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- EMDB-15414: Vaccinia C16 N-terminal domains -

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Basic information

Entry
Database: EMDB / ID: EMD-15414
TitleVaccinia C16 N-terminal domains
Map dataMap of C16 N-terminal domains in the C16-Ku70/Ku80 complex
Sample
  • Complex: C16 N-terminal domains in the C16-Ku70/Ku80 complex
    • Protein or peptide: Protein C10
Function / homologyPoxvirus C4/C10 / Poxvirus C4/C10 protein / Protein C10
Function and homology information
Biological speciesVaccinia virus / 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_15414.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of C16 N-terminal domains in the C16-Ku70/Ku80 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (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: 0.14
Minimum - Maximum-0.023858862 - 2.2646763
Average (Standard dev.)0.00071888056 (±0.014832933)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Half map: Half map 1

Fileemd_15414_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2

Fileemd_15414_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : C16 N-terminal domains in the C16-Ku70/Ku80 complex

EntireName: C16 N-terminal domains in the C16-Ku70/Ku80 complex
Components
  • Complex: C16 N-terminal domains in the C16-Ku70/Ku80 complex
    • Protein or peptide: Protein C10

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Supramolecule #1: C16 N-terminal domains in the C16-Ku70/Ku80 complex

SupramoleculeName: C16 N-terminal domains in the C16-Ku70/Ku80 complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vaccinia virus / Strain: Western Reserve
Molecular weightTheoretical: 230 KDa

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Macromolecule #1: Protein C10

MacromoleculeName: Protein C10 / type: protein_or_peptide / ID: 1 / 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
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: 81353
FSC plot (resolution estimation)

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Atomic model buiding 1

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

PDB-8ag3:
Vaccinia C16 N-terminal domains

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