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- PDB-2mme: Hybrid structure of the Shigella flexneri MxiH Type three secreti... -

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Basic information

Entry
Database: PDB / ID: 2mme
TitleHybrid structure of the Shigella flexneri MxiH Type three secretion system needle
ComponentsMxiH
KeywordsPROTEIN TRANSPORT / type-three secretion system / filamentous protein / helical assembly / Shigella flexneri / protein translocation / hybrid methods / Rosetta
Function / homologyType 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 protein secretion system complex / protein secretion by the type III secretion system / : / MxiH
Function and homology information
Biological speciesShigella flexneri (bacteria)
MethodSOLID-STATE NMR / ELECTRON MICROSCOPY / helical reconstruction / Rosetta fold-and-dock, Rosetta symmetric relax / cryo EM / Resolution: 7.7 Å
Model detailslowest energy, model1
AuthorsDemers, J.P. / Habenstein, B. / Loquet, A. / Vasa, S.K. / Becker, S. / Baker, D. / Lange, A. / Sgourakis, N.G.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Structure of a type III secretion needle at 7-Å resolution provides insights into its assembly and signaling mechanisms.
Authors: Takashi Fujii / Martin Cheung / Amandine Blanco / Takayuki Kato / Ariel J Blocker / Keiichi Namba /
Abstract: Type III secretion systems of Gram-negative bacteria form injection devices that deliver effector proteins into eukaryotic cells during infection. They span both bacterial membranes and the ...Type III secretion systems of Gram-negative bacteria form injection devices that deliver effector proteins into eukaryotic cells during infection. They span both bacterial membranes and the extracellular space to connect with the host cell plasma membrane. Their extracellular portion is a needle-like, hollow tube that serves as a secretion conduit for effector proteins. The needle of Shigella flexneri is approximately 50-nm long and 7-nm thick and is made by the helical assembly of one protein, MxiH. We provide a 7-Å resolution 3D image reconstruction of the Shigella needle by electron cryomicroscopy, which resolves α-helices and a β-hairpin that has never been observed in the crystal and solution structures of needle proteins, including MxiH. An atomic model of the needle based on the 3D-density map, in comparison with that of the bacterial-flagellar filament, provides insights into how such a thin tubular structure is stably assembled by intricate intermolecular interactions. The map also illuminates how the needle-length control protein functions as a ruler within the central channel during export of MxiH for assembly at the distal end of the needle, and how the secretion-activation signal may be transduced through a conformational change of the needle upon host-cell contact.
History
DepositionMar 14, 2014Deposition site: BMRB / Processing site: RCSB
Revision 1.0Oct 8, 2014Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2020Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Database references / Other
Category: em_image_scans / em_single_particle_entity ...em_image_scans / em_single_particle_entity / pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer / struct_ref_seq_dif
Item: _pdbx_database_status.status_code_cs / _pdbx_nmr_software.name ..._pdbx_database_status.status_code_cs / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model / _struct_ref_seq_dif.details
Revision 1.2Jun 14, 2023Group: Database references / Other / Category: database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data
Remark 0THIS ENTRY 2MME CONTAINS A STRUCTURAL MODEL FIT TO AN ELECTRON MICROSCOPY MAP (EMD-5352) DETERMINED ...THIS ENTRY 2MME CONTAINS A STRUCTURAL MODEL FIT TO AN ELECTRON MICROSCOPY MAP (EMD-5352) DETERMINED ORIGINALLY BY AUTHORS: T.FUJII, M.CHEUNG, A.BLANCO, T.KATO, A.J.BLOCKER, K.NAMBA

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Structure visualization

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  • Superimposition on EM map
  • EMDB-5352
  • Imaged by UCSF Chimera
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  • EMDB-5352
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: MxiH
B: MxiH
C: MxiH
D: MxiH
E: MxiH
F: MxiH
G: MxiH
H: MxiH
I: MxiH
J: MxiH
K: MxiH
L: MxiH
M: MxiH
N: MxiH
O: MxiH
P: MxiH
Q: MxiH
R: MxiH
S: MxiH
T: MxiH
U: MxiH
V: MxiH
W: MxiH
X: MxiH
Y: MxiH
Z: MxiH
a: MxiH
b: MxiH
c: MxiH


Theoretical massNumber of molelcules
Total (without water)272,49329
Polymers272,49329
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 5000target function
RepresentativeModel #1lowest energy

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Components

#1: Protein ...
MxiH


Mass: 9396.321 Da / Num. of mol.: 29
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria) / Strain: Serotype 6, BC-114 / Gene: mxiH / Plasmid: pET16b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6XVY0

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Experimental details

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Experiment

Experiment
Method
SOLID-STATE NMR
ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
11113C-13C (PDSD)
12115N-13C (Specific-CP)

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Sample preparation

ComponentName: Shigella flexneri MxiH Type three secretion system needle
Type: COMPLEX
Buffer solutionpH: 7.4 / Details: 20 mM Tris, pH 7.4, 150 mM NaCl, 2mM MgSO4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE
DetailsContents: 20 mg [U-100% 13C; U-100% 15N] MxiH, wet / Solvent system: wet
SampleUnits: % / Component: MxiH-1 / Isotopic labeling: [U-100% 13C; U-100% 15N]
Sample conditionspH: 5.5 / Pressure: ambient / Temperature: 278 K

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Data collection

MicroscopyModel: JEOL 3200FSC / Date: Jul 2, 2008
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k)
NMR spectrometerType: Bruker Avance / Manufacturer: Bruker / Model: AVANCE / Field strength: 850 MHz

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Processing

3D reconstructionResolution: 7.7 Å / Symmetry type: HELICAL
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms18763 0 0 0 18763
NMR software
NameDeveloperClassification
SparkyGoddarddata analysis
Rosettarefinement
RefinementMethod: Rosetta fold-and-dock, Rosetta symmetric relax / Software ordinal: 2
Details: symmetric fragment-based Monte Carlo trials followed by full-atom refinement, symmetric refinement (relax) of backbone, sidechain, and rigid-body degrees of freedom
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 5000 / Conformers submitted total number: 10

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