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- PDB-8bzo: Cryo-EM structure of CDK2-CyclinA in complex with p27 from the SC... -

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Basic information

Entry
Database: PDB / ID: 8bzo
TitleCryo-EM structure of CDK2-CyclinA in complex with p27 from the SCFSKP2 E3 ligase Complex
Components
  • Cyclin-A2
  • Cyclin-dependent kinase 2
  • Cyclin-dependent kinase inhibitor 1B
KeywordsCELL CYCLE / cyclin-dependent kinase / signalling / ubiquitination
Function / homology
Function and homology information


negative regulation of growth / cell cycle G1/S phase transition / cellular response to luteinizing hormone stimulus / G2/M DNA replication checkpoint / programmed cell death / cyclin-dependent protein serine/threonine kinase inhibitor activity / Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / cellular response to leptin stimulus / response to glucagon / male pronucleus ...negative regulation of growth / cell cycle G1/S phase transition / cellular response to luteinizing hormone stimulus / G2/M DNA replication checkpoint / programmed cell death / cyclin-dependent protein serine/threonine kinase inhibitor activity / Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / cellular response to leptin stimulus / response to glucagon / male pronucleus / cellular response to cocaine / female pronucleus / cyclin-dependent protein serine/threonine kinase regulator activity / positive regulation of DNA biosynthetic process / cellular response to insulin-like growth factor stimulus / cyclin A1-CDK2 complex / cyclin E2-CDK2 complex / cyclin E1-CDK2 complex / cyclin A2-CDK2 complex / G2 Phase / Y chromosome / cyclin-dependent protein kinase activity / regulation of heterochromatin organization / 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 / regulation of mitotic cell cycle phase transition / X chromosome / PTK6 Regulates Cell Cycle / 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 / telomere maintenance in response to DNA damage / animal organ regeneration / microtubule organizing center / regulation of DNA replication / G0 and Early G1 / cochlea development / Activation of the pre-replicative complex / cellular response to platelet-derived growth factor stimulus / Telomere Extension By Telomerase / 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 / 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 / centrosome duplication / Cyclin A:Cdk2-associated events at S phase entry / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / Cyclin A/B1/B2 associated events during G2/M transition / condensed chromosome / mitotic G1 DNA damage checkpoint signaling / cellular response to nitric oxide / post-translational protein modification / positive regulation of fibroblast proliferation / cyclin binding / positive regulation of DNA replication / negative regulation of protein localization to chromatin / regulation of mitotic cell cycle / cellular response to estradiol stimulus / G1/S transition of mitotic cell cycle / G2/M transition of mitotic cell cycle / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / meiotic cell cycle / peptidyl-serine phosphorylation / cellular senescence / DNA Damage/Telomere Stress Induced Senescence / Meiotic recombination / CDK-mediated phosphorylation and removal of Cdc6 / SCF(Skp2)-mediated degradation of p27/p21 / Transcriptional regulation of granulopoiesis / Orc1 removal from chromatin / Cyclin D associated events in G1 / Regulation of TP53 Degradation / nuclear envelope / transcription regulator complex / Factors involved in megakaryocyte development and platelet production / ciliary basal body / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / cellular response to hypoxia / Regulation of TP53 Activity through Phosphorylation / Ras protein signal transduction / DNA replication / protein phosphorylation / chromosome, telomeric region / endosome / Ub-specific processing proteases / chromatin remodeling / negative regulation of cell population proliferation / protein domain specific binding / protein serine kinase activity / cell division / DNA repair / protein serine/threonine kinase activity / centrosome
Similarity search - Function
Cyclin-dependent kinase inhibitor domain / Cyclin-dependent kinase inhibitor domain superfamily / Cyclin-dependent kinase inhibitor / Cyclin-A, N-terminal APC/C binding region / Cyclin-A N-terminal APC/C binding region / : / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin ...Cyclin-dependent kinase inhibitor domain / Cyclin-dependent kinase inhibitor domain superfamily / Cyclin-dependent kinase inhibitor / Cyclin-A, N-terminal APC/C binding region / Cyclin-A N-terminal APC/C binding region / : / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / 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 / : / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Cyclin-dependent kinase inhibitor 1B / Cyclin-A2 / Cyclin-dependent kinase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRowland, R.J. / Salamina, M. / Endicott, J.A. / Noble, M.E.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/N009738/1 and MR/V029142/1 United Kingdom
CitationJournal: Sci Rep / Year: 2023
Title: Cryo-EM structure of SKP1-SKP2-CKS1 in complex with CDK2-cyclin A-p27KIP1.
Authors: Rhianna J Rowland / Richard Heath / Daniel Maskell / Rebecca F Thompson / Neil A Ranson / James N Blaza / Jane A Endicott / Martin E M Noble / Marco Salamina /
Abstract: p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals ...p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals its recruitment to the SCF (S-phase kinase associated protein 1 (SKP1)-cullin-SKP2) E3 ubiquitin ligase complex for proteasomal degradation. The nature of p27 binding to SKP2 and CKS1 was revealed by the SKP1-SKP2-CKS1-p27 phosphopeptide crystal structure. Subsequently, a model for the hexameric CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex was proposed by overlaying an independently determined CDK2-cyclin A-p27 structure. Here we describe the experimentally determined structure of the isolated CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex at 3.4 Å global resolution using cryogenic electron microscopy. This structure supports previous analysis in which p27 was found to be structurally dynamic, transitioning from disordered to nascent secondary structure on target binding. We employed 3D variability analysis to further explore the conformational space of the hexameric complex and uncovered a previously unidentified hinge motion centred on CKS1. This flexibility gives rise to open and closed conformations of the hexameric complex that we propose may contribute to p27 regulation by facilitating recognition with SCF. This 3D variability analysis further informed particle subtraction and local refinement approaches to enhance the local resolution of the complex.
History
DepositionDec 15, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / 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: Cyclin-dependent kinase inhibitor 1B


