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- EMDB-4425: Cryo-EM structure of DNA-PKcs -

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Basic information

Entry
Database: EMDB / ID: EMD-4425
TitleCryo-EM structure of DNA-PKcs
Map dataDNA-PKcs
Sample
  • Organelle or cellular component: DNA-dependent protein kinase catalytic subunit
    • Protein or peptide: DNA-dependent protein kinase catalytic subunit
Biological speciesHomo (humans)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.7 Å
AuthorsWu Q / Blundell TL
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust200814/Z/16/Z United Kingdom
CitationJournal: Prog Biophys Mol Biol / Year: 2019
Title: Understanding the structure and role of DNA-PK in NHEJ: How X-ray diffraction and cryo-EM contribute in complementary ways.
Authors: Qian Wu / Shikang Liang / Takashi Ochi / Dimitri Y Chirgadze / Juha T Huiskonen / Tom L Blundell /
Abstract: DNA double-strand breaks (DSBs), generated by ionizing radiation, reactive oxygen species and DNA replication across nicks, are the most severe DNA damage in eukaryotic cells. Non-Homologous End ...DNA double-strand breaks (DSBs), generated by ionizing radiation, reactive oxygen species and DNA replication across nicks, are the most severe DNA damage in eukaryotic cells. Non-Homologous End Joining repairs DNA double-strand breaks directly without a template and so can take place at any point in the cell cycle. Ku70/80 heterodimers rapidly assemble around broken DNA ends, allowing DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase, to be recruited and facilitating synapsis of broken DNA ends. This then provides a stage for end-processing and ligation. Here we review progress leading in 2017 to the medium resolution X-ray structure of DNA-PKcs, a single polypeptide chain of 4128 amino acids. This was followed quickly by chain tracing of cryo-EM structures of DNA-PKcs in complex with Ku and DNA. We discuss how combination of structural information from X-ray and cryo-EM studies can produce a working model for complex multicomponent molecular assemblies such as those found in DNA-double-strand-break repair.
History
DepositionNov 16, 2018-
Header (metadata) releaseMay 8, 2019-
Map releaseMay 8, 2019-
UpdateOct 30, 2019-
Current statusOct 30, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0906
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0906
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4425.png

Downloads & links

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Map

FileDownload / File: emd_4425.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDNA-PKcs
Voxel sizeX=Y=Z: 1.43 Å
Density
Contour LevelBy AUTHOR: 0.0906 / Movie #1: 0.0906
Minimum - Maximum-0.23143104 - 0.48773596
Average (Standard dev.)0.0020121848 (±0.017275512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 314.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z314.600314.600314.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ307236
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.2310.4880.002

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Supplemental data

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Sample components

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Entire : DNA-dependent protein kinase catalytic subunit

EntireName: DNA-dependent protein kinase catalytic subunit
Components
  • Organelle or cellular component: DNA-dependent protein kinase catalytic subunit
    • Protein or peptide: DNA-dependent protein kinase catalytic subunit

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Supramolecule #1: DNA-dependent protein kinase catalytic subunit

SupramoleculeName: DNA-dependent protein kinase catalytic subunit / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo (humans)

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Macromolecule #1: DNA-dependent protein kinase catalytic subunit

