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- EMDB-16325: Cryo-EM structure of SKP1-SKP2-CKS1-CDK2-CyclinA-p27KIP1 Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-16325
TitleCryo-EM structure of SKP1-SKP2-CKS1-CDK2-CyclinA-p27KIP1 Complex
Map dataFull map
Sample
  • Complex: Hexameric complex of SKP1-SKP2-CKS1 with CDK2-CyclinA-p27(kip1)
    • Protein or peptide: Cyclin-dependent kinase 2
    • Protein or peptide: Cyclin-A2
    • Protein or peptide: Cyclin-dependent kinase inhibitor 1B
    • Protein or peptide: S-phase kinase-associated protein 1
    • Protein or peptide: S-phase kinase-associated protein 2
    • Protein or peptide: Cyclin-dependent kinases regulatory subunit 1
    • Protein or peptide: p27 KIP1 C-terminus
Keywordscell cycle / cyclin-dependent kinase / signalling / ubiquitination
Function / homology
Function and homology information


positive regulation of protein polyubiquitination / F-box domain binding / Phosphorylation of proteins involved in the G2/M transition by Cyclin A:Cdc2 complexes / cyclin A2-CDK1 complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / PcG protein complex / cell cycle G1/S phase transition / cellular response to luteinizing hormone stimulus / mitotic cell cycle phase transition / Cul7-RING ubiquitin ligase complex ...positive regulation of protein polyubiquitination / F-box domain binding / Phosphorylation of proteins involved in the G2/M transition by Cyclin A:Cdc2 complexes / cyclin A2-CDK1 complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / PcG protein complex / cell cycle G1/S phase transition / cellular response to luteinizing hormone stimulus / mitotic cell cycle phase transition / Cul7-RING ubiquitin ligase complex / Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / positive regulation of ubiquitin protein ligase activity / cyclin-dependent protein serine/threonine kinase inhibitor activity / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / maintenance of protein location in nucleus / cellular response to leptin stimulus / male pronucleus / cyclin-dependent protein serine/threonine kinase activator activity / female pronucleus / cellular response to cocaine / response to glucagon / positive regulation of intracellular estrogen receptor signaling pathway / cyclin-dependent protein serine/threonine kinase regulator activity / cellular response to insulin-like growth factor stimulus / SCF ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / positive regulation of DNA biosynthetic process / cochlea development / cyclin A1-CDK2 complex / cyclin E2-CDK2 complex / cyclin E1-CDK2 complex / cellular response to platelet-derived growth factor stimulus / 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 / Prolactin receptor signaling / X chromosome / PTK6 Regulates Cell Cycle / regulation of DNA replication / regulation of anaphase-promoting complex-dependent catabolic process / protein monoubiquitination / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / centriole replication / cullin family protein binding / Regulation of APC/C activators between G1/S and early anaphase / protein K63-linked ubiquitination / centrosome duplication / Telomere Extension By Telomerase / G0 and Early G1 / ubiquitin-like ligase-substrate adaptor activity / positive regulation of double-strand break repair via homologous recombination / negative regulation of mitotic cell cycle / Activation of the pre-replicative complex / cellular response to nitric oxide / cyclin-dependent protein kinase holoenzyme complex / cyclin-dependent kinase / animal organ regeneration / Nuclear events stimulated by ALK signaling in cancer / protein K48-linked ubiquitination / 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 / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / cyclin binding / post-translational protein modification / : / meiotic cell cycle / positive regulation of DNA replication / ubiquitin binding / SCF-beta-TrCP mediated degradation of Emi1 / male germ cell nucleus / NIK-->noncanonical NF-kB signaling / molecular function activator activity / cellular response to estradiol stimulus / Vpu mediated degradation of CD4 / Dectin-1 mediated noncanonical NF-kB signaling / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Degradation of GLI1 by the proteasome / Activation of NF-kappaB in B cells / Negative regulation of NOTCH4 signaling / Iron uptake and transport / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis
Similarity search - Function
Cyclin-dependent kinase inhibitor domain / Cyclin-dependent kinase inhibitor domain superfamily / Cyclin-dependent kinase inhibitor / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit / Cyclin-dependent kinases regulatory subunits signature 1. / Cyclin-dependent kinases regulatory subunits signature 2. / Cyclin-dependent kinase regulatory subunit / Leucine-rich repeat, cysteine-containing subtype ...Cyclin-dependent kinase inhibitor domain / Cyclin-dependent kinase inhibitor domain superfamily / Cyclin-dependent kinase inhibitor / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit / Cyclin-dependent kinases regulatory subunits signature 1. / Cyclin-dependent kinases regulatory subunits signature 2. / Cyclin-dependent kinase regulatory subunit / Leucine-rich repeat, cysteine-containing subtype / Leucine-rich repeat - CC (cysteine-containing) subfamily / A Receptor for Ubiquitination Targets / F-box domain profile. / F-box-like domain superfamily / F-box-like / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / Cyclin-A, N-terminal APC/C binding region / Cyclin-A N-terminal APC/C binding region / : / F-box domain / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / Cyclin, C-terminal domain / Cyclin_C / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Cyclin, N-terminal / Cyclin, N-terminal domain / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Cyclin-like superfamily / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat domain 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 / Cyclin-dependent kinases regulatory subunit 1 / S-phase kinase-associated protein 1 / S-phase kinase-associated protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 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 12, 2022-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateJul 19, 2023-
Current statusJul 19, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16325.png

