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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-11050 | |||||||||
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Title | Mec1-Ddc2 (F2244L mutant) in complex with Mg AMP-PNP | |||||||||
![]() | Mec1(F2244L)-Ddc2 map refined to 2.82 Angstroms and sharpened using a B-factor of -20 | |||||||||
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![]() | serine/threonine protein kinase / complex / DNA damage response / checkpoint control / Hydrolase | |||||||||
Function / homology | ![]() ATR-ATRIP complex / positive regulation of DNA-templated DNA replication / telomere maintenance via recombination / regulation of double-strand break repair / reciprocal meiotic recombination / nucleobase-containing compound metabolic process / nuclear chromosome / telomere maintenance via telomerase / signal transduction in response to DNA damage / telomere maintenance ...ATR-ATRIP complex / positive regulation of DNA-templated DNA replication / telomere maintenance via recombination / regulation of double-strand break repair / reciprocal meiotic recombination / nucleobase-containing compound metabolic process / nuclear chromosome / telomere maintenance via telomerase / signal transduction in response to DNA damage / telomere maintenance / DNA damage checkpoint signaling / establishment of protein localization / chromatin organization / chromosome / DNA recombination / DNA replication / damaged DNA binding / non-specific serine/threonine protein kinase / protein kinase activity / DNA repair / protein serine kinase activity / protein serine/threonine kinase activity / mitochondrion / ATP binding / nucleus / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.82 Å | |||||||||
![]() | Yates LA / Zhang X | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Mechanism of auto-inhibition and activation of Mec1 checkpoint kinase. Authors: Elias A Tannous / Luke A Yates / Xiaodong Zhang / Peter M Burgers / ![]() ![]() Abstract: In response to DNA damage or replication fork stalling, the basal activity of Mec1 is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1 ...In response to DNA damage or replication fork stalling, the basal activity of Mec1 is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1 dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo-electron microscopy, we determine the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å and the wild type at 3.8 Å, both in complex with Mg-AMP-PNP. These structures yield a near-complete atomic model for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 124.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 18.3 KB 18.3 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 10.4 KB | Display | ![]() |
Images | ![]() | 242.8 KB | ||