MacromoleculeName: DNA-dependent protein kinase catalytic subunit / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo (humans)
SequenceString: MAGSGAGVRC SLLRLQETLS AADRCGAALA GHQLIRGLGQ ECVLSSSPAV LALQTSLVFS RDFGLLVFV RKSLNSIEFR ECREEILKFL CIFLEKMGQK IAPYSVEIKN TCTSVYTKDR A AKCKIPAL DLLIKLLQTF RSSRLMDEFK IGELFSKFYG ELALKKKIPD ...String:
MAGSGAGVRC SLLRLQETLS AADRCGAALA GHQLIRGLGQ ECVLSSSPAV LALQTSLVFS RDFGLLVFV RKSLNSIEFR ECREEILKFL CIFLEKMGQK IAPYSVEIKN TCTSVYTKDR A AKCKIPAL DLLIKLLQTF RSSRLMDEFK IGELFSKFYG ELALKKKIPD TVLEKVYELL GL LGEVHPS EMINNAENLF RAFLGELKTQ MTSAVREPKL PVLAGCLKGL SSLLCNFTKS MEE DPQTSR EIFNFVLKAI RPQIDLKRYA VPSAGLRLFA LHASQFSTCL LDNYVSLFEV LLKW CAHTN VELKKAALSA LESFLKQVSN MVAKNAEMHK NKLQYFMEQF YGIIRNVDSN NKELS IAIR GYGLFAGPCK VINAKDVDFM YVELIQRCKQ MFLTQTDTGD DRVYQMPSFL QSVASV LLY LDTVPEVYTP VLEHLVVMQI DSFPQYSPKM QLVCCRAIVK VFLALAAKGP VLRNCIS TV VHQGLIRICS KPVVLPKGPE SESEDHRASG EVRTGKWKVP TYKDYVDLFR HLLSSDQM M DSILADEAFF SVNSSSESLN HLLYDEFVKS VLKIVEKLDL TLEIQTVGEQ ENGDEAPGV WMIPTSDPAA NLHPAKPKDF SAFINLVEFC REILPEKQAE FFEPWVYSFS YELILQSTRL PLISGFYKL LSITVRNAKK IKYFEGVSPK SLKHSPEDPE KYSCFALFVK FGKEVAVKMK Q YKDELLAS CLTFLLSLPH NIIELDVRAY VPALQMAFKL GLSYTPLAEV GLNALEEWSI YI DRHVMQP YYKDILPCLD GYLKTSALSD ETKNNWEVSA LSRAAQKGFN KVVLKHLKKT KNL SSNEAI SLEEIRIRVV QMLGSLGGQI NKNLLTVTSS DEMMKSYVAW DREKRLSFAV PFRE MKPVI FLDVFLPRVT ELALTASDRQ TKVAACELLH SMVMFMLGKA TQMPEGGQGA PPMYQ LYKR TFPVLLRLAC DVDQVTRQLY EPLVMQLIHW FTNNKKFESQ DTVALLEAIL DGIVDP VDS TLRDFCGRCI REFLKWSIKQ ITPQQQEKSP VNTKSLFKRL YSLALHPNAF KRLGASL AF NNIYREFREE ESLVEQFVFE ALVIYMESLA LAHADEKSLG TIQQCCDAID HLCRIIEK K HVSLNKAKKR RLPRGFPPSA SLCLLDLVKW LLAHCGRPQT ECRHKSIELF YKFVPLLPG NRSPNLWLKD VLKEEGVSFL INTFEGGGCG QPSGILAQPT LLYLRGPFSL QATLCWLDLL LAALECYNT FIGERTVGAL QVLGTEAQSS LLKAVAFFLE SIAMHDIIAA EKCFGTGAAG N RTSPQEGE RYNYSKCTVV VRIMEFTTTL LNTSPEGWKL LKKDLCNTHL MRVLVQTLCE PA SIGFNIG DVQVMAHLPD VCVNLMKALK MSPYKDILET HLREKITAQS IEELCAVNLY GPD AQVDRS RLAAVVSACK QLHRAGLLHN ILPSQSTDLH HSVGTELLSL VYKGIAPGDE RQCL PSLDL SCKQLASGLL ELAFAFGGLC ERLVSLLLNP AVLSTASLGS SQGSVIHFSH GEYFY SLFS ETINTELLKN LDLAVLELMQ SSVDNTKMVS AVLNGMLDQS FRERANQKHQ GLKLAT TIL QHWKKCDSWW AKDSPLETKM AVLALLAKIL QIDSSVSFNT SHGSFPEVFT TYISLLA DT KLDLHLKGQA VTLLPFFTSL TGGSLEELRR VLEQLIVAHF PMQSREFPPG TPRFNNYV D CMKKFLDALE LSQSPMLLEL MTEVLCREQQ HVMEELFQSS FRRIARRGSC VTQVGLLES VYEMFRKDDP RLSFTRQSFV DRSLLTLLWH CSLDALREFF STIVVDAIDV LKSRFTKLNE STFDTQITK KMGYYKILDV MYSRLPKDDV HAKESKINQV FHGSCITEGN ELTKTLIKLC Y DAFTENMA GENQLLERRR LYHCAAYNCA ISVICCVFNE LKFYQGFLFS EKPEKNLLIF EN LIDLKRR YNFPVEVEVP MERKKKYIEI RKEAREAANG DSDGPSYMSS LSYLADSTLS EEM SQFDFS TGVQSYSYSS QDPRPATGRF RRREQRDPTV HDDVLELEMD ELNRHECMAP LTAL VKHMH RSLGPPQGEE DSVPRDLPSW MKFLHGKLGN PIVPLNIRLF LAKLVINTEE VFRPY AKHW LSPLLQLAAS