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- EMDB-15416: Vaccinia C16 protein bound to Ku70/Ku80 -

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Basic information

Entry
Database: EMDB / ID: EMD-15416
TitleVaccinia C16 protein bound to Ku70/Ku80
Map dataMap of C16 vaccinia protein bound to Ku70/Ku80
Sample
  • Complex: Vaccinia C16 protein bound to Ku70/Ku80
    • Protein or peptide: Ku70-Xrcc6
    • Protein or peptide: X-ray repair cross-complementing protein 5
    • Protein or peptide: Protein C10
Function / homology
Function and homology information


Ku70:Ku80 complex / negative regulation of t-circle formation / DNA end binding / double-strand break repair via classical nonhomologous end joining / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / cellular response to X-ray / nonhomologous end joining complex / 5'-deoxyribose-5-phosphate lyase activity ...Ku70:Ku80 complex / negative regulation of t-circle formation / DNA end binding / double-strand break repair via classical nonhomologous end joining / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / cellular response to X-ray / nonhomologous end joining complex / 5'-deoxyribose-5-phosphate lyase activity / DNA ligation / regulation of smooth muscle cell proliferation / nuclear telomere cap complex / Cytosolic sensors of pathogen-associated DNA / IRF3-mediated induction of type I IFN / recombinational repair / regulation of telomere maintenance / hematopoietic stem cell proliferation / positive regulation of neurogenesis / U3 snoRNA binding / cellular response to fatty acid / cellular hyperosmotic salinity response / protein localization to chromosome, telomeric region / site of DNA damage / telomeric DNA binding / 2-LTR circle formation / neurogenesis / ATP-dependent activity, acting on DNA / positive regulation of catalytic activity / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / hematopoietic stem cell differentiation / enzyme activator activity / positive regulation of protein kinase activity / small-subunit processome / DNA helicase activity / positive regulation of telomere maintenance via telomerase / activation of innate immune response / cyclin binding / telomere maintenance / positive regulation of telomerase activity / cellular response to leukemia inhibitory factor / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Nonhomologous End-Joining (NHEJ) / protein-DNA complex / cellular response to gamma radiation / double-strand break repair / double-strand break repair via nonhomologous end joining / scaffold protein binding / brain development / double-stranded DNA binding / secretory granule lumen / DNA recombination / ficolin-1-rich granule lumen / chromosome, telomeric region / : / transcription regulator complex / damaged DNA binding / transcription cis-regulatory region binding / response to xenobiotic stimulus / ribonucleoprotein complex / DNA damage response / negative regulation of DNA-templated transcription / ubiquitin protein ligase binding / protein-containing complex binding / nucleolus / Neutrophil degranulation / innate immune response / ATP hydrolysis activity / positive regulation of DNA-templated transcription / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / RNA binding / extracellular region / nucleoplasm / ATP binding / membrane / nucleus / plasma membrane / cytosol
Similarity search - Function
Poxvirus C4/C10 / Poxvirus C4/C10 protein / Ku70, bridge and pillars domain superfamily / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta ...Poxvirus C4/C10 / Poxvirus C4/C10 protein / Ku70, bridge and pillars domain superfamily / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 beta-barrel domain / Ku70/Ku80 C-terminal arm / Ku70/Ku80 N-terminal alpha/beta domain / Ku70 and Ku80 are 70kDa and 80kDa subunits of the Lupus Ku autoantigen / Ku70/Ku80 beta-barrel domain / SPOC-like, C-terminal domain superfamily / SAP domain superfamily / SAP domain / SAP motif profile. / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / von Willebrand factor (vWF) type A domain / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily
Similarity search - Domain/homology
Protein C10 / X-ray repair cross-complementing protein 6 / X-ray repair cross-complementing protein 5
Similarity search - Component
Biological speciesHomo sapiens (human) / Vaccinia virus Western Reserve
Methodsingle particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsRivera-Calzada A / Arribas-Bosacoma R / Pearl LH / Llorca O
Funding support Spain, United Kingdom, 6 items
OrganizationGrant numberCountry
Agencia Estatal de Investigacion (AEI)AEI/10.13039/501100011033 Spain
Ministerio de Ciencia e Innovacion (MCIN)PID2020-114429RB-I00 Spain
Autonomous Community of MadridY2018/BIO-4747 Spain
Autonomous Community of MadridP2018/NMT-4443 Spain
Cancer Research UKC302/A14532 United Kingdom
Cancer Research UKC302/A24386 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for the inactivation of cytosolic DNA sensing by the vaccinia virus.
Authors: Angel Rivera-Calzada / Raquel Arribas-Bosacoma / Alba Ruiz-Ramos / Paloma Escudero-Bravo / Jasminka Boskovic / Rafael Fernandez-Leiro / Antony W Oliver / Laurence H Pearl / Oscar Llorca /
Abstract: Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as ...Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 - Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA.
History
DepositionJul 19, 2022-
Header (metadata) releaseNov 9, 2022-
Map releaseNov 9, 2022-
UpdateNov 30, 2022-
Current statusNov 30, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_15416.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of C16 vaccinia protein bound to Ku70/Ku80
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 304 pix.
= 253.84 Å
0.84 Å/pix.
x 304 pix.
= 253.84 Å
0.84 Å/pix.
x 304 pix.
= 253.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 5.0
Minimum - Maximum-3.0549178 - 90.36707
Average (Standard dev.)0.010092188 (±1.0516663)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 253.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Vaccinia C16 protein bound to Ku70/Ku80

