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- EMDB-18407: Cryo-EM structure of SidH from Legionella pneumophila in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-18407
TitleCryo-EM structure of SidH from Legionella pneumophila in complex with LubX
Map dataEM Map of Legionella effector SidH bound to metaeffector LubX
Sample
  • Complex: Quaternary complex of SidH with E.coli tRNA, EF-Tu and LubX
    • Protein or peptide: Elongation factor TuEF-Tu
    • RNA: t-RNA
    • Protein or peptide: Protein SidH
    • Protein or peptide: E3 ubiquitin--protein ligase
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
KeywordsSidH / Legionella / tRNA-binding / EF-Tu-binding / RNA BINDING PROTEIN
Function / homology
Function and homology information


guanosine tetraphosphate binding / translation elongation factor activity / ubiquitin-protein transferase activity / protein ubiquitination / GTPase activity / GTP binding / cytoplasm
Similarity search - Function
U-box domain / U-box domain profile. / Modified RING finger domain / U-box domain / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal ...U-box domain / U-box domain profile. / Modified RING finger domain / U-box domain / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / 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 / Zinc finger, RING/FYVE/PHD-type / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
E3 ubiquitin--protein ligase / Elongation factor Tu / SidH
Similarity search - Component
Biological speciesLegionella pneumophila (bacteria) / Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSharma R / Adams M / Bhogaraju S
Funding support France, 1 items
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 7, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18407.png

Downloads & links

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Map

FileDownload / File: emd_18407.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM Map of Legionella effector SidH bound to metaeffector LubX
Voxel sizeX=Y=Z: 0.827 Å
Density
Contour LevelBy AUTHOR: 0.0301
Minimum - Maximum-0.19360532 - 0.4717938
Average (Standard dev.)0.00013525634 (±0.0064209388)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 330.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map 1

Fileemd_18407_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_18407_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Quaternary complex of SidH with E.coli tRNA, EF-Tu and LubX

EntireName: Quaternary complex of SidH with E.coli tRNA, EF-Tu and LubX
Components
  • Complex: Quaternary complex of SidH with E.coli tRNA, EF-Tu and LubX
    • Protein or peptide: Elongation factor TuEF-Tu
    • RNA: t-RNA
    • Protein or peptide: Protein SidH
    • Protein or peptide: E3 ubiquitin--protein ligase
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate

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Supramolecule #1: Quaternary complex of SidH with E.coli tRNA, EF-Tu and LubX

SupramoleculeName: Quaternary complex of SidH with E.coli tRNA, EF-Tu and LubX
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Legionella pneumophila (bacteria)

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Macromolecule #1: Elongation factor Tu

MacromoleculeName: Elongation factor Tu / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 43.370492 KDa
SequenceString: MSKEKFERTK PHVNVGTIGH VDHGKTTLTA AITTVLAKTY GGAARAFDQI DNAPEEKARG ITINTSHVEY DTPTRHYAHV DCPGHADYV KNMITGAAQM DGAILVVAAT DGPMPQTREH ILLGRQVGVP YIIVFLNKCD MVDDEELLEL VEMEVRELLS Q YDFPGDDT ...String:
MSKEKFERTK PHVNVGTIGH VDHGKTTLTA AITTVLAKTY GGAARAFDQI DNAPEEKARG ITINTSHVEY DTPTRHYAHV DCPGHADYV KNMITGAAQM DGAILVVAAT DGPMPQTREH ILLGRQVGVP YIIVFLNKCD MVDDEELLEL VEMEVRELLS Q YDFPGDDT PIVRGSALKA LEGDAEWEAK ILELAGFLDS YIPEPERAID KPFLLPIEDV FSISGRGTVV TGRVERGIIK VG EEVEIVG IKETQKSTCT GVEMFRKLLD EGRAGENVGV LLRGIKREEI ERGQVLAKPG TIKPHTKFES EVYILSKDEG GRH TPFFKG YRPQFYFRTT DVTGTIELPE GVEMVMPGDN IKMVVTLIHP IAMDDGLRFA IREGGRTVGA GVVAKVLS

