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- PDB-9fgq: Structure of human APC3loop 375-381 bound to the NCP -

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Entry
Database: PDB / ID: 9fgq
TitleStructure of human APC3loop 375-381 bound to the NCP
Components
  • Cell division cycle protein 27 homolog
  • DNA (131-MER)
  • DNA (132-MER)
  • Histone H2A type 2-A
  • Histone H2B type 1-B
  • Histone H3.1
  • Histone H4
KeywordsCELL CYCLE / Arginine anchor / NCP / APC3 / Complex
Function / homology
Function and homology information


Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / protein branched polyubiquitination / metaphase/anaphase transition of mitotic cell cycle / regulation of meiotic cell cycle / anaphase-promoting complex-dependent catabolic process / Phosphorylation of the APC/C ...Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / protein branched polyubiquitination / metaphase/anaphase transition of mitotic cell cycle / regulation of meiotic cell cycle / anaphase-promoting complex-dependent catabolic process / Phosphorylation of the APC/C / protein K11-linked ubiquitination / Regulation of APC/C activators between G1/S and early anaphase / Transcriptional Regulation by VENTX / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / protein K48-linked ubiquitination / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Deposition of new CENPA-containing nucleosomes at the centromere / telomere organization / Inhibition of DNA recombination at telomere / Meiotic synapsis / Interleukin-7 signaling / RNA Polymerase I Promoter Opening / APC/C:Cdc20 mediated degradation of Cyclin B / APC-Cdc20 mediated degradation of Nek2A / Assembly of the ORC complex at the origin of replication / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / SUMOylation of chromatin organization proteins / regulation of mitotic cell cycle / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / SIRT1 negatively regulates rRNA expression / epigenetic regulation of gene expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / Assembly of the pre-replicative complex / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / G2/M DNA damage checkpoint / HDMs demethylate histones / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / NoRC negatively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / B-WICH complex positively regulates rRNA expression / PKMTs methylate histone lysines / Meiotic recombination / Pre-NOTCH Transcription and Translation / CDK-mediated phosphorylation and removal of Cdc6 / spindle / Metalloprotease DUBs / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / HCMV Early Events / neuron projection development / mitotic spindle / structural constituent of chromatin / Separation of Sister Chromatids / UCH proteinases / nucleosome / Antigen processing: Ubiquitination & Proteasome degradation / heterochromatin formation / E3 ubiquitin ligases ubiquitinate target proteins / nucleosome assembly / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / HATs acetylate histones / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Factors involved in megakaryocyte development and platelet production / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / protein phosphatase binding / Oxidative Stress Induced Senescence / gene expression / Estrogen-dependent gene expression / chromosome, telomeric region / Ub-specific processing proteases / protein ubiquitination / cadherin binding
Similarity search - Function
Anaphase-promoting complex, cyclosome, subunit 3 / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / : / Histone H2B signature. / Histone H2B ...Anaphase-promoting complex, cyclosome, subunit 3 / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / : / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Cell division cycle protein 27 homolog / Histone H2B type 1-B / Histone H4 / Histone H3.1 / Histone H2A type 2-A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsYoung, R.V.C. / Muhammad, R. / Alfieri, C.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
The Institute of Cancer Research (ICR) United Kingdom
CitationJournal: EMBO J / Year: 2024
Title: Spatial control of the APC/C ensures the rapid degradation of cyclin B1.
Authors: Luca Cirillo / Rose Young / Sapthaswaran Veerapathiran / Annalisa Roberti / Molly Martin / Azzah Abubacar / Camilla Perosa / Catherine Coates / Reyhan Muhammad / Theodoros I Roumeliotis / ...Authors: Luca Cirillo / Rose Young / Sapthaswaran Veerapathiran / Annalisa Roberti / Molly Martin / Azzah Abubacar / Camilla Perosa / Catherine Coates / Reyhan Muhammad / Theodoros I Roumeliotis / Jyoti S Choudhary / Claudio Alfieri / Jonathon Pines /
Abstract: The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is effected by the Anaphase Promoting Complex/Cyclosome (APC/C) ...The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is effected by the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The SAC prevents the APC/C from recognising Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live-cell imaging, in vitro reconstitution biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross-correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an 'arginine-anchor' in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the arginine anchor on Cyclin B1 reduces its interaction with the APC/C and delays its degradation: cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes promote the efficient interaction between Cyclin B1 and the APC/C to ensure the timely degradation of Cyclin B1 and genomic stability.
History
DepositionMay 24, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
K: Cell division cycle protein 27 homolog
L: Cell division cycle protein 27 homolog
A: Histone H3.1
B: Histone H4
C: Histone H2A type 2-A
D: Histone H2B type 1-B
E: Histone H3.1
F: Histone H4
G: Histone H2A type 2-A
H: Histone H2B type 1-B
I: DNA (132-MER)
J: DNA (131-MER)