EntireName: Vaccinia C16 protein bound to Ku70/Ku80
Components
  • Complex: Vaccinia C16 protein bound to Ku70/Ku80
    • Protein or peptide: Ku70-Xrcc6
    • Protein or peptide: X-ray repair cross-complementing protein 5
    • Protein or peptide: Protein C10

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Supramolecule #1: Vaccinia C16 protein bound to Ku70/Ku80

SupramoleculeName: Vaccinia C16 protein bound to Ku70/Ku80 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 230 KDa

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Macromolecule #1: Ku70-Xrcc6

MacromoleculeName: Ku70-Xrcc6 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 74.234703 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSAWSHPQFE KGSAGSAAGS GAGWSHPQFE KLEVLFQGPG GSMSGWESYY KTEGDEEAEE EQEENLEASG DYKYSGRDSL IFLVDASKA MFESQSEDEL TPFDMSIQCI QSVYISKIIS SDRDLLAVVF YGTEKDKNSV NFKNIYVLQE LDNPGAKRIL E LDQFKGQQ ...String:
MSAWSHPQFE KGSAGSAAGS GAGWSHPQFE KLEVLFQGPG GSMSGWESYY KTEGDEEAEE EQEENLEASG DYKYSGRDSL IFLVDASKA MFESQSEDEL TPFDMSIQCI QSVYISKIIS SDRDLLAVVF YGTEKDKNSV NFKNIYVLQE LDNPGAKRIL E LDQFKGQQ GQKRFQDMMG HGSDYSLSEV LWVCANLFSD VQFKMSHKRI MLFTNEDNPH GNDSAKASRA RTKAGDLRDT GI FLDLMHL KKPGGFDISL FYRDIISIAE DEDLRVHFEE SSKLEDLLRK VRAKETRKRA LSRLKLKLNK DIVISVGIYN LVQ KALKPP PIKLYRETNE PVKTKTRTFN TSTGGLLLPS DTKRSQIYGS RQIILEKEET EELKRFDDPG LMLMGFKPLV LLKK HHYLR PSLFVYPEES LVIGSSTLFS ALLIKCLEKE VAALCRYTPR RNIPPYFVAL VPQEEELDDQ KIQVTPPGFQ LVFLP FADD KRKMPFTEKI MATPEQVGKM KAIVEKLRFT YRSDSFENPV LQQHFRNLEA LALDLMEPEQ AVDLTLPKVE AMNKRL GSL VDEFKELVYP PDYNPEGKVT KRKHDNEGSG SKRPKVEYSE EELKTHISKG TLGKFTVPML KEACRAYGLK SGLKKQE LL EALTKHFQD

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Macromolecule #2: X-ray repair cross-complementing protein 5

