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- PDB-6f0x: Cryo-EM structure of TRIP13 in complex with ATP gamma S, p31comet... -

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Basic information

Entry
Database: PDB / ID: 6f0x
TitleCryo-EM structure of TRIP13 in complex with ATP gamma S, p31comet, C-Mad2 and Cdc20
Components
  • Cell division cycle protein 20 homologCell cycle
  • MAD2L1-binding protein
  • Mitotic spindle assembly checkpoint protein MAD2A
  • Pachytene checkpoint protein 2 homolog
KeywordsCELL CYCLE / AAA+ ATPase / remodeller / spindle assembly checkpoint (SAC) / mitosis / chromosome segregation
Function / homology
Function and homology information


deactivation of mitotic spindle assembly checkpoint / metaphase/anaphase transition of cell cycle / mitotic spindle assembly checkpoint MAD1-MAD2 complex / metaphase/anaphase transition of meiosis I / Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components / mitotic checkpoint complex / positive regulation of anaphase-promoting complex-dependent catabolic process / meiotic recombination checkpoint signaling / positive regulation of mitotic cell cycle spindle assembly checkpoint / establishment of centrosome localization ...deactivation of mitotic spindle assembly checkpoint / metaphase/anaphase transition of cell cycle / mitotic spindle assembly checkpoint MAD1-MAD2 complex / metaphase/anaphase transition of meiosis I / Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components / mitotic checkpoint complex / positive regulation of anaphase-promoting complex-dependent catabolic process / meiotic recombination checkpoint signaling / positive regulation of mitotic cell cycle spindle assembly checkpoint / establishment of centrosome localization / regulation of meiotic nuclear division / positive regulation of synapse maturation / 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 / synaptonemal complex assembly / Phosphorylation of Emi1 / anaphase-promoting complex / anaphase-promoting complex-dependent catabolic process / regulation of meiotic cell cycle / positive regulation of mitotic metaphase/anaphase transition / regulation of exit from mitosis / positive regulation of synaptic plasticity / nuclear pore nuclear basket / anaphase-promoting complex binding / ubiquitin ligase activator activity / positive regulation of ubiquitin protein ligase activity / oocyte maturation / reciprocal meiotic recombination / female meiosis I / negative regulation of ubiquitin protein ligase activity / mitotic sister chromatid cohesion / oogenesis / mitotic spindle assembly checkpoint signaling / male meiosis I / spermatid development / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / Regulation of APC/C activators between G1/S and early anaphase / negative regulation of mitotic cell cycle / mitotic spindle assembly / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / APC/C:Cdc20 mediated degradation of Cyclin B / regulation of mitotic cell cycle / APC-Cdc20 mediated degradation of Nek2A / APC/C:Cdc20 mediated degradation of Securin / male germ cell nucleus / SCF-beta-TrCP mediated degradation of Emi1 / RHO GTPases Activate Formins / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / transcription coregulator activity / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / negative regulation of protein catabolic process / mitotic spindle / spindle / kinetochore / spindle pole / Separation of Sister Chromatids / double-strand break repair / Antigen processing: Ubiquitination & Proteasome degradation / chromosome / nervous system development / spermatogenesis / nuclear membrane / transcription by RNA polymerase II / cell differentiation / protein ubiquitination / Ub-specific processing proteases / cell division / centrosome / nucleolus / negative regulation of apoptotic process / perinuclear region of cytoplasm / ATP hydrolysis activity / protein homodimerization activity / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Mad1/Cdc20-bound-Mad2 binding protein / Mad1 and Cdc20-bound-Mad2 binding / Pachytene checkpoint protein 2-like / The WD repeat Cdc20/Fizzy family / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily / Anaphase-promoting complex subunit 4, WD40 domain ...Mad1/Cdc20-bound-Mad2 binding protein / Mad1 and Cdc20-bound-Mad2 binding / Pachytene checkpoint protein 2-like / The WD repeat Cdc20/Fizzy family / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 4 WD40 domain / ClpA/B family / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Cell division cycle protein 20 homolog / Mitotic spindle assembly checkpoint protein MAD2A / MAD2L1-binding protein / Pachytene checkpoint protein 2 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsAlfieri, C. / Chang, L. / Barford, D.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Cancer Research UKC576/A14109 United Kingdom
CitationJournal: Nature / Year: 2018
Title: Mechanism for remodelling of the cell cycle checkpoint protein MAD2 by the ATPase TRIP13.
Authors: Claudio Alfieri / Leifu Chang / David Barford /
Abstract: The maintenance of genome stability during mitosis is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), an inhibitor of the anaphase- ...The maintenance of genome stability during mitosis is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex (APC/C, also known as the cyclosome). Unattached kinetochores control MCC assembly by catalysing a change in the topology of the β-sheet of MAD2 (an MCC subunit), thereby generating the active closed MAD2 (C-MAD2) conformer. Disassembly of free MCC, which is required for SAC inactivation and chromosome segregation, is an ATP-dependent process driven by the AAA+ ATPase TRIP13. In combination with p31, an SAC antagonist, TRIP13 remodels C-MAD2 into inactive open MAD2 (O-MAD2). Here, we present a mechanism that explains how TRIP13-p31 disassembles the MCC. Cryo-electron microscopy structures of the TRIP13-p31-C-MAD2-CDC20 complex reveal that p31 recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 (MAD2) to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodelling. Molecular modelling suggests that by gripping MAD2 within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2 to push upwards on, and simultaneously rotate, the globular domains of the p31-C-MAD2 complex. This unwinds a region of the αA helix of C-MAD2 that is required to stabilize the C-MAD2 β-sheet, thus destabilizing C-MAD2 in favour of O-MAD2 and dissociating MAD2 from p31. Our study provides insights into how specific substrates are recruited to AAA+ ATPases through adaptor proteins and suggests a model of how translocation through the axial pore of AAA+ ATPases is coupled to protein remodelling.
History
DepositionNov 20, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 2, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 4, 2018Group: Data collection / Database references / Category: citation
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.title / _citation.year
Revision 1.2Jul 18, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Dec 2, 2020Group: Derived calculations / Category: struct_conn
Item: _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag ..._struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: Pachytene checkpoint protein 2 homolog
B: Pachytene checkpoint protein 2 homolog
C: Pachytene checkpoint protein 2 homolog
D: Pachytene checkpoint protein 2 homolog
E: Pachytene checkpoint protein 2 homolog
F: Pachytene checkpoint protein 2 homolog
P: MAD2L1-binding protein
Q: Cell division cycle protein 20 homolog
Z: Mitotic spindle assembly checkpoint protein MAD2A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)403,67314
Polymers401,0579
Non-polymers2,6165
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36110 Å2
ΔGint-150 kcal/mol
Surface area110220 Å2
MethodPISA