Theoretical massNumber of molelcules
Total (without water)336,67712
Polymers336,67712
Non-polymers00
Water1,35175
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 10 molecules KLAEBFCGDH

#1: Protein Cell division cycle protein 27 homolog / Anaphase-promoting complex subunit 3 / APC3 / CDC27 homolog / CDC27Hs / H-NUC


Mass: 32788.488 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: This is a disorder loop of human APC3 residues 177-446 fused to a SpyTag via a 27 residue GSA linker.
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC27, ANAPC3, D0S1430E, D17S978E / Plasmid: pFastBac1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P30260
#2: Protein Histone H3.1 / Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone ...Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone H3/i / Histone H3/j / Histone H3/k / Histone H3/l


Mass: 15437.167 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Human histone H3.1 / Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H3A, H3FA, HIST1H3B, H3FL, HIST1H3C, H3FC, HIST1H3D, H3FB, HIST1H3E, H3FD, HIST1H3F, H3FI, HIST1H3G, H3FH, HIST1H3H, H3FK, HIST1H3I, H3FF, HIST1H3J, H3FJ
Production host: Escherichia coli (E. coli) / References: UniProt: P68431
#3: Protein Histone H4


Mass: 11394.426 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Human histone H4 / Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, ...Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, H4FE, HIST1H4K, H4/D, H4FD, HIST1H4L, H4/K, H4FK, HIST2H4A, H4/N, H4F2, H4FN, HIST2H4, HIST2H4B, H4/O, H4FO, HIST4H4
Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#4: Protein Histone H2A type 2-A / H2A-clustered histone 18 / H2A-clustered histone 19 / Histone H2A.2 / Histone H2A/o


Mass: 14125.549 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: This is a H2A/H2B fusion protein with a SpyCatcher tag attached
Source: (gene. exp.) Homo sapiens (human)
Gene: H2AC18, H2AFO, HIST2H2AA, HIST2H2AA3, H2AC19, HIST2H2AA4
Plasmid: pET-14b / Production host: Escherichia coli (E. coli) / References: UniProt: Q6FI13
#5: Protein Histone H2B type 1-B / H2B-clustered histone 3 / Histone H2B.1 / Histone H2B.f / H2B/f


Mass: 29445.771 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: This is a H2A/H2B fusion protein with a SpyCatcher tag attached,This is a H2A/H2B fusion protein with a SpyCatcher tag attached
Source: (gene. exp.) Homo sapiens (human) / Gene: H2BC3, H2BFF, HIST1H2BB / Plasmid: pET-14b / Production host: Escherichia coli (E. coli) / References: UniProt: P33778

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DNA chain , 2 types, 2 molecules IJ

#6: DNA chain DNA (132-MER)


Mass: 64911.336 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The Widom 147 bp sequence with 32 nucleotides of DNA on either side
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#7: DNA chain DNA (131-MER)


Mass: 65382.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Widom 147 DNA sequence flanked with 32 nucleotides on either side
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Non-polymers , 1 types, 75 molecules

#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 75 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationN

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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: APC3 motif bound to the NCP acidic patch / Type: COMPLEX
Details: Recombinant protein sample of residues 375-381 of APC3
Entity ID: #1-#3, #6-#7 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 20 mM HEPEs pH8.0, 50 mM NaCl, 0.5 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPEsC8H18N2O4S1
250 mMSodium chlorideNaCl1
30.5 mMTCEPC9H15O6P1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Image recordingElectron dose: 62 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
13RELION5.0-beta-latest3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2698450
Details: Template base particel picking and Topaz trained particle picking
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 414277 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 50.81 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003412177
ELECTRON MICROSCOPYf_angle_d0.561717554
ELECTRON MICROSCOPYf_chiral_restr0.0351995
ELECTRON MICROSCOPYf_plane_restr0.00411308
ELECTRON MICROSCOPYf_dihedral_angle_d30.59653494

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