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- EMDB-28738: DNAPKcs-Ku-DNA subcomplex in PAXX-LR-ATP complex -

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Basic information

Entry
Database: EMDB / ID: EMD-28738
TitleDNAPKcs-Ku-DNA subcomplex in PAXX-LR-ATP complex
Map dataDKB in PAXX-LR-ATP
Sample
  • Complex: NHEJ Long-range complex with ATP
    • Protein or peptide: XRCC6_HUMAN
    • Protein or peptide: XRCC5_HUMAN X-ray repair cross-complementing protein 5
    • Protein or peptide: PRKDC_HUMAN DNA-dependent protein kinase catalytic subunit
    • DNA: DNA(31-MER)
    • DNA: DNA(30-MER)
KeywordsComplex / Kinase / NHEJ / DNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsChen S / He Y
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 GM135651 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Sci Adv / Year: 2023
Title: Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ.
Authors: Siyu Chen / Alex Vogt / Linda Lee / Tasmin Naila / Ryan McKeown / Alan E Tomkinson / Susan P Lees-Miller / Yuan He /
Abstract: DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). ...DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)-free, short-range state to align the DSB ends (Chen ). Using single-particle cryo-electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.
History
DepositionOct 31, 2022-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28738.png

Downloads & links

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Map

FileDownload / File: emd_28738.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDKB in PAXX-LR-ATP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.11 Å/pix.
x 200 pix.
= 422.4 Å		2.11 Å/pix.
x 200 pix.
= 422.4 Å		2.11 Å/pix.
x 200 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.112 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.035
Minimum - Maximum-0.09920758 - 0.19448526
Average (Standard dev.)0.00021079226 (±0.0080622705)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 422.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half1

Fileemd_28738_half_map_1.map
Annotationhalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half2

Fileemd_28738_half_map_2.map
Annotationhalf2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NHEJ Long-range complex with ATP

EntireName: NHEJ Long-range complex with ATP
Components
  • Complex: NHEJ Long-range complex with ATP
    • Protein or peptide: XRCC6_HUMAN
    • Protein or peptide: XRCC5_HUMAN X-ray repair cross-complementing protein 5
    • Protein or peptide: PRKDC_HUMAN DNA-dependent protein kinase catalytic subunit
    • DNA: DNA(31-MER)
    • DNA: DNA(30-MER)

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Supramolecule #1: NHEJ Long-range complex with ATP

SupramoleculeName: NHEJ Long-range complex with ATP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 1.75 MDa

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Macromolecule #1: XRCC6_HUMAN

MacromoleculeName: XRCC6_HUMAN / type: protein_or_peptide / ID: 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)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MSGWESYYKT EGDEEAEEEQ EENLEASGDY KYSGRDSLIF LVDASKAMFE SQSEDELTPF DMSIQCIQSV YISKIISSDR DLLAVVFYGT EKDKNSVNFK NIYVLQELDN PGAKRILELD QFKGQQGQKR FQDMMGHGSD YSLSEVLWVC ANLFSDVQFK MSHKRIMLFT ...String:
MSGWESYYKT EGDEEAEEEQ EENLEASGDY KYSGRDSLIF LVDASKAMFE SQSEDELTPF DMSIQCIQSV YISKIISSDR DLLAVVFYGT EKDKNSVNFK NIYVLQELDN PGAKRILELD QFKGQQGQKR FQDMMGHGSD YSLSEVLWVC ANLFSDVQFK MSHKRIMLFT NEDNPHGNDS AKASRARTKA GDLRDTGIFL DLMHLKKPGG FDISLFYRDI ISIAEDEDLR VHFEESSKLE DLLRKVRAKE TRKRALSRLK LKLNKDIVIS VGIYNLVQKA LKPPPIKLYR ETNEPVKTKT RTFNTSTGGL LLPSDTKRSQ IYGSRQIILE KEETEELKRF DDPGLMLMGF KPLVLLKKHH YLRPSLFVYP EESLVIGSST LFSALLIKCL EKEVAALCRY TPRRNIPPYF VALVPQEEEL DDQKIQVTPP GFQLVFLPFA DDKRKMPFTE KIMATPEQVG KMKAIVEKLR FTYRSDSFEN PVLQQHFRNL EALALDLMEP EQAVDLTLPK VEAMNKRLGS LVDEFKELVY PPDYNPEGKV TKRKHDNEGS GSKRPKVEYS EEELKTHISK GTLGKFTVPM LKEACRAYGL KSGLKKQELL EALTKHFQD

