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- PDB-6suq: Crystal Structure of TcdB2-TccC3-TEV -

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Basic information

Entry
Database: PDB / ID: 6suq
TitleCrystal Structure of TcdB2-TccC3-TEV
ComponentsTcdB2,TccC3,Genome polyprotein
KeywordsTOXIN / Toxins / Tc Toxins
Function / homology
Function and homology information


nuclear-inclusion-a endopeptidase / helper-component proteinase / hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides / host cell cytoplasmic vesicle / Hydrolases; Acting on peptide bonds (peptidases) / helical viral capsid / serine-type peptidase activity / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / RNA-directed RNA polymerase ...nuclear-inclusion-a endopeptidase / helper-component proteinase / hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides / host cell cytoplasmic vesicle / Hydrolases; Acting on peptide bonds (peptidases) / helical viral capsid / serine-type peptidase activity / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / DNA-templated transcription / host cell nucleus / structural molecule activity / proteolysis / RNA binding / extracellular region / ATP binding / cytoplasm
Similarity search - Function
Helper component proteinase / Peptidase S30, polyprotein P1, potyvirus / Polyprotein, Potyviridae / Helper-component proteinase (HC-Pro) cysteine protease (CPD) domain / Potyviral polyprotein protein 3 / Helper-component proteinase (HC-Pro) cysteine protease (CPD) domain superfamily / Helper component proteinase / Potyvirus P1 protease / Potyviridae polyprotein / Protein P3 of Potyviral polyprotein ...Helper component proteinase / Peptidase S30, polyprotein P1, potyvirus / Polyprotein, Potyviridae / Helper-component proteinase (HC-Pro) cysteine protease (CPD) domain / Potyviral polyprotein protein 3 / Helper-component proteinase (HC-Pro) cysteine protease (CPD) domain superfamily / Helper component proteinase / Potyvirus P1 protease / Potyviridae polyprotein / Protein P3 of Potyviral polyprotein / Helper-component proteinase (HC-Pro) cysteine protease (CPD) domain profile. / Potyviridae P1 protease domain profile. / Potyvirus NIa protease (NIa-pro) domain / Peptidase family C4 / Potyvirus NIa protease (NIa-pro) domain profile. / Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / Potyvirus coat protein / Potyvirus coat protein / Rhs repeat-associated core / Integrin alpha, N-terminal / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Genome polyprotein / TccC3 / TcdB2
Similarity search - Component
Biological speciesPhotorhabdus luminescens (bacteria)
Tobacco etch virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.7 Å
AuthorsRoderer, D. / Schubert, E. / Sitsel, O. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
European Research Council615984 Germany
CitationJournal: Nat Commun / Year: 2019
Title: Towards the application of Tc toxins as a universal protein translocation system.
Authors: Daniel Roderer / Evelyn Schubert / Oleg Sitsel / Stefan Raunser /
Abstract: Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that ...Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system.
History
DepositionSep 16, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TcdB2,TccC3,Genome polyprotein


Theoretical massNumber of molelcules
Total (without water)272,9661
Polymers272,9661
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area91050 Å2
MethodPISA
Unit cell
Length a, b, c (Å)234.430, 234.430, 143.190
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein TcdB2,TccC3,Genome polyprotein


Mass: 272965.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus luminescens (bacteria), (gene. exp.) Tobacco etch virus
Gene: tcdB2, TccC3 / Production host: Escherichia coli (E. coli)
References: UniProt: Q8GF99, UniProt: Q8GF97, UniProt: P04517, Hydrolases; Acting on peptide bonds (peptidases), helper-component proteinase, Hydrolases; Acting on acid anhydrides; Acting on acid ...References: UniProt: Q8GF99, UniProt: Q8GF97, UniProt: P04517, Hydrolases; Acting on peptide bonds (peptidases), helper-component proteinase, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement, nuclear-inclusion-a endopeptidase, RNA-directed RNA polymerase

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.71 Å3/Da / Density % sol: 73.88 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: 0.1 M sodium chloride, 0.1 M magnesium chloride, 0.1 M tri-sodium citrate pH 5.5, 12 % PEG 4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 0.99985 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Aug 18, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.99985 Å / Relative weight: 1
ReflectionResolution: 3.7→47.73 Å / Num. obs: 48613 / % possible obs: 100 % / Redundancy: 20 % / CC1/2: 0.972 / Net I/σ(I): 5.26
Reflection shellResolution: 3.7→3.832 Å / Mean I/σ(I) obs: 2.14 / Num. unique obs: 4808 / CC1/2: 0.707

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Processing

Software
NameVersionClassification
PHENIX1.16-3549refinement
XDSVERSION Mar 15, 2019 BUILT=20190315data reduction
XSCALEVERSION Mar 15, 2019 BUILT=20190315data scaling
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4O9X

4o9x


Resolution: 3.7→47.73 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflection
Rfree0.302 --
Rwork0.269 --
obs-48591 99.89 %
Refinement stepCycle: LAST / Resolution: 3.7→47.73 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17025 0 0 0 17025

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