MacromoleculeName: X-ray repair cross-complementing protein 5 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 85.546484 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAHHHHHHHH HHGALEVLFQ GPHMVRSGNK AAVVLCMDVG FTMSNSIPGI ESPFEQAKKV ITMFVQRQVF AENKDEIALV LFGTDGTDN PLSGGDQYQN ITVHRHLMLP DFDLLEDIES KIQPGSQQAD FLDALIVSMD VIQHETIGKK FEKRHIEIFT D LSSRFSKS ...String:
MAHHHHHHHH HHGALEVLFQ GPHMVRSGNK AAVVLCMDVG FTMSNSIPGI ESPFEQAKKV ITMFVQRQVF AENKDEIALV LFGTDGTDN PLSGGDQYQN ITVHRHLMLP DFDLLEDIES KIQPGSQQAD FLDALIVSMD VIQHETIGKK FEKRHIEIFT D LSSRFSKS QLDIIIHSLK KCDISLQFFL PFSLGKEDGS GDRGDGPFRL GGHGPSFPLK GITEQQKEGL EIVKMVMISL EG EDGLDEI YSFSESLRKL CVFKKIERHS IHWPCRLTIG SNLSIRIAAY KSILQERVKK TWTVVDAKTL KKEDIQKETV YCL NDDDET EVLKEDIIQG FRYGSDIVPF SKVDEEQMKY KSEGKCFSVL GFCKSSQVQR RFFMGNQVLK VFAARDDEAA AVAL SSLIH ALDDLDMVAI VRYAYDKRAN PQVGVAFPHI KHNYECLVYV QLPFMEDLRQ YMFSSLKNSK KYAPTEAQLN AVDAL IDSM SLAKKDEKTD TLEDLFPTTK IPNPRFQRLF QCLLHRALHP REPLPPIQQH IWNMLNPPAE VTTKSQIPLS KIKTLF PLI EAKKKDQVTA QEIFQDNHED GPTAKKLKTE QGGAHFSVSS LAEGSVTSVG SVNPAENFRV LVKQKKASFE EASNQLI NH IEQFLDTNET PYFMKSIDCI RAFREEAIKF SEEQRFNNFL KALQEKVEIK QLNHFWEIVV QDGITLITKE EASGSSVT A EEAKKFLAPK DKPSGDTAAV FEEGGDVDDL LDMI

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Macromolecule #3: Protein C10

MacromoleculeName: Protein C10 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus Western Reserve / Strain: Western Reserve
Molecular weightTheoretical: 42.47668 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDIYDDKGLQ TIKLFNNEFD CIRNDIRELF KHVTDSDSIQ LPMEDNSDII ENIRKILYRR LKNVECVDID STITFMKYDP NDDNKRTCS NWVPLTNNYM EYCLVIYLET PICGGKIKLY HPTGNIKSDK DIMFAKTLDF KSKKVLTGRK TIAVLDISVS Y NRSMTTIH ...String:
MDIYDDKGLQ TIKLFNNEFD CIRNDIRELF KHVTDSDSIQ LPMEDNSDII ENIRKILYRR LKNVECVDID STITFMKYDP NDDNKRTCS NWVPLTNNYM EYCLVIYLET PICGGKIKLY HPTGNIKSDK DIMFAKTLDF KSKKVLTGRK TIAVLDISVS Y NRSMTTIH YNDDVDIDIH TDKNGKELCY CYITIDDHYL VDVETIGVIV NRSGKCLLVN NHLGIGIVKD KRISDSFGDV CM DTIFDFS EARELFSLTN DDNRNIAWDT DKLDDDTDIW TPVTEDDYKF LSRLVLYAKS QSDTVFDYYV LTGDTEPPTV FIF KVTRFY FNMPKGGENL YFQGWSHPQF EKGGGSGGGS GGSSAWSHPQ FEK

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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

Concentration0.4 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
50.0 mMHEPES-KOHHEPES-KOH
250.0 mMNaClSodium chlorideNaClSodium chloride
1.0 mMEDTAEthylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid
0.5 mMTCEPTCEP
0.5 mMPMSFPMSF
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 13216 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 12495814
Startup modelType of model: OTHER / Details: Ab intio model generated from the data set
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware: (Name: RELION, cryoSPARC)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 152123

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Atomic model buiding 1

Initial model
PDB IDChain

chain_id: A

chain_id: B
DetailsInitial model for chains C and D was generated using AlphaFold2
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8ag5:
Vaccinia C16 protein bound to Ku70/Ku80

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