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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 / Transferase
Function/homologySerine/threonine-protein kinase ATM / regulation of cellular response to gamma radiation / regulation of microglial cell activation / positive regulation of DNA catabolic process / signal transduction involved in mitotic G2 DNA damage checkpoint / positive regulation of telomerase catalytic core complex assembly / establishment of RNA localization to telomere / Telomere-length maintenance and DNA damage repair / Telomere-length maintenance and DNA damage repair / positive regulation of telomere maintenance via telomere lengthening ...Serine/threonine-protein kinase ATM / regulation of cellular response to gamma radiation / regulation of microglial cell activation / positive regulation of DNA catabolic process / signal transduction involved in mitotic G2 DNA damage checkpoint / positive regulation of telomerase catalytic core complex assembly / establishment of RNA localization to telomere / Telomere-length maintenance and DNA damage repair / Telomere-length maintenance and DNA damage repair / positive regulation of telomere maintenance via telomere lengthening / DNA-dependent protein kinase activity / cellular response to nitrosative stress / Sensing of DNA Double Strand Breaks / positive regulation of DNA damage response, signal transduction by p53 class mediator / establishment of protein-containing complex localization to telomere / negative regulation of telomere capping / pre-B cell allelic exclusion / meiotic telomere clustering / negative regulation of TORC1 signaling / cellular response to X-ray / immunoglobulin production / histone mRNA catabolic process / lipoprotein catabolic process / female meiotic nuclear division / male meiotic nuclear division / V(D)J recombination / PIK-related kinase, FAT / oocyte development / regulation of telomere maintenance via telomerase / peptidyl-serine autophosphorylation / FAT domain profile. / FATC domain profile. / PIK-related kinase / FATC domain / positive regulation of histone phosphorylation / reciprocal meiotic recombination / DNA repair complex / FAT domain / FATC domain / strand displacement / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Homologous DNA Pairing and Strand Exchange / DNA double-strand break processing / Resolution of D-loop Structures through Holliday Junction Intermediates / somitogenesis / response to ionizing radiation / mitotic spindle assembly checkpoint / HDR through Single Strand Annealing (SSA) / DNA synthesis involved in DNA repair / DNA damage induced protein phosphorylation / TP53 Regulates Transcription of Caspase Activators and Caspases / Phosphatidylinositol 3- and 4-kinases signature 1. / Presynaptic phase of homologous DNA pairing and strand exchange / negative regulation of B cell proliferation / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases family profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / TP53 Regulates Transcription of Genes Involved in Cytochrome C Release / Phosphatidylinositol 3-/4-kinase, catalytic domain / determination of adult lifespan / replicative senescence / Regulation of HSF1-mediated heat shock response / histone phosphorylation / regulation of autophagy / ovarian follicle development / Nonhomologous End-Joining (NHEJ) / post-embryonic development / positive regulation of telomere maintenance via telomerase / telomere maintenance / TP53 Regulates Transcription of DNA Repair Genes / Phosphatidylinositol 3- and 4-kinase / 1-phosphatidylinositol-3-kinase activity / double-strand break repair via nonhomologous end joining / Autodegradation of the E3 ubiquitin ligase COP1 / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / thymus development / HDR through Homologous Recombination (HRR) / Stabilization of p53 / double-strand break repair via homologous recombination / G2/M DNA damage checkpoint / multicellular organism growth / DNA Damage/Telomere Stress Induced Senescence / cellular response to gamma radiation / Regulation of TP53 Activity through Methylation / brain development / regulation of cellular response to heat / Meiotic recombination / spindle / positive regulation of neuron apoptotic process / neuron apoptotic process / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / cell cycle arrest / intrinsic apoptotic signaling pathway in response to DNA damage / Regulation of TP53 Degradation / Processing of DNA double-strand break ends / protein N-terminus binding / heart development / Armadillo-type fold / DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest
Function and homology information
Specimen sourceHomo sapiens / / human
MethodElectron microscopy (5.7 Å resolution / Particle / Single particle) / Transmission electron microscopy
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.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 13, 2017 / Release: May 17, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 17, 2017Structure modelrepositoryInitial release
1.1May 31, 2017Structure modelDatabase references
1.2Jul 26, 2017Structure modelData collection / Experimental preparationem_imaging_optics / em_sample_support / em_software_em_imaging_optics.energyfilter_name / _em_software.details / _em_software.name
1.3Aug 30, 2017Structure modelAuthor supporting evidence / Data collectionem_imaging_optics / pdbx_audit_support_em_imaging_optics.energyfilter_name / _pdbx_audit_support.funding_organization
1.4Jan 31, 2018Structure modelData processing / Experimental preparationem_sample_support / em_software_em_sample_support.grid_type / _em_software.details / _em_software.name
1.5Feb 7, 2018Structure modelData collectionem_imaging_optics_em_imaging_optics.energyfilter_lower

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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
Polyers352,3941
Non-polymers00
Water0
1


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

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Components

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


Mass: 352393.969 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens / / human / Cell line: HEK293 / Gene: ATM / Plasmid name: pDEST12.2-OriP / Cell line (production host): HEK293 / Production host: Homo sapiens
References: UniProt:Q13315, EC:2.7.11.1 (non-specific serine/threonine protein kinase)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Dimeric human ATM (Ataxia telangiectasia mutated) kinase
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.705 deg. / Units: MEGADALTONS / Experimental value: YES
Source (natural)Organism: Homo sapiens
Source (recombinant)Cell: HEK293 / Organism: Homo sapiens / Plasmid: pDEST12.2-OriP
Buffer solutionpH: 8
Buffer component
IDConc.UnitsNameFormulaBuffer ID
125mMHEPES pH 7.51
225mMTris pH 8.81
3150mMsodium chlorideNaCl1
40.01%Tween 201
52mMTCEP1
SpecimenConc.: 0.6 mg/ml
Details: The sample was purified by anti-FLAG affinity chromatography followed by overnight dialysis and a final gel-filtration.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
IDSpecimen IDGrid materialGrid mesh sizeGrid type
11GOLD300Quantifoil R1.2/1.3
21GOLD300Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 kelvins
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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 97902 / Calibrated magnification: 35714 / Nominal defocus max: 4000 nm / Nominal defocus min: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 80.15 kelvins
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) / Number of grids imaged: 4 / Number 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
14RELION1.4RECONSTRUCTION
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNumber of particles selected: 371671
SymmetryPoint symmetry: C1
3D reconstructionResolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 60556 / Number of class averages: 2 / Symmetry type: POINT
Atomic model buildingRef space: 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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