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- EMDB-16327: Cryo-EM structure of SKP1-SKP2-CKS1 from the SCFSKP2 E3 ligase complex -

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Entry
Database: EMDB / ID: EMD-16327
TitleCryo-EM structure of SKP1-SKP2-CKS1 from the SCFSKP2 E3 ligase complex
Map data
Sample
  • Complex: Locally refined complex of SKP1-SKP2-CKS1-p27 from the hexametric SCFSKP2 E3 Ligase complex
    • 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: Cyclin-dependent kinase inhibitor 1B
Keywordscell cycle / cyclin-dependent kinase / signalling / ubiquitinationCell cycle
Function / homology
Function and homology information


cyclin-dependent protein kinase activating kinase regulator activity / regulation of lens fiber cell differentiation / negative regulation of cardiac muscle tissue regeneration / negative regulation of cyclin-dependent protein kinase activity / autophagic cell death / positive regulation of protein polyubiquitination / negative regulation of epithelial cell proliferation involved in prostate gland development / FOXO-mediated transcription of cell cycle genes / F-box domain binding / Aberrant regulation of mitotic exit in cancer due to RB1 defects ...cyclin-dependent protein kinase activating kinase regulator activity / regulation of lens fiber cell differentiation / negative regulation of cardiac muscle tissue regeneration / negative regulation of cyclin-dependent protein kinase activity / autophagic cell death / positive regulation of protein polyubiquitination / negative regulation of epithelial cell proliferation involved in prostate gland development / FOXO-mediated transcription of cell cycle genes / F-box domain binding / Aberrant regulation of mitotic exit in cancer due to RB1 defects / PcG protein complex / regulation of cell cycle G1/S phase transition / regulation of exit from mitosis / epithelial cell proliferation involved in prostate gland development / negative regulation of phosphorylation / mitotic cell cycle phase transition / negative regulation of epithelial cell apoptotic process / negative regulation of cyclin-dependent protein serine/threonine kinase activity / ubiquitin ligase activator activity / Cul7-RING ubiquitin ligase complex / positive regulation of ubiquitin protein ligase activity / maintenance of protein location in nucleus / cyclin-dependent protein serine/threonine kinase inhibitor activity / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / nuclear export / RHO GTPases activate CIT / cyclin-dependent protein serine/threonine kinase activator activity / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / AKT phosphorylates targets in the cytosol / Cul4A-RING E3 ubiquitin ligase complex / epithelial cell apoptotic process / SCF ubiquitin ligase complex / cellular response to antibiotic / positive regulation of intracellular estrogen receptor signaling pathway / negative regulation of kinase activity / ubiquitin ligase complex scaffold activity / molecular function inhibitor activity / protein kinase inhibitor activity / cellular response to lithium ion / p53-Dependent G1 DNA Damage Response / Prolactin receptor signaling / PTK6 Regulates Cell Cycle / protein monoubiquitination / regulation of cyclin-dependent protein serine/threonine kinase activity / regulation of G1/S transition of mitotic cell cycle / Constitutive Signaling by AKT1 E17K in Cancer / cullin family protein binding / inner ear development / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / negative regulation of vascular associated smooth muscle cell proliferation / ubiquitin-like ligase-substrate adaptor activity / negative regulation of mitotic cell cycle / cellular response to organic cyclic compound / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / cyclin-dependent protein kinase holoenzyme complex / protein K48-linked ubiquitination / response to amino acid / localization / response to cadmium ion / response to glucose / TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest / Nuclear events stimulated by ALK signaling in cancer / Cyclin E associated events during G1/S transition / DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest / Cyclin A:Cdk2-associated events at S phase entry / positive regulation of microtubule polymerization / regulation of cell migration / Notch signaling pathway / Hsp70 protein binding / regulation of mitotic cell cycle / FLT3 Signaling / Regulation of BACH1 activity / cyclin binding / MAP3K8 (TPL2)-dependent MAPK1/3 activation / positive regulation of DNA replication / placenta development / ubiquitin binding / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / molecular function activator activity / Vpu mediated degradation of CD4 / Dectin-1 mediated noncanonical NF-kB signaling / sensory perception of sound / 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 / G1/S transition of mitotic cell cycle / 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 / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / Degradation of beta-catenin by the destruction complex / potassium ion transport / DNA Damage/Telomere Stress Induced Senescence / negative regulation of cell growth / response to peptide hormone / NOTCH1 Intracellular Domain Regulates Transcription / CLEC7A (Dectin-1) signaling
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 / F-box domain / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
Cyclin-dependent kinase inhibitor 1B / 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: 3.5 Å
AuthorsRowland RJ / Salamina M / Endicott JA / Noble MEM
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 13, 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_16327.png

Downloads & links

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Map

FileDownload / File: emd_16327.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.149
Minimum - Maximum-1.5415878 - 2.1927319
Average (Standard dev.)-0.0006498481 (±0.028245226)
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_16327_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16327_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16327_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Locally refined complex of SKP1-SKP2-CKS1-p27 from the hexametric...

EntireName: Locally refined complex of SKP1-SKP2-CKS1-p27 from the hexametric SCFSKP2 E3 Ligase complex
Components
  • Complex: Locally refined complex of SKP1-SKP2-CKS1-p27 from the hexametric SCFSKP2 E3 Ligase complex
    • 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: Cyclin-dependent kinase inhibitor 1B

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Supramolecule #1: Locally refined complex of SKP1-SKP2-CKS1-p27 from the hexametric...

SupramoleculeName: Locally refined complex of SKP1-SKP2-CKS1-p27 from the hexametric SCFSKP2 E3 Ligase complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 66 KDa

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

MacromoleculeName: S-phase kinase-associated protein 1 / type: protein_or_peptide / ID: 1 / 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 #2: S-phase kinase-associated protein 2

MacromoleculeName: S-phase kinase-associated protein 2 / type: protein_or_peptide / ID: 2 / 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 #3: Cyclin-dependent kinases regulatory subunit 1

MacromoleculeName: Cyclin-dependent kinases regulatory subunit 1 / type: protein_or_peptide / ID: 3 / 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 #4: Cyclin-dependent kinase inhibitor 1B

MacromoleculeName: Cyclin-dependent kinase inhibitor 1B / type: protein_or_peptide / ID: 4 / Details: DGSPNAGSVEQ(TPO)PKK / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.188303 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSNVRVSNGS PSLERMDARQ AEHPKPSACR NLFGPVDHEE LTRDLEKHCR DMEEASQRKW NFDFQNHKPL EGKYEWQEVE KGSLPEFYY RPPRPPKGAC KVPAQESQDV SGSRPAAPLI GAPANSEDTH LVDPKTDPSD SQTGLAEQCA GIRKRPATDD S STQNKRAN ...String:
MSNVRVSNGS PSLERMDARQ AEHPKPSACR NLFGPVDHEE LTRDLEKHCR DMEEASQRKW NFDFQNHKPL EGKYEWQEVE KGSLPEFYY RPPRPPKGAC KVPAQESQDV SGSRPAAPLI GAPANSEDTH LVDPKTDPSD SQTGLAEQCA GIRKRPATDD S STQNKRAN RTEENVSDGS PNAGSVEQ(TPO)P KKPGLRRRQT

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

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

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Software - details: homogenous refinement
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Software - details: local refinement
Final reconstructionNumber classes used: 1 / Applied 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: Particle subtraction followed by local refinement
Number images used: 136325
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: 122
Output model

PDB-8byl:
Cryo-EM structure of SKP1-SKP2-CKS1 from the SCFSKP2 E3 ligase complex

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