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- EMDB-70166: In-situ structure of the injectisome of Shigella flexneri without... -

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Entry
Database: EMDB / ID: EMD-70166
TitleIn-situ structure of the injectisome of Shigella flexneri without needle from mxiG linker deletion 111-124 mutant
Map dataIn-situ structure of the injectisome of Shigella flexneri without needle from mxiG linker deletion 111-124 mutant
Sample
  • Cell: Shigella flexneri
KeywordsInjectisome / Shigella flexneri / Complex / T3SS / CELL INVASION
Biological speciesShigella flexneri 5a str. M90T (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 39.65 Å
AuthorsTachiyama S / Liu J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI172097 United States
CitationJournal: Front Cell Infect Microbiol / Year: 2025
Title: A flexible peptide linking the periplasmic and cytoplasmic domains of MxiG controls type III secretion signaling and stable sorting platform assembly in .
Authors: Shoichi Tachiyama / Meena Muthuramalingam / Sean K Whittier / Yunjie Chang / Jian Yue / Waleed Younis / Wendy L Picking / Jun Liu / William D Picking /
Abstract: uses its type III secretion system (T3SS) to invade human enterocytes. The T3SS injectisome is controlled by proteins at the tip of an exposed needle that sense host cell contact. Substrate ... uses its type III secretion system (T3SS) to invade human enterocytes. The T3SS injectisome is controlled by proteins at the tip of an exposed needle that sense host cell contact. Substrate selection and powering of secretion is controlled by a cytoplasmic assembly called the sorting platform (SP). The SP possesses six pod structures linked to a central ATPase via radial spokes. The SP associates with the injectisome inner membrane ring (IR) via the adaptor protein MxiK. The major IR component is MxiG, whose globular periplasmic domain (MxiG) packs with MxiJ in a 24-fold symmetry. MxiG also has a transmembrane helix attached to a small cytoplasmic domain (MxiG) via a flexible linker peptide. Change from the IR's 24-fold symmetry to six-fold symmetry for the SP in occurs via MxiG pairs that associate with MxiK. The intervening pairs shift to the center of the IR/SP assembly, which is distinct from what is seen for . This implicates the linker in dynamic motions at the IR-SP interface, but the functional importance of the linker is unknown. Using a library of mutants, we found that the linker can accept diverse mutations without eliminating injectisome function. However, some mutants were found to give rise to subpopulations able to form needles and secrete effectors in the absence of a stably assembled SP. Mutants lacking the entire linker could not secrete any effector proteins (e.g. the IpaD tip protein) and had no T3SS-related virulence functions, however, there were subpopulations that could still secrete MxiH and assemble visible needles. In contrast, a very short linker could export IpaD to the needle tip, but could not rapidly respond to external secretion signals and were thus unable to quickly enter epithelial cells. These findings implicate the MxiG linker in signaling processes that are sensed at the needle tip. Our findings suggest that the native MxiG linker peptide has evolved to maximize T3SS function at steps beyond needle formation, while needle formation can occur even when the SP is highly destabilized.
History
DepositionApr 11, 2025-
Header (metadata) releaseSep 3, 2025-
Map releaseSep 3, 2025-
UpdateSep 3, 2025-
Current statusSep 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70166.png

Downloads & links

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Map

FileDownload / File: emd_70166.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn-situ structure of the injectisome of Shigella flexneri without needle from mxiG linker deletion 111-124 mutant
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.3 Å/pix.
x 200 pix.
= 859.2 Å		4.3 Å/pix.
x 200 pix.
= 859.2 Å		4.3 Å/pix.
x 200 pix.
= 859.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.296 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.061
Minimum - Maximum-0.3075495 - 0.3749086
Average (Standard dev.)-0.00000000014061 (±0.04041263)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 859.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map of the injectisome of Shigella flexneri...

Fileemd_70166_half_map_1.map
AnnotationHalf map of the injectisome of Shigella flexneri without needle from mxiG linker deletion 111-124 mutant
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the injectisome of Shigella flexneri...

Fileemd_70166_half_map_2.map
AnnotationHalf map of the injectisome of Shigella flexneri without needle from mxiG linker deletion 111-124 mutant
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Shigella flexneri

EntireName: Shigella flexneri (bacteria)
Components
  • Cell: Shigella flexneri

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Supramolecule #1: Shigella flexneri

SupramoleculeName: Shigella flexneri / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Shigella flexneri 5a str. M90T (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.96 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 39.65 Å / Resolution method: FSC 0.5 CUT-OFF / Number subtomograms used: 695
ExtractionNumber tomograms: 191 / Number images used: 1554
CTF correctionType: PHASE FLIPPING ONLY
Final angle assignmentType: OTHER

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