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- PDB-8c4c: F-actin decorated by SipA497-669 -

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Basic information

Entry
Database: PDB / ID: 8c4c
TitleF-actin decorated by SipA497-669
Components
  • Actin, alpha skeletal muscle
  • Cell invasion protein SipA
KeywordsCELL INVASION / Salmonella invasion
Function / homology
Function and homology information


Striated Muscle Contraction / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle fiber development / stress fiber / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / 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, 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 / Actin, alpha skeletal muscle / Cell invasion protein SipA
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Gallus gallus (chicken)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsYuan, B. / Wald, J. / Marlovits, T.C.
Funding support Germany, 1items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)417,82636
Polymers413,45412
Non-polymers4,37224
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 42109.973 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken)
References: UniProt: P68139, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Protein
Cell invasion protein SipA / Effector protein SipA


Mass: 19143.604 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)
Strain: LT2 / Gene: sipA, sspA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8VQB5
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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
1F-actin polymerized by SipA497-669 under low-salt conditionsCOMPLEX#1-#20MULTIPLE SOURCES
2actin binding domain Sipa497-669COMPLEX#21RECOMBINANT
3F-actinActinCOMPLEX#11NATURAL
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22
33
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
32Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)99287LT2
43Gallus gallus (chicken)9031
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 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
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 3 sec. / Electron dose: 70 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: RELION / Version: 3.1 / Category: 3D reconstruction
CTF correctionType: NONE
Helical symmerty
IDImage processing-IDAngular rotation/subunit (°)Axial rise/subunit (Å)Axial symmetry
11-166.3928.14C1
21-166.3928.14C1
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130720 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 63.3 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002329040
ELECTRON MICROSCOPYf_angle_d0.519939384
ELECTRON MICROSCOPYf_chiral_restr0.04074368
ELECTRON MICROSCOPYf_plane_restr0.00315040
ELECTRON MICROSCOPYf_dihedral_angle_d6.39254008

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