ENNGGEGIHY MVVEIVATIL SWTGLATPTG VPKDEVLANR LLNFLM KHV FHPKRAVFRH NLEIIKTLVE CWKDCLSIPY RLIFEKFSGK DPNSKDNSVG IQLLGIV MA NDLPPYDPQC GIQSSEYFQA LVNNMSFVRY KEVYAAAAEV LGLILRYVME RKNILEES L CELVAKQLKQ HQNTMEDKFI VCLNKVTKSF PPLADRFMNA VFFLLPKFHG VLKTLCLEV VLCRVEGMTE LYFQLKSKDF VQVMRHRDDE RQKVCLDIIY KMMPKLKPVE LRELLNPVVE FVSHPSTTC REQMYNILMW IHDNYRDPES ETDNDSQEIF KLAKDVLIQG LIDENPGLQL I IRNFWSHE TRLPSNTLDR LLALNSLYSP KIEVHFLSLA TNFLLEMTSM SPDYPNPMFE HP LSECEFQ EYTIDSDWRF RSTVLTPMFV ETQASQGTLQ TRTQEGSLSA RWPVAGQIRA TQQ QHDFTL TQTADGRSSF DWLTGSSTDP LVDHTSPSSD SLLFAHKRSE RLQRAPLKSV GPDF GKKRL GLPGDEVDNK VKGAAGRTDL LRLRRRFMRD QEKLSLMYAR KGVAEQKREK EIKSE LKMK QDAQVVLYRS YRHGDLPDIQ IKHSSLITPL QAVAQRDPII AKQLFSSLFS GILKEM DKF KTLSEKNNIT QKLLQDFNRF LNTTFSFFPP FVSCIQDISC QHAALLSLDP AAVSAGC LA SLQQPVGIRL LEEALLRLLP AELPAKRVRG KARLPPDVLR WVELAKLYRS IGEYDVLR G IFTSEIGTKQ ITQSALLAEA RSDYSEAAKQ YDEALNKQDW VDGEPTEAEK DFWELASLD CYNHLAEWKS LEYCSTASID SENPPDLNKI WSEPFYQETY LPYMIRSKLK LLLQGEADQS LLTFIDKAM HGELQKAILE LHYSQELSLL YLLQDDVDRA KYYIQNGIQS FMQNYSSIDV L LHQSRLTK LQSVQALTEI QEFISFISKQ GNLSSQVPLK RLLNTWTNRY PDAKMDPMNI WD DIITNRC FFLSKIEEKL TPLPEDNSMN VDQDGDPSDR MEVQEQEEDI SSLIRSCKFS MKM KMIDSA RKQNNFSLAM KLLKELHKES KTRDDWLVSW VQSYCRLSHC RSRSQGCSEQ VLTV LKTVS LLDENNVSSY LSKNILAFRD QNILLGTTYR IIANALSSEP ACLAEIEEDK ARRIL ELSG SSSEDSEKVI AGLYQRAFQH LSEAVQAAEE EAQPPSWSCG PAAGVIDAYM TLADFC DQQ LRKEEENASV IDSAELQAYP ALVVEKMLKA LKLNSNEARL KFPRLLQIIE RYPEETL SL MTKEISSVPC WQFISWISHM VALLDKDQAV AVQHSVEEIT DNYPQAIVYP FIISSESY S FKDTSTGHKN KEFVARIKSK LDQGGVIQDF INALDQLSNP ELLFKDWSND VRAELAKTP VNKKNIEKMY ERMYAALGDP KAPGLGAFRR KFIQTFGKEF DKHFGKGGSK LLRMKLSDFN DITNMLLLK MNKDSKPPGN LKECSPWMSD FKVEFLRNEL EIPGQYDGRG KPLPEYHVRI A GFDERVTV MASLRRPKRI IIRGHDEREH PFLVKGGEDL RQDQRVEQLF QVMNGILAQD SA CSQRALQ LRTYSVVPMT SRLGLIEWLE NTVTLKDLLL NTMSQEEKAA YLSDPRAPPC EYK DWLTKM SGKHDVGAYM LMYKGANRTE TVTSFRKRES KVPADLLKRA FVRMSTSPEA FLAL RSHFA SSHALICISH WILGIGDRHL NNFMVAMETG GVIGIDFGHA FGSATQFLPV PELMP FRLT RQFINLMLPM KETGLMYSIM VHALRAFRSD PGLLTNTMDV FVKEPSFDWK NFEQKM LKK GGSWIQEINV AEKNWYPRQK ICYAKRKLAG ANPAVITCDE LLLGHEKAPA FRDYVAV AR GSKDHNIRAQ EPESGLSEET QVKCLMDQAT DPNILGRTWE GWEPWM

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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

Concentration0.2 mg/mL
BufferpH: 7.6
Details: 20 mM Hepes pH 7.6, 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, 5 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
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: FEI FALCON II (4k x 4k) / Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: MotionCorr2
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 129117
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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