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

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Basic information

Entry
Database: PDB / ID: 8uee
TitleAtomic structure of Salmonella SipA/F-actin complex by cryo-EM
Components
  • Actin, alpha skeletal muscle
  • Cell invasion protein SipA
KeywordsCELL INVASION / actin / Salmonella / type III secretion system / SipA
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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Cell invasion protein SipA / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsNiedzialkowska, E. / Runyan, L. / Kudryashova, E. / Kudryashov, D.S. / Egelman, E.H.
Funding support United States, 2items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
F: Actin, alpha skeletal muscle
H: Actin, alpha skeletal muscle
I: Actin, alpha skeletal muscle
J: Actin, alpha skeletal muscle
K: Actin, alpha skeletal muscle
L: Actin, alpha skeletal muscle
M: Actin, alpha skeletal muscle
B: Cell invasion protein SipA
C: Cell invasion protein SipA
D: Cell invasion protein SipA
A: Cell invasion protein SipA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)410,61032
Polymers406,78511
Non-polymers3,82521
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 11 molecules FHIJKLMBCDA

#1: Protein
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41875.633 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Protein
Cell invasion protein SipA / Effector protein SipA


Mass: 28413.857 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Gene: sipA, sspA, STM2882 / Production host: Escherichia coli (E. coli) / References: UniProt: P0CL52

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Non-polymers , 4 types, 22 molecules

#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Salmonella SipA/F-actin complexCOMPLEX#1-#20MULTIPLE SOURCES
2Actin, alpha skeletal muscleCOMPLEX1NATURAL
3SipACOMPLEX1RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21
33
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Oryctolagus cuniculus (rabbit)9986
31Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)99287
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: Buffer composition: 25 mM TRIS-H-Cl pH 8.0 2 mM DTT
Buffer componentConc.: 25 mM / Name: TRIS / Formula: TRIS-HCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 291 K
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 5.58 sec. / Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12452

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.0.2CTF correction
7UCSF ChimeraX1.3model fitting
8Coot0.9.8.1model fitting
10PHENIX1.3model refinement
11Coot0.9.8.1model refinement
12cryoSPARC4.0.2initial Euler assignment
13cryoSPARC4.0.2final Euler assignment
14cryoSPARC4.0.2classification
15cryoSPARC4.0.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 53.656 ° / Axial rise/subunit: 112.075 Å / Axial symmetry: C1
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1548993
Details: Reconstruction algorithm: helical refinement and non-uniform refinement in cryoSPARC that is similar to IHRSR algorithm
Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00426476
ELECTRON MICROSCOPYf_angle_d0.60435905
ELECTRON MICROSCOPYf_dihedral_angle_d9.9793659
ELECTRON MICROSCOPYf_chiral_restr0.0463993
ELECTRON MICROSCOPYf_plane_restr0.0044597

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