Downloads & links

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Map

FileDownload / File: emd_16325.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.159
Minimum - Maximum-1.4381531 - 2.1350794
Average (Standard dev.)-0.00039038886 (±0.028598841)
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_16325_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_16325_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_16325_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : Hexameric complex of SKP1-SKP2-CKS1 with CDK2-CyclinA-p27(kip1)

EntireName: Hexameric complex of SKP1-SKP2-CKS1 with CDK2-CyclinA-p27(kip1)
Components
  • Complex: Hexameric complex of SKP1-SKP2-CKS1 with CDK2-CyclinA-p27(kip1)
    • Protein or peptide: Cyclin-dependent kinase 2
    • Protein or peptide: Cyclin-A2
    • Protein or peptide: Cyclin-dependent kinase inhibitor 1B
    • Protein or peptide: S-phase kinase-associated protein 1
    • Protein or peptide: S-phase kinase-associated protein 2
    • Protein or peptide: Cyclin-dependent kinases regulatory subunit 1
    • Protein or peptide: p27 KIP1 C-terminus

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Supramolecule #1: Hexameric complex of SKP1-SKP2-CKS1 with CDK2-CyclinA-p27(kip1)

SupramoleculeName: Hexameric complex of SKP1-SKP2-CKS1 with CDK2-CyclinA-p27(kip1)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 175 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: 33.994398 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MENFQKVEKI GEGTYGVVYK ARNKLTGEVV ALKKIRLDTE TEGVPSTAIR EISLLKELNH PNIVKLLDVI HTENKLYLVF EFLHQDLKK FMDASALTGI PLPLIKSYLF QLLQGLAFCH SHRVLHRDLK PQNLLINTEG AIKLADFGLA RAFGVPVRT (TPO)THEVVTLWY ...String:
MENFQKVEKI GEGTYGVVYK ARNKLTGEVV ALKKIRLDTE TEGVPSTAIR EISLLKELNH PNIVKLLDVI HTENKLYLVF EFLHQDLKK FMDASALTGI PLPLIKSYLF QLLQGLAFCH SHRVLHRDLK PQNLLINTEG AIKLADFGLA RAFGVPVRT (TPO)THEVVTLWY RAPEILLGCK YYSTAVDIWS LGCIFAEMVT RRALFPGDSE IDQLFRIFRT LGTPDEVVWP GVTSMP DYK PSFPKWARQD FSKVVPPLDE DGRSLLSQML HYDPNKRISA KAALAHPFFQ DVTKPVPHLR L

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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Macromolecule #4: S-phase kinase-associated protein 1

MacromoleculeName: S-phase kinase-associated protein 1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.679965 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MPSIKLQSSD GEIFEVDVEI AKQSVTIKTM LEDLGMDDEG DDDPVPLPNV NAAILKKVIQ WCTHHKDDPP PPEDDENKEK RTDDIPVWD QEFLKVDQGT LFELILAANY LDIKGLLDVT CKTVANMIKG KTPEEIRKTF NIKNDFTEEE EAQVRKENQW C EEK

UniProtKB: S-phase kinase-associated protein 1

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Macromolecule #5: S-phase kinase-associated protein 2

MacromoleculeName: S-phase kinase-associated protein 2 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.817785 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHRKHLQEIP DLSSNVATSF TWGWDSSKTS ELLSGMGVSA LEKEEPDSEN IPQELLSNLG HPESPPRKRL KSKGSDKDFV IVRRPKLNR ENFPGVSWDS LPDELLLGIF SCLCLPELLK VSGVCKRWYR LASDESLWQT LDLTGKNLHP DVTGRLLSQG V IAFRCPRS ...String:
MHRKHLQEIP DLSSNVATSF TWGWDSSKTS ELLSGMGVSA LEKEEPDSEN IPQELLSNLG HPESPPRKRL KSKGSDKDFV IVRRPKLNR ENFPGVSWDS LPDELLLGIF SCLCLPELLK VSGVCKRWYR LASDESLWQT LDLTGKNLHP DVTGRLLSQG V IAFRCPRS FMDQPLAEHF SPFRVQHMDL SNSVIEVSTL HGILSQCSKL QNLSLEGLRL SDPIVNTLAK NSNLVRLNLS GC SGFSEFA LQTLLSSCSR LDELNLSWCF DFTEKHVQVA VAHVSETITQ LNLSGYRKNL QKSDLSTLVR RCPNLVHLDL SDS VMLKND CFQEFFQLNY LQHLSLSRCY DIIPETLLEL GEIPTLKTLQ VFGIVPDGTL QLLKEALPHL QINCSHFTTI ARPT IGNKK NQEIWGIKCR LTLQKPSCL

UniProtKB: S-phase kinase-associated protein 2

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Macromolecule #6: Cyclin-dependent kinases regulatory subunit 1

MacromoleculeName: Cyclin-dependent kinases regulatory subunit 1 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.679211 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSHKQIYYSD KYDDEEFEYR HVMLPKDIAK LVPKTHLMSE SEWRNLGVQQ SQGWVHYMIH EPEPHILLFR RPLPKKPKK

UniProtKB: Cyclin-dependent kinases regulatory subunit 1

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Macromolecule #7: p27 KIP1 C-terminus

MacromoleculeName: p27 KIP1 C-terminus / type: protein_or_peptide / ID: 7 / Details: C-terminus of p27 KIP1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.126154 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
AGSVEQ(TPO)PKK

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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:

2ast


Details: Using PDB 2AST and 1JSU
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / 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 classificationNumber classes: 3 / Software - Name: cryoSPARC / Software - details: heterogenous refinement
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-8bya:
Cryo-EM structure of SKP1-SKP2-CKS1-CDK2-CyclinA-p27KIP1 Complex

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