UniProtKB: Elongation factor Tu

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Macromolecule #3: Protein SidH

MacromoleculeName: Protein SidH / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 255.113719 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSM KRTIETYIIY LKEDLKIADT CKTIKDGLLK SITDKTHFSE ELATYFERDN PNAPFKVNTT DPTQVAVLK KLLNALENAE KSFRAIENID ISRDRYTAMI AKDAVMVSYK AVHEIYAALQ LINHSNSDIQ DIVGPHIQKL L PQMALASK ...String:
MGSSHHHHHH SSGLVPRGSM KRTIETYIIY LKEDLKIADT CKTIKDGLLK SITDKTHFSE ELATYFERDN PNAPFKVNTT DPTQVAVLK KLLNALENAE KSFRAIENID ISRDRYTAMI AKDAVMVSYK AVHEIYAALQ LINHSNSDIQ DIVGPHIQKL L PQMALASK ALGNFAPEHP EESAGAVLAG VVNMLPTEKP TESESLGKLS NLIFELPHYF EELQKLIATG ASGIATKSIT SA EDYQSAM IKKANETKYY FEQLSSKSGL LAIPSYLSIV KRLIAHSTDL VNAGAPLTKQ AYLDAVAKLE DIKHNILPQL ISE LEMVEE SMGLKPGLLT DPALEQMNKY YTQLAEQVDN IAKAAGVLDT VSDYSDSIGG KIVRFLAGDS KKLDVGPKLT PAPD LGVLM DDVFIQKRRS NQESRLNEAR LSSEDKSVLA AANRFFDKIG SYNSIHKAWS KWSLANISQS EKDALIKEYK QFQPH FAAL YPDIDKLVVD ALTQPTGSNI VSRLYSSDYK QLWSSDHFKQ VLSCKDSVLS SIQQSLAQSE FKAKLIEKTM SHSEET AYS MNNKTTNLTT RVQPFEPLKF TLEDDKPVEY YHKRVIAASN QILELERAQK GVAEFFNYIQ KKYPHENPSF DSLDESD KE FLRKAYKTFQ PQLLALKHDD INTRLVSSLT SSKPTDPPLR LTDLVSLKSG INDYLNEKIS DLNQDKTTLL DKEEEARE E QYAKNPLVAK GAELEKQTLF GQMSKLKLSK SVDDFFNKKF QTYLKDNLSP EVWKQLSSNG ETLDFDKIPY LEFHKDSPE VAMYKQLINS MHYMKNGLEK LESLNDYGDP NNIYHRTRFV MTTFNALVMN ICFSKYYVME AGNNPGLKAI VQEGLDLLKP LEGMPLIGD YLKTTEKQEP PKQNIITAWK KQQAVVESGL SKGPKPKTDQ QLISEQLGKI QEAIDNFDGD LEVSDSAREK I KTQIGEFA KGISGLSFGP GSVKKILAAL TKLETQLSNL DKESPEVTLG KLKDIHSELN AQFRAAAEYT EYHSGQKFGS YS NNISTIV SNFCNGLVSN LPLKQEPAPK VKAPEKPVTP VITGTTNPHE VVFGTKHEEF NSVYQPYLLL KRITDEFRDQ NNP YKPSFD ELKEEAVSYY DKIQPLLESV DPKFDKNFIA KHKESSTLLK AIDEVMSMRQ RINNPESSFA KLKDLHLEGD FEKE ENKEK FRQLYKEIQP YLHKIDSTYD QTQFLEGLKT AKDFSGALQR IMNEENALQQ STSLKDTSYL QLIAESLYQI PVKLN KLKA EPDTPEPSKE EIDANVKAFV EGLNGLSFGP GSVKKILSTA AKLQMQLSDI GKEGRELTMG RLKEIQAEFG TILMAA ADN AEFHLGLKPG TYSRTVSERF EKFYSSLIVN LPLEKDQTGL ELLIDTTSTQ KRLAREMERL ESVKEDTSAI DTKKSIF GT EHEQFSTLYQ PYASLRHIAK DIEDGVHMNL YERTLEELKE EASNEYKKIQ PYLAKINPEF TEDYISKTDG EYSLLHAI D RVFEERHKIN KPSSPFDKLR DLYLDGDFEK EENKEQFLQL YAELQPHLIK INYQYDLAYF LRELQTPEDF KAATERIIN DESKLQELIT GLDDTKRLKV KLCEERIGYF IDLLKKQELE VGPEKIQAFK EKIFFNYIHA NVNSALDAKI GSHAEQFLQF IEKDFLDKK NEILEKITID QDMEEEIAKA IDRIAPDIIN NKIESFKKLL FDSYIQSDIK NSLHNELGIY TSLFIDKISP E IHLHQSEI LGNVAFDSKM GAEIGSKINA ITPGILLNNN PLKEAYVDLN NTLKEINTLL DEENKKTRDN PCRNEKIAKL TS LKDRLSD LDSIPKENTV EFLKKMQEET RSSLKSLESN DALINIYDVL NSLKETIENG SDLPEIKKDK LQMISDVQNI LSN FDKNPA ERLNLAVQIL NDSNPEVLSK TKGNFLIGEA FKGQVFTNYI NTKISEQLNN ELGPYGKVFL KQIMPDFIAK KSEI IKEIA IDNMETGLET QFKIHAPAIF EKNKELKAAY EQLNVHLKEV QSLIEAEEKK PKGNPCREEK IAALRSHQSQ LMNTQ RIPD HETLRFLQEQ NKSAKSFMGK LEKYDTMISV YDSLTEIREH VSNHKSLSKE IKDEKIQEIS KMEDMLKTTS KEPSIR LAE VKAHGLSDQC KNVLLKNSDN FLVSFFKTLF SKLFNIKNEN ETLVSSFKQR LQNIKGPEPV ATPMETPENE APLVNAN IT RF

