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- EMDB-16344: Cryo-EM structure of CDK2-CyclinA in complex with p27 from the SC... -

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Entry
Database: EMDB / ID: EMD-16344
TitleCryo-EM structure of CDK2-CyclinA in complex with p27 from the SCFSKP2 E3 ligase Complex
Map datafull map
Sample
  • Complex: Complex of CDK2-CyclinA-p27(kip1)from the SCFSKP2 E3 ligase complex
    • Protein or peptide: Cyclin-dependent kinase 2
    • Protein or peptide: Cyclin-A2
    • Protein or peptide: Cyclin-dependent kinase inhibitor 1B
Keywordscell cycle / cyclin-dependent kinase / signalling / ubiquitination
Function / homology
Function and homology information


negative regulation of growth / : / cyclin A2-CDK1 complex / cell cycle G1/S phase transition / cellular response to luteinizing hormone stimulus / 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 / programmed cell death / male pronucleus ...negative regulation of growth / : / cyclin A2-CDK1 complex / cell cycle G1/S phase transition / cellular response to luteinizing hormone stimulus / 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 / programmed cell death / male pronucleus / female pronucleus / cellular response to cocaine / response to glucagon / cyclin-dependent protein serine/threonine kinase regulator activity / positive regulation of DNA biosynthetic process / regulation of mitotic cell cycle phase transition / cellular response to insulin-like growth factor stimulus / 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 / Y chromosome / cyclin-dependent protein kinase activity / 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) / centriole replication / Regulation of APC/C activators between G1/S and early anaphase / telomere maintenance in response to DNA damage / regulation of DNA replication / microtubule organizing center / centrosome duplication / G0 and Early G1 / cochlea development / animal organ regeneration / Telomere Extension By Telomerase / 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 / Activation of ATR in response to replication stress / Regulation of MITF-M-dependent genes involved in cell cycle and proliferation / Cyclin E associated events during G1/S transition / Cajal body / Cyclin A:Cdk2-associated events at S phase entry / Cyclin A/B1/B2 associated events during G2/M transition / cyclin-dependent protein kinase holoenzyme complex / regulation of G2/M transition of mitotic cell cycle / condensed chromosome / cellular response to platelet-derived growth factor stimulus / mitotic G1 DNA damage checkpoint signaling / cellular response to nitric oxide / post-translational protein modification / regulation of mitotic cell cycle / cyclin binding / positive regulation of DNA replication / male germ cell nucleus / meiotic cell cycle / cellular response to estradiol stimulus / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / G1/S transition of mitotic cell cycle / peptidyl-serine phosphorylation / potassium ion transport / DNA Damage/Telomere Stress Induced Senescence / Meiotic recombination / CDK-mediated phosphorylation and removal of Cdc6 / G2/M transition of mitotic cell cycle / SCF(Skp2)-mediated degradation of p27/p21 / positive regulation of fibroblast proliferation / Transcriptional regulation of granulopoiesis / Orc1 removal from chromatin / Cyclin D associated events in G1 / cellular senescence / Regulation of TP53 Degradation / nuclear envelope / Factors involved in megakaryocyte development and platelet production / regulation of gene expression / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / transcription regulator complex / cellular response to hypoxia / Regulation of TP53 Activity through Phosphorylation / Ras protein signal transduction / DNA replication / chromosome, telomeric region / protein phosphorylation / endosome / Ub-specific processing proteases / chromatin remodeling / protein domain specific binding / negative regulation of cell population proliferation / cell division / protein serine kinase activity / DNA repair
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 / : / 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 inhibitor 1B / Cyclin-A2 / Cyclin-dependent kinase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRowland RJ / Salamina M / Endicott JA / Noble ME
Funding support United Kingdom, 1 items
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, 2022-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16344.png

Downloads & links

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Map

FileDownload / File: emd_16344.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 300 pix.
= 321. Å		1.07 Å/pix.
x 300 pix.
= 321. Å		1.07 Å/pix.
x 300 pix.
= 321. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.5772796 - 1.8392177
Average (Standard dev.)-0.00065121794 (±0.029441096)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16344_msk_1.map
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Half map: half map A

Fileemd_16344_half_map_1.map
Annotationhalf map A
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Half map: half map B

Fileemd_16344_half_map_2.map
Annotationhalf map B
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Sample components

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Entire : Complex of CDK2-CyclinA-p27(kip1)from the SCFSKP2 E3 ligase complex

EntireName: Complex of CDK2-CyclinA-p27(kip1)from the SCFSKP2 E3 ligase complex
Components
  • Complex: Complex of CDK2-CyclinA-p27(kip1)from the SCFSKP2 E3 ligase complex
    • Protein or peptide: Cyclin-dependent kinase 2
    • Protein or peptide: Cyclin-A2
    • Protein or peptide: Cyclin-dependent kinase inhibitor 1B

