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- PDB-8roz: Cryo-EM structure of CDK2-cyclin A in complex with CDC25A -

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Basic information

Entry
Database: PDB / ID: 8roz
TitleCryo-EM structure of CDK2-cyclin A in complex with CDC25A
Components
  • Cyclin-A2
  • Cyclin-dependent kinase 2
  • M-phase inducer phosphatase 1
KeywordsCELL CYCLE / cell-cycle / CDK / phosphatase / cancer
Function / homology
Function and homology information


positive regulation of G2/MI transition of meiotic cell cycle / cell cycle G1/S phase transition / mitotic cell cycle phase transition / Transcription of E2F targets under negative control by DREAM complex / Polo-like kinase mediated events / cyclin-dependent protein serine/threonine kinase regulator activity / regulation of cyclin-dependent protein serine/threonine kinase activity / Deregulated CDK5 triggers multiple neurodegenerative pathways in Alzheimer's disease models / cyclin A1-CDK2 complex / cyclin E2-CDK2 complex ...positive regulation of G2/MI transition of meiotic cell cycle / cell cycle G1/S phase transition / mitotic cell cycle phase transition / Transcription of E2F targets under negative control by DREAM complex / Polo-like kinase mediated events / cyclin-dependent protein serine/threonine kinase regulator activity / regulation of cyclin-dependent protein serine/threonine kinase activity / Deregulated CDK5 triggers multiple neurodegenerative pathways in Alzheimer's disease models / cyclin A1-CDK2 complex / cyclin E2-CDK2 complex / cyclin E1-CDK2 complex / cyclin A2-CDK2 complex / positive regulation of DNA-templated DNA replication initiation / G2 Phase / cyclin-dependent protein kinase activity / Y chromosome / Phosphorylation of proteins involved in G1/S transition by active Cyclin E:Cdk2 complexes / positive regulation of heterochromatin formation / p53-Dependent G1 DNA Damage Response / X chromosome / PTK6 Regulates Cell Cycle / regulation of DNA replication / regulation of anaphase-promoting complex-dependent catabolic process / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / centriole replication / Regulation of APC/C activators between G1/S and early anaphase / phosphoprotein phosphatase activity / centrosome duplication / Telomere Extension By Telomerase / G0 and Early G1 / Activation of the pre-replicative complex / cellular response to nitric oxide / cyclin-dependent protein kinase holoenzyme complex / cyclin-dependent kinase / cyclin-dependent protein serine/threonine kinase activity / TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest / Activation of ATR in response to replication stress / Cyclin E associated events during G1/S transition / Cyclin A/B1/B2 associated events during G2/M transition / Cajal body / Cyclin A:Cdk2-associated events at S phase entry / condensed chromosome / mitotic G1 DNA damage checkpoint signaling / regulation of mitotic cell cycle / regulation of G2/M transition of mitotic cell cycle / positive regulation of G2/M transition of mitotic cell cycle / cyclin binding / protein-tyrosine-phosphatase / post-translational protein modification / : / meiotic cell cycle / positive regulation of DNA replication / protein tyrosine phosphatase activity / male germ cell nucleus / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / potassium ion transport / response to radiation / DNA Damage/Telomere Stress Induced Senescence / CDK-mediated phosphorylation and removal of Cdc6 / Meiotic recombination / SCF(Skp2)-mediated degradation of p27/p21 / Orc1 removal from chromatin / G1/S transition of mitotic cell cycle / Transcriptional regulation of granulopoiesis / G2/M transition of mitotic cell cycle / Cyclin D associated events in G1 / cellular response to UV / cellular senescence / Regulation of TP53 Degradation / nuclear envelope / Factors involved in megakaryocyte development and platelet production / Processing of DNA double-strand break ends / protein-folding chaperone binding / Senescence-Associated Secretory Phenotype (SASP) / regulation of gene expression / peptidyl-serine phosphorylation / DNA replication / Regulation of TP53 Activity through Phosphorylation / cell population proliferation / transcription regulator complex / Ras protein signal transduction / chromosome, telomeric region / Ub-specific processing proteases / endosome / chromatin remodeling / protein domain specific binding / cell division / protein phosphorylation / DNA repair / protein serine kinase activity / protein serine/threonine kinase activity / DNA-templated transcription / centrosome / positive regulation of cell population proliferation / protein kinase binding / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / magnesium ion binding / signal transduction / nucleoplasm
Similarity search - Function
M-phase inducer phosphatase / M-phase inducer phosphatase / Cyclin-A, N-terminal APC/C binding region / Cyclin-A N-terminal APC/C binding region / : / Rhodanese Homology Domain / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin ...M-phase inducer phosphatase / M-phase inducer phosphatase / Cyclin-A, N-terminal APC/C binding region / Cyclin-A N-terminal APC/C binding region / : / Rhodanese Homology Domain / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / Rhodanese-like domain / Rhodanese domain profile. / Rhodanese-like domain superfamily / Cyclin, C-terminal domain / Cyclin_C / Rhodanese-like domain / Cyclin, N-terminal / Cyclin, N-terminal domain / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Cyclin-like superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Cyclin-dependent kinase 2 / Cyclin-A2 / M-phase inducer phosphatase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsRowland, R.J. / Noble, M.E.M. / Endicott, J.A.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/V029142/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N009738/1 United Kingdom
CitationJournal: To Be Published
Title: Cryo-EM structure of the CDK2-cyclin A-CDC25A Complex
Authors: Rowland, R.J. / Korolchuk, S. / Salamina, M. / Ault, J.R. / Hart, S. / Turkenburg, J.P. / Blaza, J.N. / Noble, M.E.M. / Endicott, J.A.
History
DepositionJan 12, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cyclin-dependent kinase 2
B: Cyclin-A2
C: M-phase inducer phosphatase 1