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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
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MVRSGNKAAV VLCMDVGFTM SNSIPGIESP FEQAKKVITM FVQRQVFAEN KDEIALVLFG TDGTDNPLSG GDQYQNITVH RHLMLPDFDL LEDIESKIQP GSQQADFLDA LIVSMDVIQH ETIGKKFEKR HIEIFTDLSS RFSKSQLDII IHSLKKCDIS LQFFLPFSLG ...String:
MVRSGNKAAV VLCMDVGFTM SNSIPGIESP FEQAKKVITM FVQRQVFAEN KDEIALVLFG TDGTDNPLSG GDQYQNITVH RHLMLPDFDL LEDIESKIQP GSQQADFLDA LIVSMDVIQH ETIGKKFEKR HIEIFTDLSS RFSKSQLDII IHSLKKCDIS LQFFLPFSLG KEDGSGDRGD GPFRLGGHGP SFPLKGITEQ QKEGLEIVKM VMISLEGEDG LDEIYSFSES LRKLCVFKKI ERHSIHWPCR LTIGSNLSIR IAAYKSILQE RVKKTWTVVD AKTLKKEDIQ KETVYCLNDD DETEVLKEDI IQGFRYGSDI VPFSKVDEEQ MKYKSEGKCF SVLGFCKSSQ VQRRFFMGNQ VLKVFAARDD EAAAVALSSL IHALDDLDMV AIVRYAYDKR ANPQVGVAFP HIKHNYECLV YVQLPFMEDL RQYMFSSLKN SKKYAPTEAQ LNAVDALIDS MSLAKKDEKT DTLEDLFPTT KIPNPRFQRL FQCLLHRALH PREPLPPIQQ HIWNMLNPPA EVTTKSQIPL SKIKTLFPLI EAKKKDQVTA QEIFQDNHED GPTAKKLKTE QGGAHFSVSS LAEGSVTSVG SVNPAENFRV LVKQKKASFE EASNQLINHI EQFLDTNETP YFMKSIDCIR AFREEAIKFS EEQRFNNFLK ALQEKVEIKQ LNHFWEIVVQ DGITLITKEE ASGSSVTAEE AKKFLAPKDK PSGDTAAVFE EGGDVDDLLD MI

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Macromolecule #3: PRKDC_HUMAN DNA-dependent protein kinase catalytic subunit

