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- EMDB-11056: Mec1-Ddc2 (wild-type) apo -

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Basic information

Entry
Database: EMDB / ID: EMD-11056
TitleMec1-Ddc2 (wild-type) apo
Map dataMec1-Ddc2 (wild-type) apo reconstruction refined to 4.3 Angstroms resolution and sharpened with a b-factor -200
Sample
  • Complex: Mec1-Ddc2 heterotetramer (dimer of heterodimers)
Function / homology
Function and homology information


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 / DNA recombination / DNA replication / damaged DNA binding / non-specific serine/threonine protein kinase / protein kinase activity / phosphorylation / DNA repair / protein serine kinase activity / 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 / PIK-related kinase, FAT / FAT domain / FATC domain / FATC / FATC domain ...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 / PIK-related kinase, FAT / FAT domain / FATC 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 (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 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 19, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateJan 27, 2021-
Current statusJan 27, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.32
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.32
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11056.png

Downloads & links

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Map

FileDownload / File: emd_11056.map.gz / Format: CCP4 / Size: 233.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMec1-Ddc2 (wild-type) apo reconstruction refined to 4.3 Angstroms resolution and sharpened with a b-factor -200
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 2.32 / Movie #1: 2.32
Minimum - Maximum-6.1894403 - 11.562079
Average (Standard dev.)0.003143511 (±0.46343762)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions394394394
Spacing394394394
CellA=B=C: 413.69998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z394394394
origin x/y/z0.0000.0000.000
length x/y/z413.700413.700413.700
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS394394394
D min/max/mean-6.18911.5620.003

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Supplemental data

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Sample components

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Entire : Mec1-Ddc2 heterotetramer (dimer of heterodimers)

EntireName: Mec1-Ddc2 heterotetramer (dimer of heterodimers)
Components
  • Complex: Mec1-Ddc2 heterotetramer (dimer of heterodimers)

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Supramolecule #1: Mec1-Ddc2 heterotetramer (dimer of heterodimers)

SupramoleculeName: Mec1-Ddc2 heterotetramer (dimer of heterodimers) / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 720 KDa

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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 - 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: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 15097 / Average exposure time: 0.5 sec. / Average electron dose: 58.5 e/Å2
Details: ~2/3 of the data were collected at -30 degree stage tilt
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 75000
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: 2000000 / Details: low resolution template-based picking
CTF correctionSoftware - Name: Gctf
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: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: cisTEM, RELION (ver. 3.0)) / Number images used: 132193
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6z3a

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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