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- EMDB-11050: Mec1-Ddc2 (F2244L mutant) in complex with Mg AMP-PNP -

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Basic information

Entry
Database: EMDB / ID: EMD-11050
TitleMec1-Ddc2 (F2244L mutant) in complex with Mg AMP-PNP
Map dataMec1(F2244L)-Ddc2 map refined to 2.82 Angstroms and sharpened using a B-factor of -20
Sample
  • Complex: Mec1-Ddc2
    • Protein or peptide: DNA damage checkpoint protein LCD1
    • Protein or peptide: Serine/threonine-protein kinase MEC1
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: water
Keywordsserine/threonine protein kinase / complex / DNA damage response / checkpoint control / Hydrolase
Function / homology
Function and homology information


ATR-ATRIP complex / positive regulation of DNA-templated DNA replication / regulation of double-strand break repair / telomere maintenance via recombination / reciprocal meiotic recombination / nucleobase-containing compound metabolic process / nuclear chromosome / signal transduction in response to DNA damage / telomere maintenance via telomerase / telomere maintenance ...ATR-ATRIP complex / positive regulation of DNA-templated DNA replication / regulation of double-strand break repair / telomere maintenance via recombination / reciprocal meiotic recombination / nucleobase-containing compound metabolic process / nuclear chromosome / signal transduction in response to DNA damage / telomere maintenance via telomerase / telomere maintenance / DNA damage checkpoint signaling / establishment of protein localization / chromatin organization / chromosome / DNA recombination / damaged DNA binding / eukaryotic translation initiation factor 2alpha kinase activity / DNA replication / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H2BS36 kinase activity / histone H3S57 kinase activity / histone H2AT120 kinase activity / AMP-activated protein kinase activity / histone H2AS1 kinase activity / histone H3T6 kinase activity / histone H3T11 kinase activity / histone H3T45 kinase activity / non-specific serine/threonine protein kinase / protein kinase activity / protein serine kinase activity / DNA repair / protein serine/threonine kinase activity / mitochondrion / ATP binding / nucleus / cytoplasm
Similarity search - Function
UME domain / UME (NUC010) domain / Domain in UVSB PI-3 kinase, MEI-41 and ESR-1 / DNA damage checkpoint protein, Lcd1 / DNA damage checkpoint protein / : / FATC domain / PIK-related kinase, FAT / FAT domain / FATC ...UME domain / UME (NUC010) domain / Domain in UVSB PI-3 kinase, MEI-41 and ESR-1 / DNA damage checkpoint protein, Lcd1 / DNA damage checkpoint protein / : / FATC domain / PIK-related kinase, FAT / FAT domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Serine/threonine-protein kinase MEC1 / DNA damage checkpoint protein LCD1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsYates LA / Zhang X
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust210658/Z/18/Z United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2021
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
DepositionMay 18, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.64
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.64
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6z2w
  • Surface level: 3.64
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11050.png

Downloads & links

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Map

FileDownload / File: emd_11050.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMec1(F2244L)-Ddc2 map refined to 2.82 Angstroms and sharpened using a B-factor of -20
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 328 pix.
= 347.68 Å		1.06 Å/pix.
x 328 pix.
= 347.68 Å		1.06 Å/pix.
x 328 pix.
= 347.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.64 / Movie #1: 3.64
Minimum - Maximum-9.919715 - 18.272696
Average (Standard dev.)0.021896932 (±0.9161645)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions328328328
Spacing328328328
CellA=B=C: 347.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z328328328
origin x/y/z0.0000.0000.000
length x/y/z347.680347.680347.680
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS328328328
D min/max/mean-9.92018.2730.022

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Supplemental data

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Sample components

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Entire : Mec1-Ddc2

EntireName: Mec1-Ddc2
Components
  • Complex: Mec1-Ddc2
    • Protein or peptide: DNA damage checkpoint protein LCD1
    • Protein or peptide: Serine/threonine-protein kinase MEC1
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Mec1-Ddc2

SupramoleculeName: 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
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 720 KDa

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Macromolecule #1: DNA damage checkpoint protein LCD1

MacromoleculeName: DNA damage checkpoint protein LCD1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 86.533594 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: 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

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Macromolecule #2: Serine/threonine-protein kinase MEC1

MacromoleculeName: 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
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 273.646812 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: 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

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 4 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 7.4
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 81000
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: 900000 / Details: Template-based picking
Startup modelType of model: EMDB MAP
EMDB ID:

4085

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 53581
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: RELION (ver. 3.0), cisTEM)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5x6o


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6z2w:
Mec1-Ddc2 (F2244L mutant) in complex with Mg AMP-PNP

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