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- EMDB-75999: In-situ structure of the Shigella injectisome from mxiK L46D mutant -

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Entry
Database: EMDB / ID: EMD-75999
TitleIn-situ structure of the Shigella injectisome from mxiK L46D mutant
Map dataIn-situ structure of the Shigella injectisome from mxiK L46D mutant
Sample
  • Cell: Shigella flexneri
KeywordsInjectisome / Shigella flexneri / Complex / T3SS / CELL INVASION
Biological speciesShigella flexneri (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 37.4 Å
AuthorsTachiyama S / Liu J
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI172097 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI087946 United States
CitationJournal: PLoS Pathog / Year: 2026
Title: The pod components of the Shigella T3SS sorting platform accommodate multiple copies of Spa33 (SctQ).
Authors: Sean K Whittier / Shoichi Tachiyama / Samira Heydari / Wendy L Picking / Jun Liu / William D Picking /
Abstract: The bacterial type III secretion system (T3SS) uses a membrane-embedded injectisome assembly to export effector proteins into host cells. While atomic-level structural details have been revealed for ...The bacterial type III secretion system (T3SS) uses a membrane-embedded injectisome assembly to export effector proteins into host cells. While atomic-level structural details have been revealed for much of the T3SS apparatus, the model of the cytoplasmic sorting platform remains largely low-resolution. A central structural element of the sorting platform is the so-called "pod" protein, SctQ, which anchors the sorting platform to the inner membrane via interaction with the adaptor protein SctK, and connects to the central ATPase via the spoke protein SctL. SctQ proteins also interact with alternatively translated homodimers of their C-terminal SPOA2 domains. Low resolution electron density maps have provided an outline of the sorting platform architecture, and fluorescence microscopy studies have suggested a 1:4:2 SctK:SctQ:SctL stoichiometry. While there are experimental and AlphaFold structures of individual components and complexes of the sorting platform pod, there is currently no model for the pod structure that adequately fits the electron density or accounts for the proposed stoichiometry. Here we use AlphaFold to generate a model of the upper portion of the Shigella pod complex in which two copies of the SctQ protein, Spa33, bind the adaptor protein MxiK, with each copy of Spa33 bound to an alternately translated SPOA2-SPOA2 domain. We show through mutation of energetically critical interface residues, predicted by computational mutant scanning, that both Spa33 binding sites on MxiK are required for T3SS activity in Shigella flexneri, as well as binding of Spa33 to the SPOA2-SPOA2 homodimer. We find that this model fits well to the upper two-thirds of the pod electron density, albeit in a manner that places the protein components slightly closer to the inner membrane than traditionally presented. Further, cryogenic electron tomography shows Spa33/MxiK interface mutant sorting platforms are destabilized and lack clear density for the SctL spoke protein MxiN and central ATPase Spa47, while Spa33/SPOA2-SPOA2 interaction mutants are further disrupted and lack clear lower pod density. The density for the lower portion of the sorting platform pod, thought to arise from Spa33 and MxiN, remains unmodeled, largely due to the inability of AlphaFold to confidently predict the structure of MxiN or MxiN complexes. However, we show that, allowing for potential conformational rearrangements, the pod can accommodate three full-length copies of Spa33 in a manner that fills some of the lower electron density. How a potential fourth copy of Spa33 could occupy the remaining density with MxiN remains unclear.
History
DepositionMar 11, 2026-
Header (metadata) releaseSep 16, 2026-
Map releaseSep 16, 2026-
UpdateOct 7, 2026-
Current statusOct 7, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_75999.png

Downloads & links

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Map

FileDownload / File: emd_75999.map.gz / Format: CCP4 / Size: 18.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn-situ structure of the Shigella injectisome from mxiK L46D mutant
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.3 Å/pix.
x 170 pix.
= 730.32 Å		4.3 Å/pix.
x 170 pix.
= 730.32 Å		4.3 Å/pix.
x 170 pix.
= 730.32 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: 0.0396
Minimum - Maximum-0.5331467 - 0.49730754
Average (Standard dev.)0.0000000002502 (±0.05459372)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions170170170
Spacing170170170
CellA=B=C: 730.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map of the Shigella injectisome from mxiK L46D mutant

Fileemd_75999_half_map_1.map
AnnotationHalf map of the Shigella injectisome from mxiK L46D mutant
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the Shigella injectisome from mxiK L46D mutant

Fileemd_75999_half_map_2.map
AnnotationHalf map of the Shigella injectisome from mxiK L46D 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 (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-PROPANE

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Electron microscopy

MicroscopeTFS KRIOS
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: 37.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: I3 / Number subtomograms used: 527
ExtractionNumber tomograms: 167 / Number images used: 528 / Software: (Name: I3, IMOD)
CTF correctionType: PHASE FLIPPING ONLY
Final angle assignmentType: OTHER

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