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- EMDB-26381: Cryo-EM structure of Shiga toxin 2 in complex with the native rib... -

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Basic information

Entry
Database: EMDB / ID: EMD-26381
TitleCryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk
Map data
Sample
  • Complex: Shiga toxin 2a in complex with the native ribosomal P-stalk
    • Complex: Shiga toxin 2a
      • Protein or peptide: Shiga toxin 2a subunit A (Stx2A)
      • Protein or peptide: Shiga toxin 2a subunit B (Stx2B)
    • Complex: Native ribosomal P-stalk
      • Protein or peptide: C-terminal domain (CTD) from the Ribosomal P-stalk
  • Ligand: water
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) / Baker's yeast (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsKulczyk AW
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26381.png

Downloads & links

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Map

FileDownload / File: emd_26381.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8248 Å
Density
Contour LevelBy AUTHOR: 0.006
Minimum - Maximum-0.017936839 - 0.053724695
Average (Standard dev.)6.0928298e-05 (±0.0009717562)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_26381_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26381_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Shiga toxin 2a in complex with the native ribosomal P-stalk

EntireName: Shiga toxin 2a in complex with the native ribosomal P-stalk
Components
  • Complex: Shiga toxin 2a in complex with the native ribosomal P-stalk
    • Complex: Shiga toxin 2a
      • Protein or peptide: Shiga toxin 2a subunit A (Stx2A)
      • Protein or peptide: Shiga toxin 2a subunit B (Stx2B)
    • Complex: Native ribosomal P-stalk
      • Protein or peptide: C-terminal domain (CTD) from the Ribosomal P-stalk
  • Ligand: water

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Supramolecule #1: Shiga toxin 2a in complex with the native ribosomal P-stalk

SupramoleculeName: Shiga toxin 2a in complex with the native ribosomal P-stalk
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 100 KDa

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Supramolecule #2: Shiga toxin 2a

SupramoleculeName: Shiga toxin 2a / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1-#2 / Details: 72 KDa Stx2a holotoxin
Source (natural)Organism: Shigella dysenteriae (bacteria)

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Supramolecule #3: Native ribosomal P-stalk

SupramoleculeName: Native ribosomal P-stalk / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #3 / Details: 56 kDa native ribosomal P-stalk
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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

MacromoleculeName: Shiga toxin 2a subunit A (Stx2A) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: rRNA N-glycosylase
Source (natural)Organism: Shigella dysenteriae (bacteria)
Molecular weightTheoretical: 33.214188 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: REFTIDFSTQ QSYVSSLNSI RTEISTPLEH ISQGTTSVSV INHTPPGSYF AVDIRGLDVY QARFDHLRLI IEQNNLYVAG FVNTATNTF YRFSDFTHIS VPGVTTVSMT TDSSYTTLQR VAALERSGMQ ISRHSLVSSY LALMEFSGNT MTRDASRAVL R FVTVTAEA ...String:
REFTIDFSTQ QSYVSSLNSI RTEISTPLEH ISQGTTSVSV INHTPPGSYF AVDIRGLDVY QARFDHLRLI IEQNNLYVAG FVNTATNTF YRFSDFTHIS VPGVTTVSMT TDSSYTTLQR VAALERSGMQ ISRHSLVSSY LALMEFSGNT MTRDASRAVL R FVTVTAEA LRFRQIQREF RQALSETAPV YTMTPGDVDL TLNWGRISNV LPEYRGEDGV RVGRISFNNI SAILGTVAVI LN CHHQGAR SVRAVNEDSQ PECQITGDRP VIKINNTLWE SNTAAAFLNR KSQFLYTTGK

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Macromolecule #2: Shiga toxin 2a subunit B (Stx2B)

MacromoleculeName: Shiga toxin 2a subunit B (Stx2B) / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Shigella dysenteriae (bacteria)
Molecular weightTheoretical: 7.82459 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ADCAKGKIEF SKYNEDDTFT VKVDGKEYWT SRWNLQPLLQ SAQLTGMTVT IKSSTCESGS GFAEVQFNND

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Macromolecule #3: C-terminal domain (CTD) from the Ribosomal P-stalk

MacromoleculeName: C-terminal domain (CTD) from the Ribosomal P-stalk / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast)
Molecular weightTheoretical: 654.712 Da
SequenceString:
GFGLFD

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 23 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 112924
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-7u6v:
Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk

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