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- EMDB-23514: Proximal Ku in NHEJ Short-range synaptic complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23514
TitleProximal Ku in NHEJ Short-range synaptic complex
Map dataProximal Ku in NHEJ Short-range synaptic complex
Sample
  • Complex: Proximal Ku in NHEJ Short-range synaptic complex
    • Protein or peptide: XRCC6_HUMAN X-ray repair cross-complementing protein 6
    • Protein or peptide: XRCC5_HUMAN X-ray repair cross-complementing protein 5
    • DNA: DNA (5'-D(P*TP*AP*TP*AP*TP*AP*CP*TP*AP*AP*GP*AP*AP*C)-3')
    • DNA: DNA (26-MER)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 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
American Cancer SocietyIRG-15-173-2 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P01CA092584 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 19, 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: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23514.png

Downloads & links

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Map

FileDownload / File: emd_23514.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationProximal Ku in NHEJ Short-range synaptic complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.66 Å/pix.
x 192 pix.
= 318.72 Å		1.66 Å/pix.
x 192 pix.
= 318.72 Å		1.66 Å/pix.
x 192 pix.
= 318.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.007482051 - 1.3629305
Average (Standard dev.)0.0010013101 (±0.017696045)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.661.661.66
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z318.720318.720318.720
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.0071.3630.001

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Supplemental data

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Sample components

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Entire : Proximal Ku in NHEJ Short-range synaptic complex

EntireName: Proximal Ku in NHEJ Short-range synaptic complex
Components
  • Complex: Proximal Ku in NHEJ Short-range synaptic complex
    • Protein or peptide: XRCC6_HUMAN X-ray repair cross-complementing protein 6
    • Protein or peptide: XRCC5_HUMAN X-ray repair cross-complementing protein 5
    • DNA: DNA (5'-D(P*TP*AP*TP*AP*TP*AP*CP*TP*AP*AP*GP*AP*AP*C)-3')
    • DNA: DNA (26-MER)

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Supramolecule #1: Proximal Ku in NHEJ Short-range synaptic complex

SupramoleculeName: Proximal Ku in NHEJ Short-range synaptic complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Ku
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: XRCC6_HUMAN X-ray repair cross-complementing protein 6

MacromoleculeName: XRCC6_HUMAN X-ray repair cross-complementing protein 6
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MSGWESYYKT EGDEEAEEEQ EENLEASGDY KYSGRDSLIF LVDASKAMFE SQSEDELTPF DMSIQCIQSV YISKIISSDR DLLAVVFYG TEKDKNSVNF KNIYVLQELD NPGAKRILEL DQFKGQQGQK RFQDMMGHGS DYSLSEVLWV CANLFSDVQF K MSHKRIML ...String:
MSGWESYYKT EGDEEAEEEQ EENLEASGDY KYSGRDSLIF LVDASKAMFE SQSEDELTPF DMSIQCIQSV YISKIISSDR DLLAVVFYG TEKDKNSVNF KNIYVLQELD NPGAKRILEL DQFKGQQGQK RFQDMMGHGS DYSLSEVLWV CANLFSDVQF K MSHKRIML FTNEDNPHGN DSAKASRART KAGDLRDTGI FLDLMHLKKP GGFDISLFYR DIISIAEDED LRVHFEESSK LE DLLRKVR AKETRKRALS RLKLKLNKDI VISVGIYNLV QKALKPPPIK LYRETNEPVK TKTRTFNTST GGLLLPSDTK RSQ IYGSRQ IILEKEETEE LKRFDDPGLM LMGFKPLVLL KKHHYLRPSL FVYPEESLVI GSSTLFSALL IKCLEKEVAA LCRY TPRRN IPPYFVALVP QEEELDDQKI QVTPPGFQLV FLPFADDKRK MPFTEKIMAT PEQVGKMKAI VEKLRFTYRS DSFEN PVLQ QHFRNLEALA LDLMEPEQAV DLTLPKVEAM NKRLGSLVDE FKELVYPPDY NPEGKVTKRK HDNEGSGSKR PKVEYS EEE LKTHISKGTL GKFTVPMLKE ACRAYGLKSG LKKQELL

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Macromolecule #2: XRCC5_HUMAN X-ray repair cross-complementing protein 5

MacromoleculeName: XRCC5_HUMAN X-ray repair cross-complementing protein 5
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MVRSGNKAAV VLCMDVGFTM SNSIPGIESP FEQAKKVITM FVQRQVFAEN KDEIALVLFG TDGTDNPLSG GDQYQNITVH RHLMLPDFD LLEDIESKIQ PGSQQADFLD ALIVSMDVIQ HETIGKKFEK RHIEIFTDLS SRFSKSQLDI IIHSLKKCDI S LQFFLPFS ...String:
MVRSGNKAAV VLCMDVGFTM SNSIPGIESP FEQAKKVITM FVQRQVFAEN KDEIALVLFG TDGTDNPLSG GDQYQNITVH RHLMLPDFD LLEDIESKIQ PGSQQADFLD ALIVSMDVIQ HETIGKKFEK RHIEIFTDLS SRFSKSQLDI IIHSLKKCDI S LQFFLPFS LGKEDGSGDR GDGPFRLGGH GPSFPLKGIT EQQKEGLEIV KMVMISLEGE DGLDEIYSFS ESLRKLCVFK KI ERHSIHW PCRLTIGSNL SIRIAAYKSI LQERVKKTWT VVDAKTLKKE DIQKETVYCL NDDDETEVLK EDIIQGFRYG SDI VPFSKV DEEQMKYKSE GKCFSVLGFC KSSQVQRRFF MGNQVLKVFA ARDDEAAAVA LSSLIHALDD LDMVAIVRYA YDKR ANPQV GVAFPHIKHN YECLVYVQLP FMEDLRQYMF SSLKNSKKYA PTEAQLNAVD ALIDSMSLAK KDEKTDTLED LFPTT KIPN PRFQRLFQCL LHRALHPREP LPPIQQHIWN MLNPPAEVTT KSQIPLSKIK TLFPLIEAKK KDQVTAQEIF QDNHED GPT AKKLKTEQGG AHFSVSSLAE GSVTSVGSVN PAENFRVLVK QKKASFEEAS NQLINHIEQF LDTNETPYFM KSIDCIR AF REEAIKFSEE QRFNNFLKAL QEKVEIKQLN HFWEIVVQDG ITLITKEEAS GSSVTAEEAK KFLAPKDKPS GDTAAVFE E GGDVDDLLDM I

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Macromolecule #3: DNA (5'-D(P*TP*AP*TP*AP*TP*AP*CP*TP*AP*AP*GP*AP*AP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*AP*TP*AP*TP*AP*CP*TP*AP*AP*GP*AP*AP*C)-3')
type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
(DT)(DA)(DT)(DA)(DT)(DA)(DC)(DT)(DA)(DA) (DG)(DA)(DA)(DC)

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Macromolecule #4: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
(DC)(DG)(DT)(DT)(DT)(DC)(DA)(DT)(DT)(DG) (DT)(DT)(DG)(DT)(DT)(DC)(DT)(DT)(DA)(DG) (DT)(DA)(DT)(DA)(DT)(DA)

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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 mMPotassium chlorideKCl
10.0 mMMagnesium chlorideMgCl2
2.5 %glycerol
1.0 mMDTT
0.05 %NP-40
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 200 / 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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 2 / Number real images: 32723 / Average exposure time: 0.0426 sec. / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 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: 1.5 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1766936
CTF correctionSoftware: (Name: Gctf, CTFFIND)
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: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 57170
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: EMAN, cryoSPARC)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 60000 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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