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- EMDB-18383: Cryo-EM structure of SidH from Legionella pneumophila -

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Basic information

Entry
Database: EMDB / ID: EMD-18383
TitleCryo-EM structure of SidH from Legionella pneumophila
Map dataLocal resolution filtered EM map of SidH from Legionella pneumophila
Sample
  • Complex: Ternary complex of SidH with E.coli tRNA and EF-Tu
    • Protein or peptide: Elongation factor TuEF-Tu
    • RNA: tRNATransfer RNA
    • Protein or peptide: Protein SidH
  • 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 / GTPase activity / GTP binding / cytoplasm
Similarity search - Function
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 ...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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
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: 2.7 Å
AuthorsSharma R / Weis F / 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 4, 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_18383.png

Downloads & links

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Map

FileDownload / File: emd_18383.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal resolution filtered EM map of SidH from Legionella pneumophila
Voxel sizeX=Y=Z: 0.8127 Å
Density
Contour LevelBy AUTHOR: 0.0459
Minimum - Maximum-0.19696686 - 0.60937387
Average (Standard dev.)0.00016639363 (±0.0056591476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 390.09598 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: SidH map after focussed classification on DUF domain

Fileemd_18383_additional_1.map
AnnotationSidH map after focussed classification on DUF domain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap 1

Fileemd_18383_half_map_1.map
Annotationhalfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap2

Fileemd_18383_half_map_2.map
Annotationhalfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of SidH with E.coli tRNA and EF-Tu

EntireName: Ternary complex of SidH with E.coli tRNA and EF-Tu
Components
  • Complex: Ternary complex of SidH with E.coli tRNA and EF-Tu
    • Protein or peptide: Elongation factor TuEF-Tu
    • RNA: tRNATransfer RNA
    • Protein or peptide: Protein SidH
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate

