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- PDB-7u6v: Cryo-EM structure of Shiga toxin 2 in complex with the native rib... -

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Basic information

Entry
Database: PDB / ID: 7u6v
TitleCryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk
Components
  • C-terminal domain (CTD) from the Ribosomal P-stalk
  • Shiga toxin 2a subunit A (Stx2A)
  • Shiga toxin 2a subunit B (Stx2B)
KeywordsTOXIN / Shiga toxin 2 / Ribosomal P-stalk / Complex
Function / homology
Function and homology information


rRNA N-glycosylase / rRNA N-glycosylase activity / toxin activity / negative regulation of translation
Similarity search - Function
Shiga-like toxin, subunit A / Ribosome-inactivating protein conserved site / Shiga/ricin ribosomal inactivating toxins active site signature. / Ribosome-inactivating protein / Ribosome-inactivating protein, subdomain 1 / Ribosome-inactivating protein, subdomain 2 / Ribosome-inactivating protein superfamily / Ribosome inactivating protein
Similarity search - Domain/homology
Biological speciesShigella dysenteriae (bacteria)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsKulczyk, A.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI141635 United States
CitationJournal: J Biol Chem / Year: 2023
Title: Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction.
Authors: Arkadiusz W Kulczyk / Carlos Oscar S Sorzano / Przemysław Grela / Marek Tchórzewski / Nilgun E Tumer / Xiao-Ping Li /
Abstract: Shiga toxin 2a (Stx2a) is the virulence factor of enterohemorrhagic Escherichia coli. The catalytic A1 subunit of Stx2a (Stx2A1) interacts with the ribosomal P-stalk for loading onto the ribosome and ...Shiga toxin 2a (Stx2a) is the virulence factor of enterohemorrhagic Escherichia coli. The catalytic A1 subunit of Stx2a (Stx2A1) interacts with the ribosomal P-stalk for loading onto the ribosome and depurination of the sarcin-ricin loop, which halts protein synthesis. Because of the intrinsic flexibility of the P-stalk, a structure of the Stx2a-P-stalk complex is currently unknown. We demonstrated that the native P-stalk pentamer binds to Stx2a with nanomolar affinity, and we employed cryo-EM to determine a structure of the 72 kDa Stx2a complexed with the P-stalk. The structure identifies Stx2A1 residues involved in binding and reveals that Stx2a is anchored to the P-stalk via only the last six amino acids from the C-terminal domain of a single P-protein. For the first time, the cryo-EM structure shows the loop connecting Stx2A1 and Stx2A2, which is critical for activation of the toxin. Our principal component analysis of the cryo-EM data reveals the intrinsic dynamics of the Stx2a-P-stalk interaction, including conformational changes in the P-stalk binding site occurring upon complex formation. Our computational analysis unveils the propensity for structural rearrangements within the C-terminal domain, with its C-terminal six amino acids transitioning from a random coil to an α-helix upon binding to Stx2a. In conclusion, our cryo-EM structure sheds new light into the dynamics of the Stx2a-P-stalk interaction and indicates that the binding interface between Stx2a and the P-stalk is the potential target for drug discovery.
History
DepositionMar 6, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Shiga toxin 2a subunit A (Stx2A)
B: Shiga toxin 2a subunit B (Stx2B)
C: Shiga toxin 2a subunit B (Stx2B)
D: Shiga toxin 2a subunit B (Stx2B)
E: Shiga toxin 2a subunit B (Stx2B)
F: Shiga toxin 2a subunit B (Stx2B)
P: C-terminal domain (CTD) from the Ribosomal P-stalk


Theoretical massNumber of molelcules
Total (without water)72,9927
Polymers72,9927
Non-polymers00
Water41423
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Shiga toxin 2a subunit A (Stx2A)


Mass: 33214.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella dysenteriae (bacteria) / Gene: stxA2 / Production host: Escherichia coli (E. coli) / References: UniProt: G8GWP6, rRNA N-glycosylase
#2: Protein
Shiga toxin 2a subunit B (Stx2B)


Mass: 7824.590 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella dysenteriae (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein/peptide C-terminal domain (CTD) from the Ribosomal P-stalk


Mass: 654.712 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 23 / Source method: isolated from a natural source / Formula: H2O

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Shiga toxin 2a in complex with the native ribosomal P-stalkCOMPLEX#1-#30MULTIPLE SOURCES
2Shiga toxin 2aCOMPLEX#1-#21RECOMBINANT72 KDa Stx2a holotoxin
3Native ribosomal P-stalkCOMPLEX#31NATURAL56 kDa native ribosomal P-stalk
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Shigella dysenteriae (bacteria)622
23Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 1.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
9PHENIX1.18model refinement
10cryoSPARC3initial Euler assignment
11RELION3final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 112924 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025173
ELECTRON MICROSCOPYf_angle_d0.4577012
ELECTRON MICROSCOPYf_dihedral_angle_d4.225695
ELECTRON MICROSCOPYf_chiral_restr0.041787
ELECTRON MICROSCOPYf_plane_restr0.002908

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