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- PDB-4d3e: Tetramer of IpaD, modified from 2J0O, fitted into negative stain ... -

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Entry
Database: PDB / ID: 4d3e
TitleTetramer of IpaD, modified from 2J0O, fitted into negative stain electron microscopy reconstruction of the wild type tip complex from the type III secretion system of Shigella flexneri
ComponentsINVASIN IPAD
KeywordsCELL INVASION / TIP COMPLEX / TYPE III SECRETION SYSTEM / SHIGELLA FLEXNERI / WILD TYPE / IPAD
Function / homology
Function and homology information


effector-mediated activation of programmed cell death in host / extracellular region
Similarity search - Function
IpaD-like / IpaD-like / Type III secretion systems tip complex components / BipD-like superfamily / Type III secretion systems tip complex components / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Biological speciesSHIGELLA FLEXNERI 5A STR. M90T (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 24 Å
AuthorsCheung, M. / Shen, D.-K. / Makino, F. / Kato, T. / Roehrich, D. / Martinez-Argudo, I. / Walker, M.L. / Murillo, I. / Liu, X. / Pain, M. ...Cheung, M. / Shen, D.-K. / Makino, F. / Kato, T. / Roehrich, D. / Martinez-Argudo, I. / Walker, M.L. / Murillo, I. / Liu, X. / Pain, M. / Brown, J. / Frazer, G. / Mantell, J. / Mina, P. / Todd, T. / Sessions, R.B. / Namba, K. / Blocker, A.J.
Citation
Journal: Mol Microbiol / Year: 2015
Title: Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex.
Authors: Martin Cheung / Da-Kang Shen / Fumiaki Makino / Takayuki Kato / A Dorothea Roehrich / Isabel Martinez-Argudo / Matthew L Walker / Isabel Murillo / Xia Liu / Maria Pain / James Brown / Gordon ...Authors: Martin Cheung / Da-Kang Shen / Fumiaki Makino / Takayuki Kato / A Dorothea Roehrich / Isabel Martinez-Argudo / Matthew L Walker / Isabel Murillo / Xia Liu / Maria Pain / James Brown / Gordon Frazer / Judith Mantell / Petros Mina / Thomas Todd / Richard B Sessions / Keiichi Namba / Ariel J Blocker /
Abstract: Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a ...Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.
#1: Journal: J Biol Chem / Year: 2007
Title: Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD.
Authors: Steven Johnson / Pietro Roversi / Marianela Espina / Andrew Olive / Janet E Deane / Susan Birket / Terry Field / William D Picking / Ariel J Blocker / Edouard E Galyov / Wendy L Picking / Susan M Lea /
Abstract: Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. ...Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.
History
DepositionOct 21, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 10, 2014Provider: repository / Type: Initial release
Revision 1.1Jan 14, 2015Group: Database references
Revision 1.2Aug 30, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id
Revision 1.3Jan 31, 2018Group: Data processing / Category: em_software / Item: _em_software.details / _em_software.name
Revision 1.4Apr 29, 2020Group: Data processing / Category: em_3d_reconstruction / Item: _em_3d_reconstruction.resolution

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Assembly

Deposited unit
D: INVASIN IPAD


Theoretical massNumber of molelcules
Total (without water)23,0771
Polymers23,0771
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Number of models4

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Components

#1: Protein INVASIN IPAD / 36 KDA MEMBRANE ANTIGEN


Mass: 23076.775 Da / Num. of mol.: 1
Fragment: COILDED-COIL AND GLOBULAR DOMAIN, UNP RESIDUES 125-332
Source method: isolated from a genetically manipulated source
Details: YES [LACKING FIRST 124 AA AND WITH LAST 10 AA MODELLED INTO HELIX]
Source: (gene. exp.) SHIGELLA FLEXNERI 5A STR. M90T (bacteria)
Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: P18013

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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

ComponentName: WILD-TYPE T3SS NEEDLE COMPLEX / Type: COMPLEX / Details: FIRST ZERO OF CTF SET TO 19A FOR ALL MICROGRAPHS
Buffer solutionName: 0.1% (W/V) DDM, 150 MM NACL, 25 MM TRIS PH 8, 5 MM EDTA
pH: 8
Details: 0.1% (W/V) DDM, 150 MM NACL, 25 MM TRIS PH 8, 5 MM EDTA
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Material: uranyl acetate
Specimen supportDetails: CARBON

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20 / Date: Aug 31, 2011
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Calibrated magnification: 70754 X / Nominal defocus max: 1600 nm / Nominal defocus min: 1200 nm / Cs: 2 mm
Image recordingElectron dose: 90 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)
Image scansNum. digital images: 300
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1UCSF Chimeramodel fitting
3EMAN23D reconstruction
4SPIDER3D reconstruction
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: MIX OF SINGLE PARTICLE AND HELICAL RECONSTRUCTION METHODS
Resolution: 24 Å / Num. of particles: 2963 / Actual pixel size: 2.12 Å
Details: THIS MODEL IS RESULT OF RIGID-BODY TRANSFORMATION OF AN EXISTING PDB MODEL, CONSTRAINED BY A 24 A RESOLUTION EM MAP AND SOME CYSTEINE-MEDIATED CROSSLINKING DATA. THEREFORE, IT DOES NOT ...Details: THIS MODEL IS RESULT OF RIGID-BODY TRANSFORMATION OF AN EXISTING PDB MODEL, CONSTRAINED BY A 24 A RESOLUTION EM MAP AND SOME CYSTEINE-MEDIATED CROSSLINKING DATA. THEREFORE, IT DOES NOT SUPPORT ATOMISTIC INTERPRETATIONS BECAUSE OF LACK OF RESOLUTION.
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Details: METHOD--SEE PUBLICATION REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 2J0O

2j0o

RefinementHighest resolution: 2.12 Å
Refinement stepCycle: LAST / Highest resolution: 2.12 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1621 0 0 0 1621

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