Theoretical massNumber of molelcules
Total (without water)86,7893
Polymers86,7893
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Cyclin-dependent kinase 2 / Cell division protein kinase 2 / p33 protein kinase


Mass: 34136.449 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human cyclin dependant protein kinase 2 (CDK2) phosphorylated at Tyr15 and Thr160
Source: (gene. exp.) Homo sapiens (human) / Gene: CDK2, CDKN2 / Plasmid: PGEX6P1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): STAR / References: UniProt: P24941, cyclin-dependent kinase
#2: Protein Cyclin-A2 / Cyclin-A


Mass: 30119.799 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Bovine cyclin A2 / Source: (gene. exp.) Bos taurus (cattle) / Gene: CCNA2 / Plasmid: PGEX6P1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta pLysS / References: UniProt: P30274
#3: Protein M-phase inducer phosphatase 1 / Dual specificity phosphatase Cdc25A


Mass: 22533.123 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: M-phase inducer phosphatase 1 (CDC25A) catalytic domain
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC25A / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS / References: UniProt: P30304, protein-tyrosine-phosphatase
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: Trimeric complex of CDK2-cyclin A-CDC25A / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.086 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 7.4 / Details: 50 mM HEPES, 200 mM NaCl, 1 mM DTT, pH 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESHEPES1
2200 mMSaltNaCl1
31 mMDTTDTT1
SpecimenConc.: 1.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were glow discharged for 1min at 20 mAmp/0.26 mBar
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K

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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 FIELD / Nominal magnification: 150000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.92 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1Topazparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCinitial Euler assignmentAb initio
10cryoSPARCfinal Euler assignmentnon-uniform refinement
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIX1.20.1_4487:model refinement
CTF correctionDetails: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4981705
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 670830 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 144 / Protocol: AB INITIO MODEL / Space: REAL
Details: A model was generated using ModelAngelo. Initial local fitting was performed in ChimerX followed by real space refinement in Phenix and manual fitting in coot.
Atomic model buildingDetails: ModelAngelo / Source name: Other / Type: in silico model

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