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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 / Polo-like kinase mediated events / Transcription of E2F targets under negative control by DREAM complex / regulation of cyclin-dependent protein serine/threonine kinase activity / cyclin-dependent protein serine/threonine kinase regulator activity / Deregulated CDK5 triggers multiple neurodegenerative pathways in Alzheimer's disease models / cyclin A1-CDK2 complex / cyclin E2-CDK2 complex / cyclin E1-CDK2 complex ...positive regulation of G2/MI transition of meiotic cell cycle / cell cycle G1/S phase transition / Polo-like kinase mediated events / Transcription of E2F targets under negative control by DREAM complex / regulation of cyclin-dependent protein serine/threonine kinase activity / cyclin-dependent protein serine/threonine kinase regulator 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 / cyclin-dependent protein kinase activity / G2 Phase / 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 anaphase-promoting complex-dependent catabolic process / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / microtubule organizing center / centriole replication / Regulation of APC/C activators between G1/S and early anaphase / telomere maintenance in response to DNA damage / regulation of DNA replication / centrosome duplication / phosphoprotein phosphatase activity / Telomere Extension By Telomerase / G0 and Early G1 / Activation of the pre-replicative complex / cyclin-dependent kinase / cyclin-dependent protein serine/threonine kinase activity / TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest / Regulation of MITF-M-dependent genes involved in cell cycle and proliferation / Cajal body / 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 / Cyclin A:Cdk2-associated events at S phase entry / cyclin-dependent protein kinase holoenzyme complex / condensed chromosome / regulation of G2/M transition of mitotic cell cycle / mitotic G1 DNA damage checkpoint signaling / positive regulation of G2/M transition of mitotic cell cycle / protein tyrosine phosphatase activity / protein tyrosine phosphatase activity, metal-dependent / histone H2AXY142 phosphatase activity / protein-tyrosine-phosphatase / non-membrane spanning protein tyrosine phosphatase activity / cellular response to nitric oxide / post-translational protein modification / cyclin binding / regulation of mitotic cell cycle / male germ cell nucleus / positive regulation of DNA replication / meiotic cell cycle / Ubiquitin-Mediated Degradation of Phosphorylated Cdc25A / response to radiation / potassium ion transport / DNA Damage/Telomere Stress Induced Senescence / Meiotic recombination / CDK-mediated phosphorylation and removal of Cdc6 / SCF(Skp2)-mediated degradation of p27/p21 / G1/S transition of mitotic cell cycle / Transcriptional regulation of granulopoiesis / Orc1 removal from chromatin / G2/M transition of mitotic cell cycle / Cyclin D associated events in G1 / cellular senescence / Regulation of TP53 Degradation / nuclear envelope / peptidyl-serine phosphorylation / protein-folding chaperone binding / Factors involved in megakaryocyte development and platelet production / Processing of DNA double-strand break ends / regulation of gene expression / Senescence-Associated Secretory Phenotype (SASP) / Regulation of TP53 Activity through Phosphorylation / transcription regulator complex / Ras protein signal transduction / chromosome, telomeric region / cell population proliferation / DNA replication / endosome / Ub-specific processing proteases / protein phosphorylation / chromatin remodeling / protein domain specific binding / cell division / protein serine kinase activity / DNA repair / protein serine/threonine kinase activity / DNA-templated transcription / positive regulation of cell population proliferation / centrosome / protein kinase binding / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II
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 / : / Rhodanese-like domain / Rhodanese domain profile. / Rhodanese-like domain superfamily / Cyclin, C-terminal domain ...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 / : / Rhodanese-like domain / Rhodanese domain profile. / Rhodanese-like domain superfamily / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / Rhodanese-like domain / Cyclin, C-terminal domain / Cyclin_C / 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 (domestic 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: Nat Commun / Year: 2024
Title: Cryo-EM structure of the CDK2-cyclin A-CDC25A complex.
Authors: Rhianna J Rowland / Svitlana Korolchuk / Marco Salamina / Natalie J Tatum / James R Ault / Sam Hart / Johan P Turkenburg / James N Blaza / Martin E M Noble / Jane A Endicott /
Abstract: The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we ...The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further identify a CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.
History
DepositionJan 12, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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 (domestic 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
Has protein modificationY

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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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