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- PDB-8qfs: Cryo-EM structure of SidH from Legionella pneumophila -

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Basic information

Entry
Database: PDB / ID: 8qfs
TitleCryo-EM structure of SidH from Legionella pneumophila
Components
  • Elongation factor Tu
  • Protein SidH
  • tRNA
KeywordsRNA BINDING PROTEIN / SidH / Legionella / tRNA-binding / EF-Tu-binding
Function / homology
Function and homology information


guanosine tetraphosphate binding / translation elongation factor activity / GTPase activity / GTP binding / cytosol
Similarity search - Function
Translation elongation factor EFTu/EF1A, C-terminal / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / : / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 ...Translation elongation factor EFTu/EF1A, C-terminal / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / : / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / Elongation factor Tu / SidH
Similarity search - Component
Biological speciesEscherichia coli 'BL21-GoldpLysS AG'
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsSharma, R. / Weis, F. / Bhogaraju, S.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-21-CE11-0013 France
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for the toxicity of Legionella pneumophila effector SidH.
Authors: Rahul Sharma / Michael Adams / Simonne Griffith-Jones / Tobias Sahr / Laura Gomez-Valero / Felix Weis / Michael Hons / Sarah Gharbi / Rayene Berkane / Alexandra Stolz / Carmen Buchrieser / Sagar Bhogaraju /
Abstract: Legionella pneumophila (LP) secretes more than 300 effectors into the host cytosol to facilitate intracellular replication. One of these effectors, SidH, 253 kDa in size with no sequence similarity ...Legionella pneumophila (LP) secretes more than 300 effectors into the host cytosol to facilitate intracellular replication. One of these effectors, SidH, 253 kDa in size with no sequence similarity to proteins of known function is toxic when overexpressed in host cells. SidH is regulated by the LP metaeffector LubX which targets SidH for degradation in a temporal manner during LP infection. The mechanism underlying the toxicity of SidH and its role in LP infection are unknown. Here, we determined the cryo-EM structure of SidH at 2.7 Å revealing a unique alpha helical arrangement with no overall similarity to known protein structures. Surprisingly, purified SidH came bound to a E. coli EF-Tu/t-RNA/GTP ternary complex which could be modeled into the cryo-EM density. Mutation of residues disrupting the SidH-tRNA interface and SidH-EF-Tu interface abolish the toxicity of overexpressed SidH in human cells, a phenotype confirmed in infection of Acanthamoeba castellani. We also present the cryo-EM structure of SidH in complex with a U-box domain containing ubiquitin ligase LubX delineating the mechanism of regulation of SidH. Our data provide the basis for the toxicity of SidH and into its regulation by the metaeffector LubX.
History
DepositionSep 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Elongation factor Tu
B: tRNA
A: Protein SidH
hetero molecules


Theoretical massNumber of molelcules
Total (without water)324,3605
Polymers323,8133
Non-polymers5472
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9630 Å2
ΔGint-60 kcal/mol
Surface area77220 Å2

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Components

#1: Protein Elongation factor Tu


Mass: 43370.492 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: E2QJ06
#2: RNA chain tRNA


Mass: 24620.766 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: Protein Protein SidH


Mass: 255821.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Gene: C3927_14265 / Production host: Legionella pneumophila (bacteria) / References: UniProt: Q6RCQ4
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
Has ligand of interestN

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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: Ternary complex of SidH with E.coli tRNA and EF-Tu / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Legionella pneumophila (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 41.93 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 367741 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 79.09 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005513662
ELECTRON MICROSCOPYf_angle_d0.627718803
ELECTRON MICROSCOPYf_chiral_restr0.03822221
ELECTRON MICROSCOPYf_plane_restr0.00442190
ELECTRON MICROSCOPYf_dihedral_angle_d10.59522320

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