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- PDB-5np1: Open protomer of human ATM (Ataxia telangiectasia mutated) -

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Basic information

Entry
Database: PDB / ID: 5np1
TitleOpen protomer of human ATM (Ataxia telangiectasia mutated)
ComponentsSerine-protein kinase ATM
KeywordsTRANSFERASE / FAT / MRN / DNA-repair / HEAT-repeats
Function / homology
Function and homology information


positive regulation of DNA catabolic process / establishment of RNA localization to telomere / positive regulation of telomerase catalytic core complex assembly / cellular response to nitrosative stress / negative regulation of telomere capping / positive regulation of DNA damage response, signal transduction by p53 class mediator / establishment of protein-containing complex localization to telomere / regulation of microglial cell activation / Sensing of DNA Double Strand Breaks / positive regulation of telomere maintenance via telomere lengthening ...positive regulation of DNA catabolic process / establishment of RNA localization to telomere / positive regulation of telomerase catalytic core complex assembly / cellular response to nitrosative stress / negative regulation of telomere capping / positive regulation of DNA damage response, signal transduction by p53 class mediator / establishment of protein-containing complex localization to telomere / regulation of microglial cell activation / Sensing of DNA Double Strand Breaks / positive regulation of telomere maintenance via telomere lengthening / meiotic telomere clustering / lipoprotein catabolic process / pre-B cell allelic exclusion / DNA-dependent protein kinase activity / histone H2AXS139 kinase activity / male meiotic nuclear division / histone mRNA catabolic process / female meiotic nuclear division / regulation of telomere maintenance via telomerase / pexophagy / cellular response to X-ray / peptidyl-serine autophosphorylation / V(D)J recombination / oocyte development / Impaired BRCA2 binding to PALB2 / reciprocal meiotic recombination / DNA repair complex / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / 1-phosphatidylinositol-3-kinase activity / response to ionizing radiation / mitotic spindle assembly checkpoint signaling / TP53 Regulates Transcription of Caspase Activators and Caspases / negative regulation of B cell proliferation / mitotic G2 DNA damage checkpoint signaling / Presynaptic phase of homologous DNA pairing and strand exchange / peroxisomal matrix / TP53 Regulates Transcription of Genes Involved in Cytochrome C Release / replicative senescence / positive regulation of cell adhesion / Regulation of HSF1-mediated heat shock response / somitogenesis / regulation of cellular response to heat / DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest / cellular response to retinoic acid / signal transduction in response to DNA damage / ovarian follicle development / negative regulation of TORC1 signaling / positive regulation of telomere maintenance via telomerase / Pexophagy / telomere maintenance / post-embryonic development / thymus development / regulation of signal transduction by p53 class mediator / DNA damage checkpoint signaling / regulation of autophagy / determination of adult lifespan / TP53 Regulates Transcription of DNA Repair Genes / Nonhomologous End-Joining (NHEJ) / Stabilization of p53 / double-strand break repair via homologous recombination / Autodegradation of the E3 ubiquitin ligase COP1 / brain development / multicellular organism growth / cellular response to gamma radiation / HDR through Homologous Recombination (HRR) / G2/M DNA damage checkpoint / Regulation of TP53 Activity through Methylation / DNA Damage/Telomere Stress Induced Senescence / spindle / Meiotic recombination / cellular response to reactive oxygen species / double-strand break repair via nonhomologous end joining / positive regulation of neuron apoptotic process / double-strand break repair / intrinsic apoptotic signaling pathway in response to DNA damage / cellular senescence / Regulation of TP53 Degradation / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / heart development / Processing of DNA double-strand break ends / cytoplasmic vesicle / peptidyl-serine phosphorylation / neuron apoptotic process / regulation of apoptotic process / Regulation of TP53 Activity through Phosphorylation / protein autophosphorylation / response to hypoxia / regulation of cell cycle / non-specific serine/threonine protein kinase / positive regulation of cell migration / positive regulation of apoptotic process / protein phosphorylation / protein serine kinase activity
Similarity search - Function
Telomere-length maintenance and DNA damage repair / Serine/threonine-protein kinase ATM, plant / ATM, catalytic domain / Telomere-length maintenance and DNA damage repair / Telomere-length maintenance and DNA damage repair / PIK-related kinase, FAT / FAT domain / FATC domain / FATC / FATC domain ...Telomere-length maintenance and DNA damage repair / Serine/threonine-protein kinase ATM, plant / ATM, catalytic domain / Telomere-length maintenance and DNA damage repair / Telomere-length maintenance and DNA damage repair / 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 / Armadillo-type fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Serine-protein kinase ATM
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.7 Å
AuthorsBaretic, D. / Pollard, H.K. / Fisher, D.I. / Johnson, C.M. / Santhanam, B. / Truman, C.M. / Kouba, T. / Fersht, A.R. / Phillips, C. / Williams, R.L.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184308 United Kingdom
LMB/AstraZenecaMC_A024-5PF9H United Kingdom
CitationJournal: Sci Adv / Year: 2017
Title: Structures of closed and open conformations of dimeric human ATM.
Authors: Domagoj Baretić / Hannah K Pollard / David I Fisher / Christopher M Johnson / Balaji Santhanam / Caroline M Truman / Tomas Kouba / Alan R Fersht / Christopher Phillips / Roger L Williams /
Abstract: ATM (ataxia-telangiectasia mutated) is a phosphatidylinositol 3-kinase-related protein kinase (PIKK) best known for its role in DNA damage response. ATM also functions in oxidative stress response, ...ATM (ataxia-telangiectasia mutated) is a phosphatidylinositol 3-kinase-related protein kinase (PIKK) best known for its role in DNA damage response. ATM also functions in oxidative stress response, insulin signaling, and neurogenesis. Our electron cryomicroscopy (cryo-EM) suggests that human ATM is in a dynamic equilibrium between closed and open dimers. In the closed state, the PIKK regulatory domain blocks the peptide substrate-binding site, suggesting that this conformation may represent an inactive or basally active enzyme. The active site is held in this closed conformation by interaction with a long helical hairpin in the TRD3 (tetratricopeptide repeats domain 3) domain of the symmetry-related molecule. The open dimer has two protomers with only a limited contact interface, and it lacks the intermolecular interactions that block the peptide-binding site in the closed dimer. This suggests that the open conformation may be more active. The ATM structure shows the detailed topology of the regulator-interacting N-terminal helical solenoid. The ATM conformational dynamics shown by the structures represent an important step in understanding the enzyme regulation.
History
DepositionApr 13, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 17, 2017Provider: repository / Type: Initial release
Revision 1.1May 31, 2017Group: Database references
Revision 1.2Jul 26, 2017Group: Data collection / Experimental preparation
Category: em_imaging_optics / em_sample_support / em_software
Item: _em_imaging_optics.energyfilter_name / _em_software.details / _em_software.name
Revision 1.3Aug 30, 2017Group: Author supporting evidence / Data collection / Category: em_imaging_optics / pdbx_audit_support
Item: _em_imaging_optics.energyfilter_name / _pdbx_audit_support.funding_organization
Revision 1.4Jan 31, 2018Group: Data processing / Experimental preparation / Category: em_sample_support / em_software
Item: _em_sample_support.grid_type / _em_software.details / _em_software.name
Revision 1.5Feb 7, 2018Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.energyfilter_lower
Revision 1.6Oct 24, 2018Group: Advisory / Data collection / Derived calculations / Category: pdbx_validate_close_contact / struct_conn
Revision 1.7Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: Serine-protein kinase ATM