UniProtKB: SidH

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Macromolecule #4: E3 ubiquitin--protein ligase

MacromoleculeName: E3 ubiquitin--protein ligase / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 30.50683 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHSAG LEVLFQGPMG YRIEMATRNP FDIDHKSKYL REAALEANLS HPETTPTMLT CPIDSGFLKD PVITPEGFVY NKSSILKWL ETKKEDPQSR KPLTAKDLQP FPELLIIVNR FVETQTNYEK LKNRLVQNAR VAARQKEYTE IPDIFLCPIS K TLIKTPVI ...String:
MHHHHHHSAG LEVLFQGPMG YRIEMATRNP FDIDHKSKYL REAALEANLS HPETTPTMLT CPIDSGFLKD PVITPEGFVY NKSSILKWL ETKKEDPQSR KPLTAKDLQP FPELLIIVNR FVETQTNYEK LKNRLVQNAR VAARQKEYTE IPDIFLCPIS K TLIKTPVI TAQGKVYDQE ALSNFLIATG NKDETGKKLS IDDVVVFDEL YQQIKVYNFY RKREMQKNQI QPSVSSGFGF FS LNFLTSW LWGTEEKKEK TSSDMTY

UniProtKB: E3 ubiquitin--protein ligase

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Macromolecule #2: t-RNA

MacromoleculeName: t-RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 24.620766 KDa
SequenceString:
GCCCGGA(4SU)AG CUCAG(H2U)CGG(H2U) AGAGCAGGGG AUUGAAAAUC CCCGUG(3AU)CCU UGG(5MU)(PSU)C GAU UCCGAGUCCG GGCACCA

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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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

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 43.37 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 255829
FSC plot (resolution estimation)

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