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- EMDB-42161: Atomic structure of Salmonella SipA/F-actin complex by cryo-EM -

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Basic information

Entry
Database: EMDB / ID: EMD-42161
TitleAtomic structure of Salmonella SipA/F-actin complex by cryo-EM
Map dataSipA/F-actin complex
Sample
  • Complex: Salmonella SipA/F-actin complex
    • Complex: Actin, alpha skeletal muscle
    • Complex: SipA
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Cell invasion protein SipA
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE ION
  • Ligand: water
Keywordsactin / Salmonella / type III secretion system / SipA / CELL INVASION
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly ...cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / actin binding / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / extracellular region / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Salmonella invasion protein A, C-terminal actin-binding domain superfamily / 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 ...Salmonella invasion protein A, C-terminal actin-binding domain superfamily / 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
Cell invasion protein SipA / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsNiedzialkowska E / Runyan L / Kudryashova E / Kudryashov DS / Egelman EH
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM122510 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM114666 United States
CitationJournal: Structure / Year: 2024
Title: Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin.
Authors: Ewa Niedzialkowska / Lucas A Runyan / Elena Kudryashova / Edward H Egelman / Dmitri S Kudryashov /
Abstract: Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM ...Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to P, BeF, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.
History
DepositionOct 1, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42161.png

Downloads & links

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Map

FileDownload / File: emd_42161.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSipA/F-actin complex
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.81386405 - 1.4157671
Average (Standard dev.)0.000575365 (±0.034304623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 414.72003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_42161_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_42161_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 : Salmonella SipA/F-actin complex

EntireName: Salmonella SipA/F-actin complex
Components
  • Complex: Salmonella SipA/F-actin complex
    • Complex: Actin, alpha skeletal muscle
    • Complex: SipA
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Cell invasion protein SipA
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE ION
  • Ligand: water

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Supramolecule #1: Salmonella SipA/F-actin complex

SupramoleculeName: Salmonella SipA/F-actin complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Supramolecule #2: Actin, alpha skeletal muscle

SupramoleculeName: Actin, alpha skeletal muscle / type: complex / ID: 2 / Parent: 1

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Supramolecule #3: SipA

SupramoleculeName: SipA / type: complex / ID: 3 / Parent: 1

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 41.875633 KDa
SequenceString: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG ...String:
DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF

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 enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightTheoretical: 28.413857 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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: 7 / 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: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 5 / Number of copies: 7 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8 / Component - Concentration: 25.0 mM / Component - Formula: TRIS-HCl / Component - Name: TRIS
Details: Buffer composition: 25 mM TRIS-H-Cl pH 8.0 2 mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 291 K / Instrument: LEICA EM GP
Details: 3 uL of sample was applied on Lacey grid, then sample was blotted for 3 seconds and plunge-froze in liquid ethane.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 12452 / Average exposure time: 5.58 sec. / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 112.075 Å
Applied symmetry - Helical parameters - Δ&Phi: 53.656 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.2)
Details: Reconstruction algorithm: helical refinement and non-uniform refinement in cryoSPARC that is similar to IHRSR algorithm
Number images used: 1548993
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8d14


Details: 8D14 model was used for F-actin 1Q5Z model was used for SipA
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 4.0.2) / Details: Maximum Likelihood

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8uee:
Atomic structure of Salmonella SipA/F-actin complex by cryo-EM

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