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Open data
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Basic information
Entry | Database: PDB / ID: 6z2x | ||||||
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Title | Mec1-Ddc2 (F2244L mutant) in complex with Mg AMP-PNP (State II) | ||||||
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![]() | HYDROLASE / serine/threonine protein kinase / complex / DNA damage response / checkpoint control | ||||||
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 / chromosome / chromatin organization / DNA replication / DNA recombination / 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 | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
![]() | Yates, L.A. / 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
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 1 MB | Display | ![]() |
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PDB format | ![]() | 829.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 576 KB | Display | ![]() |
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Full document | ![]() | 624.1 KB | Display | |
Data in XML | ![]() | 107.1 KB | Display | |
Data in CIF | ![]() | 163.6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 11051MC ![]() 6z2wC ![]() 6z3aC C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 86533.594 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #2: Protein | Mass: 273646.812 Da / Num. of mol.: 2 / Mutation: F2244L Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: P38111, non-specific serine/threonine protein kinase #3: Chemical | #4: Chemical | #5: Chemical | ChemComp-MG / Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Mec1-Ddc2 / Type: COMPLEX Details: Mec1-Ddc2 expressed and purified from Yeast and incubated with Mg and AMP-PNP Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 0.72 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.5 sec. / Electron dose: 51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15902 |
Image scans | Width: 5760 / Height: 4092 |
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Processing
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 900000 / Details: Template-based picking | ||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12205 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6Z2W Accession code: 6Z2W / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Stereochemistry target values: GeoStd + Monomer Library | ||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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