Filedesc metadata | ![]() | 8.1 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6z2wMC ![]() 6z2xC ![]() 6z3aC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Mec1(F2244L)-Ddc2 map refined to 2.82 Angstroms and sharpened using a B-factor of -20 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Mec1-Ddc2
Entire | Name: Mec1-Ddc2 |
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Components |
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-Supramolecule #1: Mec1-Ddc2
Supramolecule | Name: Mec1-Ddc2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 Details: Mec1-Ddc2 expressed and purified from Yeast and incubated with Mg and AMP-PNP |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 720 KDa |
-Macromolecule #1: DNA damage checkpoint protein LCD1
Macromolecule | Name: DNA damage checkpoint protein LCD1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 86.533594 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MRRETVGEFS SDDDDDILLE LGTRPPRFTQ IPPSSAALQT QIPTTLEVTT TTLNNKQSKN DNQLVNQLNK AQGEASMLRD KINFLNIER EKEKNIQAVK VNELQVKHLQ ELAKLKQELQ KLEDEKKFLQ MEARGKSKRE VITNVKPPST TLSTNTNTIT P DSSSVAIE ...String: MRRETVGEFS SDDDDDILLE LGTRPPRFTQ IPPSSAALQT QIPTTLEVTT TTLNNKQSKN DNQLVNQLNK AQGEASMLRD KINFLNIER EKEKNIQAVK VNELQVKHLQ ELAKLKQELQ KLEDEKKFLQ MEARGKSKRE VITNVKPPST TLSTNTNTIT P DSSSVAIE AKPQSPQSKK RKISDNLLKK NMVPLNPNRI IPDETSLFLE SILLHQIIGA DLSTIEILNR LKLDYITEFK FK NFVIAKG APIGKSIVSL LLRCKKTLTL DRFIDTLLED IAVLIKEISV HPNESKLAVP FLVALMYQIV QFRPSATHNL ALK DCFLFI CDLIRIYHHV LKVPIHESNM NLHVEPQIFQ YELIDYLIIS YSFDLLEGIL RVLQSHPKQT YMEFFDENIL KSFE FVYKL ALTISYKPMV NVIFSAVEVV NIITSIILNM DNSSDLKSLI SGSWWRDCIT RLYALLEKEI KSGDVYNENV DTTTL HMSK YHDFFGLIRN IGDNELGGLI SKLIYTDRLQ SVPRVISKED IGMDSDKFTA PIIGYKMEKW LLKLKDEVLN IFENLL MIY GDDATIVNGE MLIHSSKFLS REQALMIERY VGQDSPNLDL RCHLIEHTLT IIYRLWKDHF KQLREEQIKQ VESQLIM SL WRFLVCQTET VTANEREMRD HRHLVDSLHD LTIKDQASYY EDAFEDLPEY IEEELKMQLN KRTGRIMQVK YDEKFQEM A RTILESKSFD LTTLEEADSL YISMGL UniProtKB: DNA damage checkpoint protein LCD1 |
-Macromolecule #2: Serine/threonine-protein kinase MEC1
Macromolecule | Name: Serine/threonine-protein kinase MEC1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 273.646812 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MESHVKYLDE LILAIKDLNS GVDSKVQIKK VPTDPSSSQE YAKSLKILNT LIRNLKDQRR NNIMKNDTIF SKTVSALALL LEYNPFLLV MKDSNGNFEI QRLIDDFLNI SVLNYDNYHR IWFMRRKLGS WCKACVEFYG KPAKFQLTAH FENTMNLYEQ A LTEVLLGK ...String: MESHVKYLDE LILAIKDLNS GVDSKVQIKK VPTDPSSSQE YAKSLKILNT LIRNLKDQRR NNIMKNDTIF SKTVSALALL LEYNPFLLV MKDSNGNFEI QRLIDDFLNI SVLNYDNYHR IWFMRRKLGS WCKACVEFYG KPAKFQLTAH FENTMNLYEQ A LTEVLLGK TELLKFYDTL KGLYILLYWF TSEYSTFGNS IAFLDSSLGF TKFDFNFQRL IRIVLYVFDS CELAALEYAE IQ LKYISLV VDYVCNRTIS TALDAPALVC CEQLKFVLTT MHHFLDNKYG LLDNDPTMAK GILRLYSLCI SNDFSKCFVD HFP IDQWAD FSQSEHFPFT QLTNKALSIV YFDLKRRSLP VEALKYDNKF NIWVYQSEPD SSLKNVTSPF DDRYKQLEKL RLLV LKKFN KTERGTLLKY RVNQLSPGFF QRAGNDFKLI LNEASVSIQT CFKTNNITRL