MacromoleculeName: PRKDC_HUMAN DNA-dependent protein kinase catalytic subunit
type: protein_or_peptide / ID: 3 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
SequenceString: MAGSGAGVRC SLLRLQETLS AADRCGAALA GHQLIRGLGQ ECVLSSSPAV LALQTSLVFS RDFGLLVFVR KSLNSIEFRE CREEILKFLC IFLEKMGQKI APYSVEIKNT CTSVYTKDRA AKCKIPALDL LIKLLQTFRS SRLMDEFKIG ELFSKFYGEL ALKKKIPDTV ...String:
MAGSGAGVRC SLLRLQETLS AADRCGAALA GHQLIRGLGQ ECVLSSSPAV LALQTSLVFS RDFGLLVFVR KSLNSIEFRE CREEILKFLC IFLEKMGQKI APYSVEIKNT CTSVYTKDRA AKCKIPALDL LIKLLQTFRS SRLMDEFKIG ELFSKFYGEL ALKKKIPDTV LEKVYELLGL LGEVHPSEMI NNAENLFRAF LGELKTQMTS AVREPKLPVL AGCLKGLSSL LCNFTKSMEE DPQTSREIFN FVLKAIRPQI DLKRYAVPSA GLRLFALHAS QFSTCLLDNY VSLFEVLLKW CAHTNVELKK AALSALESFL KQVSNMVAKN AEMHKNKLQY FMEQFYGIIR NVDSNNKELS IAIRGYGLFA GPCKVINAKD VDFMYVELIQ RCKQMFLTQT DTGDDRVYQM PSFLQSVASV LLYLDTVPEV YTPVLEHLVV MQIDSFPQYS PKMQLVCCRA IVKVFLALAA KGPVLRNCIS TVVHQGLIRI CSKPVVLPKG PESESEDHRA SGEVRTGKWK VPTYKDYVDL FRHLLSSDQM MDSILADEAF FSVNSSSESL NHLLYDEFVK SVLKIVEKLD LTLEIQTVGE QENGDEAPGV WMIPTSDPAA NLHPAKPKDF SAFINLVEFC REILPEKQAE FFEPWVYSFS YELILQSTRL PLISGFYKLL SITVRNAKKI KYFEGVSPKS LKHSPEDPEK YSCFALFVKF GKEVAVKMKQ YKDELLASCL TFLLSLPHNI IELDVRAYVP ALQMAFKLGL SYTPLAEVGL NALEEWSIYI DRHVMQPYYK DILPCLDGYL KTSALSDETK NNWEVSALSR AAQKGFNKVV LKHLKKTKNL SSNEAISLEE IRIRVVQMLG SLGGQINKNL LTVTSSDEMM KSYVAWDREK RLSFAVPFRE MKPVIFLDVF LPRVTELALT ASDRQTKVAA CELLHSMVMF MLGKATQMPE GGQGAPPMYQ LYKRTFPVLL RLACDVDQVT RQLYEPLVMQ LIHWFTNNKK FESQDTVALL EAILDGIVDP VDSTLRDFCG RCIREFLKWS IKQITPQQQE KSPVNTKSLF KRLYSLALHP NAFKRLGASL AFNNIYREFR EEESLVEQFV FEALVIYMES LALAHADEKS LGTIQQCCDA IDHLCRIIEK KHVSLNKAKK RRLPRGFPPS ASLCLLDLVK WLLAHCGRPQ TECRHKSIEL FYKFVPLLPG NRSPNLWLKD VLKEEGVSFL INTFEGGGCG QPSGILAQPT LLYLRGPFSL QATLCWLDLL LAALECYNTF IGERTVGALQ VLGTEAQSSL LKAVAFFLES IAMHDIIAAE KCFGTGAAGN RTSPQEGERY NYSKCTVVVR IMEFTTTLLN TSPEGWKLLK KDLCNTHLMR VLVQTLCEPA SIGFNIGDVQ VMAHLPDVCV NLMKALKMSP YKDILETHLR EKITAQSIEE LCAVNLYGPD AQVDRSRLAA VVSACKQLHR AGLLHNILPS QSTDLHHSVG TELLSLVYKG IAPGDERQCL PSLDLSCKQL ASGLLELAFA FGGLCERLVS LLLNPAVLST ASLGSSQGSV IHFSHGEYFY SLFSETINTE LLKNLDLAVL ELMQSSVDNT KMVSAVLNGM LDQSFRERAN QKHQGLKLAT TILQHWKKCD SWWAKDSPLE TKMAVLALLA KILQIDSSVS FNTSHGSFPE VFTTYISLLA DTKLDLHLKG QAVTLLPFFT SLTGGSLEEL RRVLEQLIVA HFPMQSREFP PGTPRFNNYV DCMKKFLDAL ELSQSPMLLE LMTEVLCREQ QHVMEELFQS SFRRIARRGS CVTQVGLLES VYEMFRKDDP RLSFTRQSFV DRSLLTLLWH CSLDALREFF STIVVDAIDV LKSRFTKLNE STFDTQITKK MGYYKILDVM YSRLPKDDVH AKESKINQVF HGSCITEGNE LTKTLIKLCY DAFTENMAGE NQLLERRRLY HCAAYNCAIS VICCVFNELK FYQGFLFSEK PEKNLLIFEN LIDLKRRYNF PVEVEVPMER KKKYIEIRKE AREAANGDSD GPSYMSSLSY LADSTLSEEM SQFDFSTGVQ SYSYSSQDPR PATGRFRRRE QRDPTVHDDV LELEMDELNR HECMAPLTAL VKHMHRSLGP PQGEEDSVPR DLPSWMKFLH GKLGNPIVPL NIRLFLAKLV INTEEVFRPY AKHWLSPLLQ LAASENNGGE GIHYMVVEIV ATILSWTGLA TPTGVPKDEV LANRLLNFLM KHVFHPKRAV FRHNLEIIKT LVECWKDCLS IPYRLIFEKF SGKDPNSKDN SVGIQLLGIV MANDLPPYDP QCGIQSSEYF QALVNNMSFV RYKEVYAAAA EVLGLILRYV MERKNILEES LCELVAKQLK QHQNTMEDKF IVCLNKVTKS FPPLADRFMN AVFFLLPKFH GVLKTLCLEV VLCRVEGMTE LYFQLKSKDF VQVMRHRDDE RQKVCLDIIY KMMPKLKPVE LRELLNPVVE FVSHPSTTCR EQMYNILMWI HDNYRDPESE TDNDSQEIFK LAKDVLIQGL IDENPGLQLI IRNFWSHETR LPSNTLDRLL ALNSLYSPKI EVHFLSLATN FLLEMTSMSP DYPNPMFEHP LSECEFQEYT IDSDWRFRST VLTPMFVETQ ASQGTLQTRT QEGSLSARWP VAGQIRATQQ QHDFTLTQTA DGRSSFDWLT GSSTDPLVDH TSPSSDSLLF AHKRSERLQR APLKSVGPDF GKKRLGLPGD EVDNKVKGAA GRTDLLRLRR RFMRDQEKLS LMYARKGVAE QKREKEIKSE LKMKQDAQVV LYRSYRHGDL PDIQIKHSSL ITPLQAVAQR DPIIAKQLFS SLFSGILKEM DKFKTLSEKN NITQKLLQDF NRFLNTTFSF FPPFVSCIQD ISCQHAALLS LDPAAVSAGC LASLQQPVGI RLLEEALLRL LPAELPAKRV RGKARLPPDV LRWVELAKLY RSIGEYDVLR GIFTSEIGTK QITQSALLAE ARSDYSEAAK QYDEALNKQD WVDGEPTEAE KDFWELASLD CYNHLAEWKS LEYCSTASID SENPPDLNKI WSEPFYQETY LPYMIRSKLK LLLQGEADQS LLTFIDKAMH GELQKAILEL HYSQELSLLY LLQDDVDRAK YYIQNGIQSF MQNYSSIDVL LHQSRLTKLQ SVQALTEIQE FISFISKQGN LSSQVPLKRL LNTWTNRYPD AKMDPMNIWD DIITNRCFFL SKIEEKLTPL PEDNSMNVDQ DGDPSDRMEV QEQEEDISSL IRSCKFSMKM KMIDSARKQN NFSLAMKLLK ELHKESKTRD DWLVSWVQSY CRLSHCRSRS QGCSEQVLTV LKTVSLLDEN NVSSYLSKNI LAFRDQNILL GTTYRIIANA LSSEPACLAE IEEDKARRIL ELSGSSSEDS EKVIAGLYQR AFQHLSEAVQ AAEEEAQPPS WSCGPAAGVI DAYMTLADFC DQQLRKEEEN ASVIDSAELQ AYPALVVEKM LKALKLNSNE ARLKFPRLLQ IIERYPEETL SLMTKEISSV PCWQFISWIS HMVALLDKDQ AVAVQHSVEE ITDNYPQAIV YPFIISSESY SFKDTSTGHK NKEFVARIKS KLDQGGVIQD FINALDQLSN PELLFKDWSN DVRAELAKTP VNKKNIEKMY ERMYAALGDP KAPGLGAFRR KFIQTFGKEF DKHFGKGGSK LLRMKLSDFN DITNMLLLKM NKDSKPPGNL KECSPWMSDF KVEFLRNELE IPGQYDGRGK PLPEYHVRIA GFDERVTVMA SLRRPKRIII RGHDEREHPF LVKGGEDLRQ DQRVEQLFQV MNGILAQDSA CSQRALQLRT YSVVPMTSRL GLIEWLENTV TLKDLLLNTM SQEEKAAYLS DPRAPPCEYK DWLTKMSGKH DVGAYMLMYK GANRTETVTS FRKRESKVPA DLLKRAFVRM STSPEAFLAL RSHFASSHAL ICISHWILGI GDRHLNNFMV AMETGGVIGI DFGHAFGSAT QFLPVPELMP FRLTRQFINL MLPMKETGLM YSIMVHALRA FRSDPGLLTN TMDVFVKEPS FDWKNFEQKM LKKGGSWIQE INVAEKNWYP RQKICYAKRK LAGANPAVIT CDELLLGHEK APAFRDYVAV ARGSKDHNIR AQEPESGLSE ETQVKCLMDQ ATDPNILGRT WEGWEPWM

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Macromolecule #4: DNA(31-MER)

MacromoleculeName: DNA(31-MER) / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
(DT)(DC)(DT)(DA)(DA)(DG)(DA)(DA)(DC)(DT) (DC)(DT)(DG)(DA)(DT)(DG)(DT)(DC)(DA)(DG) (DT)(DA)(DG)(DA)(DT)(DT)(DA)(DC)(DA)(DC) (DT)

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Macromolecule #5: DNA(30-MER)

MacromoleculeName: DNA(30-MER) / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
(DG)(DT)(DG)(DT)(DA)(DA)(DT)(DC)(DT)(DA) (DC)(DT)(DG)(DA)(DC)(DA)(DT)(DC)(DA)(DG) (DA)(DG)(DT)(DT)(DC)(DT)(DT)(DA)(DG) (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

Concentration2 mg/mL
BufferpH: 7.9
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 302 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 30448 / Average exposure time: 4.0 sec. / Average electron dose: 65.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 60000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 60000
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: 3785786
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7lt3

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 176398
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient

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