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- EMDB-23512: XLF, XRCC4 and LigIV C-BRCT in the NHEJ Long-range synaptic complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23512
TitleXLF, XRCC4 and LigIV C-BRCT in the NHEJ Long-range synaptic complex
Map dataXLF-XRCC4-LigIV C-BRCT in the Long-range synaptic complex in NHEJ
Sample
  • Complex: XLF, XRCC4 and LigIV C-BRCT in the NHEJ Long-range synaptic complex
    • Protein or peptide: XRCC4_HUMAN DNA repair protein XRCC4
    • Protein or peptide: NHEJ1_HUMAN Non-homologous end-joining factor 1
    • Protein or peptide: DNLI4_HUMAN DNA ligase 4
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.3 Å
AuthorsHe Y / Chen S
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM135651 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM008382 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P01CA092584 United States
American Cancer SocietyIRG-15-173-21 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM047251 United States
CitationJournal: Nature / Year: 2021
Title: Structural basis of long-range to short-range synaptic transition in NHEJ.
Authors: Siyu Chen / Linda Lee / Tasmin Naila / Susan Fishbain / Annie Wang / Alan E Tomkinson / Susan P Lees-Miller / Yuan He /
Abstract: DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) ...DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors. Here we applied single-particle cryo-electron microscopy to visualize two key DNA-protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.
History
DepositionFeb 18, 2021-
Header (metadata) releaseMay 5, 2021-
Map releaseMay 5, 2021-
UpdateMay 26, 2021-
Current statusMay 26, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23512.png

Downloads & links

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Map

FileDownload / File: emd_23512.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationXLF-XRCC4-LigIV C-BRCT in the Long-range synaptic complex in NHEJ
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 216 pix.
= 475.2 Å		2.2 Å/pix.
x 216 pix.
= 475.2 Å		2.2 Å/pix.
x 216 pix.
= 475.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.0015068693 - 1.4528048
Average (Standard dev.)0.0005106381 (±0.015609429)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-108-108-108
Dimensions216216216
Spacing216216216
CellA=B=C: 475.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z216216216
origin x/y/z0.0000.0000.000
length x/y/z475.200475.200475.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS-108-108-108
NC/NR/NS216216216
D min/max/mean-0.0021.4530.001

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Supplemental data

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Sample components

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Entire : XLF, XRCC4 and LigIV C-BRCT in the NHEJ Long-range synaptic complex

EntireName: XLF, XRCC4 and LigIV C-BRCT in the NHEJ Long-range synaptic complex
Components
  • Complex: XLF, XRCC4 and LigIV C-BRCT in the NHEJ Long-range synaptic complex
    • Protein or peptide: XRCC4_HUMAN DNA repair protein XRCC4
    • Protein or peptide: NHEJ1_HUMAN Non-homologous end-joining factor 1
    • Protein or peptide: DNLI4_HUMAN DNA ligase 4

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Supramolecule #1: XLF, XRCC4 and LigIV C-BRCT in the NHEJ Long-range synaptic complex

SupramoleculeName: XLF, XRCC4 and LigIV C-BRCT in the NHEJ Long-range synaptic complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: XLF, XRCC4, LigIV C-BRCT
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: XRCC4_HUMAN DNA repair protein XRCC4

MacromoleculeName: XRCC4_HUMAN DNA repair protein XRCC4 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MERKISRIHL VSEPSITHFL QVSWEKTLES GFVITLTDGH SAWTGTVSES EISQEADDMA MEKGKYVGEL RKALLSGAGP ADVYTFNFS KESCYFFFEK NLKDVSFRLG SFNLEKVENP AEVIRELICY CLDTIAENQA KNEHLQKENE RLLRDWNDVQ G RFEKCVSA ...String:
MERKISRIHL VSEPSITHFL QVSWEKTLES GFVITLTDGH SAWTGTVSES EISQEADDMA MEKGKYVGEL RKALLSGAGP ADVYTFNFS KESCYFFFEK NLKDVSFRLG SFNLEKVENP AEVIRELICY CLDTIAENQA KNEHLQKENE RLLRDWNDVQ G RFEKCVSA KEALETDLYK RFILVLNEKK TKIRSLHNKL LNAAQEREKD IKQEGETAIC SEMTADRDPV YDESTDEESE NQ TDLSGLA SAAVSKDDSI ISSLDVTDIA PSRKRRQRMQ RNLGTEPKMA PQENQLQEKE NSRPDSSLPE TSKKEHISAE NMS LETLRN SSPEDLFDEI

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Macromolecule #2: NHEJ1_HUMAN Non-homologous end-joining factor 1

MacromoleculeName: NHEJ1_HUMAN Non-homologous end-joining factor 1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MEELEQGLLM QPWAWLQLAE NSLLAKVFIT KQGYALLVSD LQQVWHEQVD TSVVSQRAKE LNKRLTAPPA AFLCHLDNLL RPLLKDAAH PSEATFSCDC VADALILRVR SELSGLPFYW NFHCMLASPS LVSQHLIRPL MGMSLALQCQ VRELATLLHM K DLEIQDYQ ...String:
MEELEQGLLM QPWAWLQLAE NSLLAKVFIT KQGYALLVSD LQQVWHEQVD TSVVSQRAKE LNKRLTAPPA AFLCHLDNLL RPLLKDAAH PSEATFSCDC VADALILRVR SELSGLPFYW NFHCMLASPS LVSQHLIRPL MGMSLALQCQ VRELATLLHM K DLEIQDYQ ESGATLIRDR LKTEPFEENS FLEQFMIEKL PEACSIGDGK PFVMNLQDLY MAVTTQEVQV GQKHQGAGDP HT SNSASLQ GIDSQCVNQP EQLVSSAPTL SAPEKESTGT SGPLQRPQLS KVKRKKPRGL FS

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Macromolecule #3: DNLI4_HUMAN DNA ligase 4

MacromoleculeName: DNLI4_HUMAN DNA ligase 4 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString:
TPEEMASLIA DLE YRYSWD CSPLSMFRRH TVYLDSYAVI NDLSTKNEGT RLAIKALELR FHGAKVVSCL AEGVSHVIIG EDHSRVADFK AFRR TFKRK FKILKESWVT DSIDKCELQE ENQYLI

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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: 7.9
Component:
ConcentrationNameFormula
10.0 mMHEPES
50.0 mMKCl
10.0 mMMgCl2
2.5 %glycerol
1.0 mMDTT
0.05 %NP-40
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 200.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 3200FS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 4 / Number real images: 17114 / Average exposure time: 0.3 sec. / Average electron dose: 76.5 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: JEOL / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 1119381
CTF correctionSoftware: (Name: Gctf (ver. 0.50), CTFFIND (ver. 4.1))
Startup modelType of model: INSILICO MODEL
In silico model: Negative staining map reconstructed ab-initio from Cryosparc
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 9.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 43510
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: EMAN, cryoSPARC)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 7 / Avg.num./class: 50000 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

2r9a

,

3ii6

,

6zha

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient

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