Theoretical massNumber of molelcules
Total (without water)100,2313
Polymers100,2313
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8590 Å2
ΔGint-49 kcal/mol
Surface area27060 Å2
MethodPISA

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Components

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


Mass: 33943.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Thr160 phosphorylated CDK2 / Source: (gene. exp.) Homo sapiens (human) / Gene: CDK2, CDKN2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLYSs / References: UniProt: P24941, cyclin-dependent kinase
#2: Protein Cyclin-A2 / Cyclin-A / Cyclin A


Mass: 48609.574 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCNA2, CCN1, CCNA / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLYSs / References: UniProt: P20248
#3: Protein Cyclin-dependent kinase inhibitor 1B / Cyclin-dependent kinase inhibitor p27 / p27Kip1


Mass: 17678.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: p27 kip1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLYSs / References: UniProt: O43806
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: Complex of CDK2-CyclinA-p27(kip1)from the SCFSKP2 E3 ligase complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.074 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)pLYSs
Buffer solutionpH: 7.8
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 278.15 K

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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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 9 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategoryDetails (eV)
1cryoSPARCparticle selectionblob picker
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignmenthomogeneous refinement
11cryoSPARCfinal Euler assignmentnon-uniform refinement
12cryoSPARCclassification
13cryoSPARC3D reconstructionlocal refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1110356
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 136325 / Symmetry type: POINT
Atomic model buildingB value: 121 / Protocol: RIGID BODY FIT / Space: REAL
Details: Initial fitting was performed in chimera followed by real space refinement in Phenix
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0054972
ELECTRON MICROSCOPYf_angle_d0.7296776
ELECTRON MICROSCOPYf_dihedral_angle_d4.724670
ELECTRON MICROSCOPYf_chiral_restr0.042763
ELECTRON MICROSCOPYf_plane_restr0.007862

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