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

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Basic information

Entry
Database: PDB / ID: 6z2w
TitleMec1-Ddc2 (F2244L mutant) in complex with Mg AMP-PNP
Components
  • DNA damage checkpoint protein LCD1DNA repair
  • Serine/threonine-protein kinase MEC1
KeywordsHYDROLASE / serine/threonine protein kinase / complex / DNA damage response / checkpoint control
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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Serine/threonine-protein kinase MEC1 / DNA damage checkpoint protein LCD1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsYates, L.A. / Zhang, X.
Funding support United Kingdom, 1items
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, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 11, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 18, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 30, 2020Group: Structure summary / Category: struct_keywords / Item: _struct_keywords.text
Revision 1.3Jan 20, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.4Feb 10, 2021Group: Database references / Category: citation_author / Item: _citation_author.identifier_ORCID

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
D: DNA damage checkpoint protein LCD1
C: DNA damage checkpoint protein LCD1
E: Serine/threonine-protein kinase MEC1
F: Serine/threonine-protein kinase MEC1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)721,60112
Polymers720,3614
Non-polymers1,2408
Water724
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38730 Å2
ΔGint-214 kcal/mol
Surface area224130 Å2
MethodPISA

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Components

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Protein , 2 types, 4 molecules DCEF

#1: Protein DNA damage checkpoint protein LCD1 / DNA repair / DNA damage checkpoint protein 2 / Lethal / checkpoint-defective / DNA damage-sensitive protein 1


Mass: 86533.594 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: LCD1, DDC2, PIE1, YDR499W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04377
#2: Protein Serine/threonine-protein kinase MEC1 / ATR homolog / DNA-damage checkpoint kinase MEC1 / Mitosis entry checkpoint protein 1


Mass: 273646.812 Da / Num. of mol.: 2 / Mutation: F2244L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MEC1, ESR1, SAD3, YBR136W, YBR1012 / Plasmid: pBL904 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P38111, non-specific serine/threonine protein kinase

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Non-polymers , 4 types, 12 molecules

#3: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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
Molecular weightValue: 0.72 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: PY252 / Plasmid: pBL904
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: 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
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.13_2998refinement
PHENIX1.13_2998refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13cisTEMfinal Euler assignment
14RELION3classification
15cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 900000 / Details: Template-based picking
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53581 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5X6O

5x6o

RefinementStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00847532
ELECTRON MICROSCOPYf_angle_d1.164264362
ELECTRON MICROSCOPYf_chiral_restr0.0657380
ELECTRON MICROSCOPYf_plane_restr0.00748094
ELECTRON MICROSCOPYf_dihedral_angle_d13.473328722

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