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- PDB-6rwy: Export apparatus core and inner rod of the Shigella type 3 secret... -

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Basic information

Entry
Database: PDB / ID: 6rwy
TitleExport apparatus core and inner rod of the Shigella type 3 secretion system
Components
  • (Inner rod protein) x 2
  • (Surface presentation of antigens protein ...) x 3
  • Protein MxiH
KeywordsPROTEIN TRANSPORT / type 3 secretion system / shigella / protein translocation / injectisome
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 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 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 ...Type III secretion protein SpaR/YscT / Type III secretion protein HrpO / Yop virulence translocation protein R / 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. / 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
Similarity search - Domain/homology
Surface presentation of antigens protein SpaP / Surface presentation of antigens protein SpaQ / Surface presentation of antigens protein SpaR / Type 3 secretion system needle filament protein
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.11 Å
AuthorsLunelli, M. / Kamprad, A.
Funding support2items
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, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 4, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-10046
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  • Superimposition on EM map
  • EMDB-10046
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Inner rod protein
B: Inner rod protein
C: Inner rod protein
D: Inner rod protein
E: Inner rod protein
F: Inner rod protein
G: Inner rod protein
H: Inner rod protein
I: Inner rod protein
J: Inner rod protein
K: Inner rod protein
L: Protein MxiH
M: Protein MxiH
N: Protein MxiH
O: Protein MxiH
P: Protein MxiH
Q: Protein MxiH
R: Protein MxiH
S: Protein MxiH
T: Protein MxiH
U: Protein MxiH
V: Protein MxiH
a: Surface presentation of antigens protein SpaP
b: Surface presentation of antigens protein SpaP
c: Surface presentation of antigens protein SpaP
d: Surface presentation of antigens protein SpaP
e: Surface presentation of antigens protein SpaP
f: Surface presentation of antigens protein SpaR
g: Surface presentation of antigens protein SpaQ
h: Surface presentation of antigens protein SpaQ
i: Surface presentation of antigens protein SpaQ
j: Surface presentation of antigens protein SpaQ
k: Surface presentation of antigens protein SpaQ


Theoretical massNumber of molelcules
Total (without water)379,81033
Polymers379,81033
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area88950 Å2
ΔGint-895 kcal/mol
Surface area133610 Å2
MethodPISA

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Components

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Protein , 3 types, 22 molecules AGHIJKBCDEFLMNOPQRSTUV

#1: Protein
Inner rod protein


Mass: 5039.203 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Details: Alanine polypeptide because sequence uncertain / Source: (natural) Shigella flexneri (bacteria) / Variant: M90T
#2: Protein
Inner rod protein


Mass: 6230.672 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Details: Alanine polypeptide because sequence uncertain / Source: (natural) Shigella flexneri (bacteria) / Variant: M90T
#3: Protein
Protein MxiH


Mass: 11060.279 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
Details: N-terminal strep-tag II and factor Xa cleaveage site.
Source: (gene. exp.) Shigella flexneri (bacteria) / Gene: mxiH, CP0137 / Variant: M90T / Plasmid: pASK-IBA5plus / Production host: Shigella flexneri (bacteria) / Variant (production host): M90T / References: UniProt: P0A223

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Surface presentation of antigens protein ... , 3 types, 11 molecules abcdefghijk

#4: Protein
Surface presentation of antigens protein SpaP / Spa24 protein


Mass: 24215.562 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Shigella flexneri (bacteria) / Variant: M90T / References: UniProt: P0A1L3
#5: Protein Surface presentation of antigens protein SpaR / Spa29 protein


Mass: 28513.773 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Shigella flexneri (bacteria) / Variant: M90T / References: UniProt: P0A1M6
#6: Protein
Surface presentation of antigens protein SpaQ / Protein spa9


Mass: 9433.338 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Shigella flexneri (bacteria) / Variant: M90T / References: UniProt: P0A1M4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Export apparatus core, inner rod and start of the needle of the Shigella type 3 secretion systemCOMPLEXall0MULTIPLE SOURCES
2Export apparatus coreCOMPLEX#4-#61NATURAL
3Inner rodCOMPLEX#1-#21NATURAL
4NeedleCOMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
41NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
32Shigella flexneri (bacteria)623M90T
43Shigella flexneri (bacteria)623M90T
54Shigella flexneri (bacteria)623M90T
Source (recombinant)Organism: Shigella flexneri (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Isolated needle complex in detergent solution
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 101179 X / Nominal defocus max: 4 nm / Nominal defocus min: 1.5 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 25 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5238
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 7

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Processing

SoftwareName: PHENIX / Version: dev_3409: / Classification: refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
4CTFFIND3.5CTF correction
7UCSF Chimera1.13.1model fitting
8iMODFIT1.44model fitting
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
14PHENIX3409model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 171833
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72298 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 162 / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16F2D

6f2d

A1
26F2D

6f2d

F1
36F2D

6f2d

G1
42MME

2mme

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00423012
ELECTRON MICROSCOPYf_angle_d0.80531354
ELECTRON MICROSCOPYf_dihedral_angle_d11.6113779
ELECTRON MICROSCOPYf_chiral_restr0.0433890
ELECTRON MICROSCOPYf_plane_restr0.0063985

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