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Supramolecule #1: Complex of CDK2-CyclinA-p27(kip1)from the SCFSKP2 E3 ligase complex

SupramoleculeName: Complex of CDK2-CyclinA-p27(kip1)from the SCFSKP2 E3 ligase complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 74 KDa

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Macromolecule #1: Cyclin-dependent kinase 2

MacromoleculeName: Cyclin-dependent kinase 2 / type: protein_or_peptide / ID: 1 / Details: Thr160 phosphorylated CDK2 / Number of copies: 1 / Enantiomer: LEVO / EC number: cyclin-dependent kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 33.943312 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MENFQKVEKI GEGTYGVVYK ARNKLTGEVV ALKKIRLDTE TEGVPSTAIR EISLLKELNH PNIVKLLDVI HTENKLYLVF EFLHQDLKK FMDASALTGI PLPLIKSYLF QLLQGLAFCH SHRVLHRDLK PQNLLINTEG AIKLADFGLA RAFGVPVRTY (TPO)HEVVTLWY ...String:
MENFQKVEKI GEGTYGVVYK ARNKLTGEVV ALKKIRLDTE TEGVPSTAIR EISLLKELNH PNIVKLLDVI HTENKLYLVF EFLHQDLKK FMDASALTGI PLPLIKSYLF QLLQGLAFCH SHRVLHRDLK PQNLLINTEG AIKLADFGLA RAFGVPVRTY (TPO)HEVVTLWY RAPEILLGCK YYSTAVDIWS LGCIFAEMVT RRALFPGDSE IDQLFRIFRT LGTPDEVVWP GVTSMPD YK PSFPKWARQD FSKVVPPLDE DGRSLLSQML HYDPNKRISA KAALAHPFFQ DVTKPVPHLR

UniProtKB: Cyclin-dependent kinase 2

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Macromolecule #2: Cyclin-A2

MacromoleculeName: Cyclin-A2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48.609574 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLGNSAPGPA TREAGSALLA LQQTALQEDQ ENINPEKAAP VQQPRTRAAL AVLKSGNPRG LAQQQRPKTR RVAPLKDLPV NDEHVTVPP WKANSKQPAF TIHVDEAEKE AQKKPAESQK IEREDALAFN SAISLPGPRK PLVPLDYPMD GSFESPHTMD M SIILEDEK ...String:
MLGNSAPGPA TREAGSALLA LQQTALQEDQ ENINPEKAAP VQQPRTRAAL AVLKSGNPRG LAQQQRPKTR RVAPLKDLPV NDEHVTVPP WKANSKQPAF TIHVDEAEKE AQKKPAESQK IEREDALAFN SAISLPGPRK PLVPLDYPMD GSFESPHTMD M SIILEDEK PVSVNEVPDY HEDIHTYLRE MEVKCKPKVG YMKKQPDITN SMRAILVDWL VEVGEEYKLQ NETLHLAVNY ID RFLSSMS VLRGKLQLVG TAAMLLASKF EEIYPPEVAE FVYITDDTYT KKQVLRMEHL VLKVLTFDLA APTVNQFLTQ YFL HQQPAN CKVESLAMFL GELSLIDADP YLKYLPSVIA GAAFHLALYT VTGQSWPESL IRKTGYTLES LKPCLMDLHQ TYLK APQHA QQSIREKYKN SKYHGVSLLN PPETLNL

UniProtKB: Cyclin-A2

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Macromolecule #3: Cyclin-dependent kinase inhibitor 1B

MacromoleculeName: Cyclin-dependent kinase inhibitor 1B / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.678531 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSNVRVSNGS PSLERMDARQ AEHPKPSACR NLFGPVDHEE LTRDLEKHCR DMEEASQRKW NFDFQNHKPL EGKYEWQEVE KGSLPEFYY RPPRPPKGAC KVPAQESQDG SGSRPAAPLI GAPANSEDTH LVDPKTDPSD SQTGLAEQCA GIRKRPATD

UniProtKB: Cyclin-dependent kinase inhibitor 1B

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 278.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average exposure time: 9.0 sec. / Average electron dose: 65.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1110356
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1jsu

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: local refinement / Number images used: 136325
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Software - details: homogeneous refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Software - details: non-uniform refinement
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial fitting was performed in chimera followed by real space refinement in Phenix
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 121
Output model

PDB-8bzo:
Cryo-EM structure of CDK2-CyclinA in complex with p27 from the SCFSKP2 E3 ligase Complex

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