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- PDB-8uxt: Acinetobacter baumannii Tse15 Rhs effector, toxin cleavage mutant... -

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Basic information

Entry
Database: PDB / ID: 8uxt
TitleAcinetobacter baumannii Tse15 Rhs effector, toxin cleavage mutant (D1369N, D1391N)
ComponentsTse15
KeywordsTOXIN / Rhs-effector / Rhs cargo effector / Type 6 secretion system effector / Acinetobacter baumannii toxin / YD-repeat protein / T6SS.
Function / homology
Function and homology information


Hydrolases; Acting on ester bonds / hydrolase activity
Similarity search - Function
RHS protein / RHS protein / RHS repeat / RHS Repeat / YD repeat / Rhs repeat-associated core / :
Similarity search - Domain/homology
Biological speciesAcinetobacter baumannii AB307-0294 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.77 Å
AuthorsHayes, B.K. / Venugopal, H. / McGowan, S.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1165036 Australia
National Health and Medical Research Council (NHMRC, Australia)1128981 Australia
CitationJournal: Nat Commun / Year: 2024
Title: Structure of a Rhs effector clade domain provides mechanistic insights into type VI secretion system toxin delivery.
Authors: Brooke K Hayes / Marina Harper / Hariprasad Venugopal / Jessica M Lewis / Amy Wright / Han-Chung Lee / Joel R Steele / David L Steer / Ralf B Schittenhelm / John D Boyce / Sheena McGowan /
Abstract: The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins into adjacent cells. Here we present the structure of Tse15, a T6SS ...The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins into adjacent cells. Here we present the structure of Tse15, a T6SS Rhs effector from the nosocomial pathogen Acinetobacter baumannii. Tse15 forms a triple layered β-cocoon Rhs domain with an N-terminal α-helical clade domain and an unfolded C-terminal toxin domain inside the Rhs cage. Tse15 is cleaved into three domains, through independent auto-cleavage events involving aspartyl protease activity for toxin self-cleavage and a nucleophilic glutamic acid for N-terminal clade cleavage. Proteomic analyses identified that significantly more peptides from the N-terminal clade and toxin domains were secreted than from the Rhs cage, suggesting toxin delivery often occurs without the cage. We propose the clade domain acts as an internal chaperone to mediate toxin tethering to the T6SS machinery. Conservation of the clade domain in other Gram-negative bacteria suggests this may be a common mechanism for delivery.
History
DepositionNov 10, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tse15


Theoretical massNumber of molelcules
Total (without water)181,5451
Polymers181,5451
Non-polymers00
Water00
1


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

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Components

#1: Protein Tse15


Mass: 181545.062 Da / Num. of mol.: 1 / Mutation: D1369N,D1391N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baumannii AB307-0294 (bacteria)
Gene: ABBFA_02439 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): Rosetta 2 / References: UniProt: A0A5K6CSR3
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Recombinant Tse15(D1369N, D1391N) protein / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.183 MDa / Experimental value: NO
Source (natural)Organism: Acinetobacter baumannii AB307-0294 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8 / Details: 50 mM Hepes pH 8.0; 300 mM NaCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELD / Nominal magnification: 165000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 1.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1163105 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211506
ELECTRON MICROSCOPYf_angle_d0.46315587
ELECTRON MICROSCOPYf_dihedral_angle_d4.081554
ELECTRON MICROSCOPYf_chiral_restr0.0391660
ELECTRON MICROSCOPYf_plane_restr0.0032053

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