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- EMDB-16425: F-actin decorated by SipA426-685 -

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Basic information

Entry
Database: EMDB / ID: EMD-16425
TitleF-actin decorated by SipA426-685
Map data
Sample
  • Complex: F-actin polymerized by SipA497-669 under low-salt conditions
    • Complex: actin binding domain SipA-C
      • Protein or peptide: Cell invasion protein SipA
    • Complex: F-actin
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE ION
  • Ligand: MAGNESIUM ION
KeywordsSalmonella invasion / CELL INVASION
Function / homology
Function and homology information


Striated Muscle Contraction / striated muscle thin filament / skeletal muscle thin filament assembly / stress fiber / skeletal muscle fiber development / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / actin cytoskeleton / actin binding / hydrolase activity ...Striated Muscle Contraction / striated muscle thin filament / skeletal muscle thin filament assembly / stress fiber / skeletal muscle fiber development / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / actin cytoskeleton / actin binding / hydrolase activity / extracellular region / ATP binding
Similarity search - Function
Salmonella invasion protein A, C-terminal actin-binding domain superfamily / : / Salmonella Invasion protein A, C-terminal actin binding domain / Salmonella invasion protein A, N-terminal / Salmonella invasion protein A, chaperone-binding / SipA N-terminal domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site ...Salmonella invasion protein A, C-terminal actin-binding domain superfamily / : / Salmonella Invasion protein A, C-terminal actin binding domain / Salmonella invasion protein A, N-terminal / Salmonella invasion protein A, chaperone-binding / SipA N-terminal domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Actin, alpha skeletal muscle / Cell invasion protein SipA
Similarity search - Component
Biological speciesSalmonella (bacteria) / Gallus gallus (chicken)
Methodhelical reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsYuan B / Wald J / Marlovits TC
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)numbers INST152/772-1, 152/774-1, 152/775-1, 152/776-1 and 152/777-1 FUGG. Germany
CitationJournal: Sci Adv / Year: 2023
Title: Structural basis for subversion of host cell actin cytoskeleton during infection.
Authors: Biao Yuan / Jonas Scholz / Jiri Wald / Roland Thuenauer / Rory Hennell James / Irina Ellenberg / Sabine Windhorst / Jan Faix / Thomas C Marlovits /
Abstract: Secreted bacterial type III secretion system (T3SS) proteins are essential for successful infection by many human pathogens. Both T3SS translocator SipC and effector SipA are critical for infection ...Secreted bacterial type III secretion system (T3SS) proteins are essential for successful infection by many human pathogens. Both T3SS translocator SipC and effector SipA are critical for infection by subversion of the host cell cytoskeleton, but the precise molecular interplay between them remains unknown. Here, using cryo-electron microscopy, we show that SipA binds along the F-actin grooves with a unique binding pattern. SipA stabilizes F-actin through charged interface residues and appears to prevent inorganic phosphate release through closure of the "back door" of adenosine 5'-triphosphate pocket. We also show that SipC enhances the binding of SipA to F-actin, thus demonstrating that a sequential presence of T3SS proteins in host cells is associated with a sequence of infection events-starting with actin nucleation, filament growth, and stabilization. Together, our data explain the coordinated interplay of a precisely tuned and highly effective mechanism during infection and provide a blueprint for interfering with effectors acting on actin.
History
DepositionJan 3, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16425.png

Downloads & links

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Map

FileDownload / File: emd_16425.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 512 pix.
= 422.912 Å		0.83 Å/pix.
x 512 pix.
= 422.912 Å		0.83 Å/pix.
x 512 pix.
= 422.912 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.004
Minimum - Maximum-0.036531385 - 0.105752826
Average (Standard dev.)0.0000937643 (±0.0014555953)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 422.912 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_16425_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16425_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : F-actin polymerized by SipA497-669 under low-salt conditions

EntireName: F-actin polymerized by SipA497-669 under low-salt conditions
Components
  • Complex: F-actin polymerized by SipA497-669 under low-salt conditions
    • Complex: actin binding domain SipA-C
      • Protein or peptide: Cell invasion protein SipA
    • Complex: F-actin
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: F-actin polymerized by SipA497-669 under low-salt conditions

SupramoleculeName: F-actin polymerized by SipA497-669 under low-salt conditions
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: actin binding domain SipA-C

SupramoleculeName: actin binding domain SipA-C / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2 / Details: SipA426-685
Source (natural)Organism: Salmonella (bacteria) / Strain: LT2

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Supramolecule #3: F-actin

SupramoleculeName: F-actin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 42.109973 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSS S LEKSYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQ KEITALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: Cell invasion protein SipA

MacromoleculeName: Cell invasion protein SipA / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Salmonella (bacteria)
Molecular weightTheoretical: 28.413857 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: TGETTSFDEV DGVTSKSIIG KPVQATVHGV DDNKQQSQTA EIVNVKPLAS QLAGVENVKT DTLQSDTTVI TGNKAGTTDN DNSQTDKTG PFSGLKFKQN SFLSTVPSVT NMHSMHFDAR ETFLGVIRKA LEPDTSTPFP VRRAFDGLRA EILPNDTIKS A ALKAQCSD ...String:
TGETTSFDEV DGVTSKSIIG KPVQATVHGV DDNKQQSQTA EIVNVKPLAS QLAGVENVKT DTLQSDTTVI TGNKAGTTDN DNSQTDKTG PFSGLKFKQN SFLSTVPSVT NMHSMHFDAR ETFLGVIRKA LEPDTSTPFP VRRAFDGLRA EILPNDTIKS A ALKAQCSD IDKHPELKAK METLKEVITH HPQKEKLAEI ALQFAREAGL TRLKGETDYV LSNVLDGLIG DGSWRAGPAY ES YLNKPGV DRVITTVDGL HMQR

UniProtKB: Cell invasion protein SipA

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 8 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 4 / Number of copies: 8 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8
Details: 5 mM Tris buffer, pH 7.5, 0.1 mM DTT, 0.2 mM ATP, 0.2 mM EGTA and 0.05 mM MgCl2
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 3.0 sec. / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 28.15 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.48 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 120864
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-8c4e:
F-actin decorated by SipA426-685

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