TSWTVILGRL ACLESEKFSG TLPNS TKDM DNWYVCHLCD IEKTGNPFVR INPNRPEAAG KSEIFRILHS NFLSHPNIDE FSESLLSGIL FSLHRIFSHF QPPKLT DGN GQINKSFKLV QKCFMNSNRY LRLLSTRIIP LFNISDSHNS EDEHTATLIK FLQSQKLPVV KENLVIAWTQ LTLTTSN DV FDTLLLKLID IFNSDDYSLR IMMTLQIKNM AKILKKTPYQ LLSPILPVLL RQLGKNLVER KVGFQNLIEL LGYSSKTI L DIFQRYIIPY AIIQYKSDVL SEIAKIMCDG DTSLINQMKV NLLKKNSRQI FAVALVKHGL FSLDILETLF LNRAPTFDK GYITAYLPDY KTLAEITKLY KNSVTKDASD SENANMILCS LRFLITNFEK DKRHGSKYKN INNWTDDQEQ AFQKKLQDNI LGIFQVFSS DIHDVEGRTT YYEKLRVING ISFLIIYAPK KSIISALAQI SICLQTGLGL KEVRYEAFRC WHLLVRHLND E ELSTVIDS LIAFILQKWS EFNGKLRNIV YSILDTLIKE KSDLILKLKP YTTLALVGKP ELGILARDGQ FARMVNKIRS TT DLIPIFA NNLKSSNKYV INQNLDDIEV YLRRKQTERS IDFTPKKVGQ TSDITLVLGA LLDTSHKFRN LDKDLCEKCA KCI SMIGVL DVTKHEFKRT TYSENEVYDL NDSVQTIKFL IWVINDILVP AFWQSENPSK QLFVALVIQE SLKYCGLSSE SWDM NHKEL YPNEAKLWEK FNSVSKTTIY PLLSSLYLAQ SWKEYVPLKY PSNNFKEGYK IWVKRFTLDL LKTGTTENHP LHVFS SLIR EDDGSLSNFL LPYISLDIII KAEKGTPYAD ILNGIIIEFD SIFTCNLEGM NNLQVDSLRM CYESIFRVFE YCKKWA TEF KQNYSKLHGT FIIKDTKTTN MLLRIDEFLR TTPSDLLAQR SLETDSFERS ALYLEQCYRQ NPHDKNQNGQ LLKNLQI TY EEIGDIDSLD GVLRTFATGN LVSKIEELQY SENWKLAQDC FNVLGKFSDD PKTTTRMLKS MYDHQLYSQI ISNSSFHS S DGKISLSPDV KEWYSIGLEA ANLEGNVQTL KNWVEQIESL RNIDDREVLL QYNIAKALIA ISNEDPLRTQ KYIHNSFRL IGTNFITSSK ETTLLKKQNL LMKLHSLYDL SFLSSAKDKF EYKSNTTILD YRMERIGADF VPNHYILSMR KSFDQLKMNE QADADLGKT FFTLAQLARN NARLDIASES LMHCLERRLP QAELEFAEIL WKQGENDRAL KIVQEIHEKY QENSSVNARD R AAVLLKFT EWLDLSNNSA SEQIIKQYQD IFQIDSKWDK PYYSIGLYYS RLLERKKAEG YITNGRFEYR AISYFLLAFE KN TAKVREN LPKVITFWLD IAAASISEAP GNRKEMLSKA TEDICSHVEE ALQHCPTYIW YFVLTQLLSR LLHSHQSSAQ IIM HILLSL AVEYPSHILW YITALVNSNS SKRVLRGKHI LEKYRQHSQN PHDLVSSALD LTKALTRVCL QDVKSITSRS GKSL EKDFK FDMNVAPSAM VVPVRKNLDI ISPLESNSMR GYQPFRPVVS IIRFGSSYKV FSSLKKPKQL NIIGSDGNIY GIMCK KEDV RQDNQYMQFA TTMDFLLSKD IASRKRSLGI NIYSVLSLRE DCGILEMVPN VVTLRSILST KYESLKIKYS LKSLHD RWQ HTAVDGKLEF YMEQVDKFPP ILYQWFLENF PDPINWFNAR NTYARSYAVM AMVGHILGLG DRHCENILLD IQTGKVL HV DLDCLFEKGK RLPVPEIVPF RLTPNLLDAL GIIGTEGTFK KSSEVTLALM RKNEVALMNV IETIMYDRNM DHSIQKAL K VLRNKIRGID PQDGLVLSVA GQTETLIQEA TSEDNLSKMY IGWLPFW UniProtKB: Serine/threonine-protein kinase MEC1 |
-Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 2 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ![]() ChemComp-ANP: |
-Macromolecule #4: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #6: water
Macromolecule | Name: water / type: ligand / ID: 6 / Number of copies: 4 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ![]() ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.4 |
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Grid | Model: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | TFS KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 15902 / Average exposure time: 2.5 sec. / Average electron dose: 51.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |