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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 regulator activity / negative regulation of cardiac muscle tissue regeneration / autophagic cell death / FOXO-mediated transcription of cell cycle genes / regulation of cell cycle G1/S phase transition / synaptic assembly at neuromuscular junction / F-box domain binding / cellular response to lithium ion / Aberrant regulation of mitotic exit in cancer due to RB1 defects / negative regulation of mitotic cell cycle ...cyclin-dependent protein kinase regulator activity / negative regulation of cardiac muscle tissue regeneration / autophagic cell death / FOXO-mediated transcription of cell cycle genes / regulation of cell cycle G1/S phase transition / synaptic assembly at neuromuscular junction / F-box domain binding / cellular response to lithium ion / Aberrant regulation of mitotic exit in cancer due to RB1 defects / negative regulation of mitotic cell cycle / cyclin-dependent protein serine/threonine kinase inhibitor activity / PcG protein complex / regulation of xenophagy / maintenance of protein location in nucleus / regulation of cyclin-dependent protein serine/threonine kinase activity / RHO GTPases activate CIT / nuclear export / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / Cul7-RING ubiquitin ligase complex / cyclin-dependent protein serine/threonine kinase activator activity / regulation of cell cycle process / neural crest cell differentiation / AKT phosphorylates targets in the cytosol / ubiquitin ligase activator activity / regulation of BMP signaling pathway / regulation of mitophagy / molecular function inhibitor activity / regulation of centrosome duplication / regulation of TOR signaling / SCF ubiquitin ligase complex / p53-Dependent G1 DNA Damage Response / regulation of DNA damage checkpoint / PTK6 Regulates Cell Cycle / Constitutive Signaling by AKT1 E17K in Cancer / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Prolactin receptor signaling / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / Cul4A-RING E3 ubiquitin ligase complex / protein kinase inhibitor activity / ubiquitin ligase complex scaffold activity / negative regulation of vascular associated smooth muscle cell proliferation / limb development / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / cyclin-dependent protein kinase holoenzyme complex / cullin family protein binding / TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest / Cyclin E associated events during G1/S transition / centrosome duplication / Cyclin A:Cdk2-associated events at S phase entry / protein K63-linked ubiquitination / cilium assembly / positive regulation of double-strand break repair via homologous recombination / ubiquitin-like ligase-substrate adaptor activity / regulation of G1/S transition of mitotic cell cycle / Nuclear events stimulated by ALK signaling in cancer / protein K48-linked ubiquitination / FLT3 Signaling / DNA damage response, signal transduction by p53 class mediator / cyclin binding / positive regulation of DNA replication / molecular function activator activity / regulation of mitotic cell cycle / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / G1/S transition of mitotic cell cycle / ubiquitin binding / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / Vpu mediated degradation of CD4 / negative regulation of cell growth / Dectin-1 mediated noncanonical NF-kB signaling / Degradation of CRY and PER proteins / Activation of NF-kappaB in B cells / Iron uptake and transport / Degradation of GLI1 by the proteasome / negative regulation of epithelial cell proliferation / cellular senescence / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Negative regulation of NOTCH4 signaling / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / beta-catenin binding / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / GSK3B-mediated proteasomal degradation of PD-L1(CD274) / DNA Damage/Telomere Stress Induced Senescence / NOTCH1 Intracellular Domain Regulates Transcription / regulation of circadian rhythm / Degradation of beta-catenin by the destruction complex / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / CLEC7A (Dectin-1) signaling / SCF(Skp2)-mediated degradation of p27/p21 / FCERI mediated NF-kB activation / heart development / protein polyubiquitination / Ubiquitin-Mediated Degradation of Phosphorylated Cdc25A / Interleukin-1 signaling / Orc1 removal from chromatin / positive regulation of protein catabolic process
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 / F-box-like domain superfamily / 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-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing 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)
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: 0.149
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
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Half map: #1

Fileemd_16327_half_map_1.map
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Half map: #2

Fileemd_16327_half_map_2.map
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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
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

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
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Software - details: homogenous refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Software - details: local refinement
Final 3D classificationNumber classes: 1 / Software - 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: 122
Output model

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

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