombinant expressionOrganism: Shigella flexneri (bacteria)
SequenceString:
MASWSHPQFE KIEGRMSVTV PNDDWTLSSL SETFDDGTQT LQGELTLALD KLAKNPSNPQ LLAEYQSKLS EYTLYRNAQS NTVKVIKDV DAAIIQNFR

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Macromolecule #7: Outer membrane protein MxiD

MacromoleculeName: Outer membrane protein MxiD / type: protein_or_peptide / ID: 7 / 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 #8: Lipoprotein MxiJ

MacromoleculeName: Lipoprotein MxiJ / type: protein_or_peptide / ID: 8 / 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 / 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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

10046

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 90547
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6rwy

,

6rwk

,

6rwx

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 59.2
Output model

PDB-8axk:
Type 3 secretion system export apparatus core, inner rod and needle of Shigella flexneri

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