Theoretical massNumber of molelcules
Total (without water)352,3941
Polymers352,3941
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, SEC-MALS indicates monodisperse sample of 0.7 MDa.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Serine-protein kinase ATM / Ataxia telangiectasia mutated / A-T mutated


Mass: 352393.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293 / Gene: ATM / Plasmid: pDEST12.2-OriP / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
References: UniProt: Q13315, non-specific serine/threonine protein kinase

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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: Dimeric human ATM (Ataxia telangiectasia mutated) kinase
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.705 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / Plasmid: pDEST12.2-OriP
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES pH 7.51
225 mMTris pH 8.81
3150 mMsodium chlorideNaClSodium chloride1
40.01 %Tween 201
52 mMTCEP1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was purified by anti-FLAG affinity chromatography followed by overnight dialysis and a final gel-filtration.
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid type
11GOLD300Quantifoil R1.2/1.3
21GOLD300Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: 3 uL sample/grid blotted for 12 s before plunge-freezing

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 97902 X / Calibrated magnification: 35714 X / Nominal defocus max: 4000 nm / Nominal defocus min: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 80.15 K
Image recordingAverage exposure time: 0.8 sec. / Electron dose: 2.1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 2720
EM imaging opticsEnergyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Gautomatchv0.5particle selection
3UCSFImage4image acquisition
5Gctfv0.5CTF correction
8Coot0.8.1model fitting
10PHENIX1.10.1_2155model refinementphenix_real_space
11RELION1.4initial Euler assignment
12RELION1.4final Euler assignment
13RELION1.4classification
14RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 371671
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60556 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01312184
ELECTRON MICROSCOPYf_angle_d1.38916923
ELECTRON MICROSCOPYf_dihedral_angle_d7.8577153
ELECTRON MICROSCOPYf_chiral_restr0.0532367
ELECTRON MICROSCOPYf_plane_restr0.0092419

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