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

SupramoleculeName: Ternary complex of SidH with E.coli tRNA and EF-Tu / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
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: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 255.821562 KDa
Recombinant expressionOrganism: Legionella pneumophila (bacteria)
SequenceString: MKHHHHHHHH HHSAGLEVLF QGPMKRTIET YIIYLKEDLK IADTCKTIKD GLLKSITDKT HFSEELATYF ERDNPNAPFK VNTTDPTQV AVLKKLLNAL ENAEKSFRAI ENIDISRDRY TAMIAKDAVM VSYKAVHEIY AALQLINHSN SDIQDIVGPH I QKLLPQMA ...String:
MKHHHHHHHH HHSAGLEVLF QGPMKRTIET YIIYLKEDLK IADTCKTIKD GLLKSITDKT HFSEELATYF ERDNPNAPFK VNTTDPTQV AVLKKLLNAL ENAEKSFRAI ENIDISRDRY TAMIAKDAVM VSYKAVHEIY AALQLINHSN SDIQDIVGPH I QKLLPQMA LASKALGNFA PEHPEESAGA VLAGVVNMLP TEKPTESESL GKLSNLIFEL PHYFEELQKL IATGASGIAT KS ITSAEDY QSAMIKKANE TKYYFEQLSS KSGLLAIPSY LSIVKRLIAH STDLVNAGAP LTKQAYLDAV AKLEDIKHNI LPQ LISELE MVEESMGLKP GLLTDPALEQ MNKYYTQLAE QVDNIAKAAG VLDTVSDYSD SIGGKIVRFL AGDSKKLDVG PKLT PAPDL GVLMDDVFIQ KRRSNQESRL NEARLSSEDK SVLAAANRFF DKIGSYNSIH KAWSKWSLAN ISQSEKDALI KEYKQ FQPH FAALYPDIDK LVVDALTQPT GSNIVSRLYS SDYKQLWSSD HFKQVLSCKD SVLSSIQQSL AQSEFKAKLI EKTMSH SEE TAYSMNNKTT NLTTRVQPFE PLKFTLEDDK PVEYYHKRVI AASNQILELE RAQKGVAEFF NYIQKKYPHE NPSFDSL DE SDKEFLRKAY KTFQPQLLAL KHDDINTRLV SSLTSSKPTD PPLRLTDLVS LKSGINDYLN EKISDLNQDK TTLLDKEE E AREEQYAKNP LVAKGAELEK QTLFGQMSKL KLSKSVDDFF NKKFQTYLKD NLSPEVWKQL SSNGETLDFD KIPYLEFHK DSPEVAMYKQ LINSMHYMKN GLEKLESLND YGDPNNIYHR TRFVMTTFNA LVMNICFSKY YVMEAGNNPG LKAIVQEGLD LLKPLEGMP LIGDYLKTTE KQEPPKQNII TAWKKQQAVV ESGLSKGPKP KTDQQLISEQ LGKIQEAIDN FDGDLEVSDS A REKIKTQI GEFAKGISGL SFGPGSVKKI LAALTKLETQ LSNLDKESPE VTLGKLKDIH SELNAQFRAA AEYTEYHSGQ KF GSYSNNI STIVSNFCNG LVSNLPLKQE PAPKVKAPEK PVTPVITGTT NPHEVVFGTK HEEFNSVYQP YLLLKRITDE FRD QNNPYK PSFDELKEEA VSYYDKIQPL LESVDPKFDK NFIAKHKESS TLLKAIDEVM SMRQRINNPE SSFAKLKDLH LEGD FEKEE NKEKFRQLYK EIQPYLHKID STYDQTQFLE GLKTAKDFSG ALQRIMNEEN ALQQSTSLKD TSYLQLIAES LYQIP VKLN KLKAEPDTPE PSKEEIDANV KAFVEGLNGL SFGPGSVKKI LSTAAKLQMQ LSDIGKEGRE LTMGRLKEIQ AEFGTI LMA AADNAEFHLG LKPGTYSRTV SERFEKFYSS LIVNLPLEKD QTGLELLIDT TSTQKRLARE MERLESVKED TSAIDTK KS IFGTEHEQFS TLYQPYASLR HIAKDIEDGV HMNLYERTLE ELKEEASNEY KKIQPYLAKI NPEFTEDYIS KTDGEYSL L HAIDRVFEER HKINKPSSPF DKLRDLYLDG DFEKEENKEQ FLQLYAELQP HLIKINYQYD LAYFLRELQT PEDFKAATE RIINDESKLQ ELITGLDDTK RLKVKLCEER IGYFIDLLKK QELEVGPEKI QAFKEKIFFN YIHANVNSAL DAKIGSHAEQ FLQFIEKDF LDKKNEILEK ITIDQDMEEE IAKAIDRIAP DIINNKIESF KKLLFDSYIQ SDIKNSLHNE LGIYTSLFID K ISPEIHLH QSEILGNVAF DSKMGAEIGS KINAITPGIL LNNNPLKEAY VDLNNTLKEI NTLLDEENKK TRDNPCRNEK IA KLTSLKD RLSDLDSIPK ENTVEFLKKM QEETRSSLKS LESNDALINI YDVLNSLKET IENGSDLPEI KKDKLQMISD VQN ILSNFD KNPAERLNLA VQILNDSNPE VLSKTKGNFL IGEAFKGQVF TNYINTKISE QLNNELGPYG KVFLKQIMPD FIAK KSEII KEIAIDNMET GLETQFKIHA PAIFEKNKEL KAAYEQLNVH LKEVQSLIEA EEKKPKGNPC REEKIAALRS HQSQL MNTQ RIPDHETLRF LQEQNKSAKS FMGKLEKYDT MISVYDSLTE IREHVSNHKS LSKEIKDEKI QEISKMEDML KTTSKE PSI RLAEVKAHGL SDQCKNVLLK NSDNFLVSFF KTLFSKLFNI KNENETLVSS FKQRLQNIKG PEPVATPMET PENEAPL VN ANITRF

UniProtKB: SidH

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Macromolecule #2: tRNA

MacromoleculeName: tRNA / 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 #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / 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: 2.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 41.93 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: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 367741
FSC plot (resolution estimation)

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