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Components

#1: Protein
Pachytene checkpoint protein 2 homolog / Human papillomavirus type 16 E1 protein-binding protein / HPV16 E1 protein-binding protein / ...Human papillomavirus type 16 E1 protein-binding protein / HPV16 E1 protein-binding protein / Thyroid hormone receptor interactor 13 / Thyroid receptor-interacting protein 13 / TRIP-13


Mass: 48606.664 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRIP13, PCH2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q15645
#2: Protein MAD2L1-binding protein


Mass: 31086.646 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / References: UniProt: Q15013
#3: Protein Cell division cycle protein 20 homolog / Cell cycle / p55CDC


Mass: 54796.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC20 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12834
#4: Protein Mitotic spindle assembly checkpoint protein MAD2A / HsMAD2 / Mitotic arrest deficient 2-like protein 1 / MAD2-like protein 1


Mass: 23533.883 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAD2L1, MAD2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q13257
#5: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM

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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

Component
IDNameTypeEntity IDParent-IDSource
1TRIP13 hexamer in complex with ATP gamma S, p31comet, C-Mad2 and Cdc20COMPLEX#1-#40MULTIPLE SOURCES
2MAD2L1-binding protein, Pachytene checkpoint protein 2 homologCOMPLEX#1-#21RECOMBINANT
3Cell division cycle protein 20 homolog, Mitotic spindle assembly checkpoint protein MAD2ACell cycleCOMPLEX#3-#41RECOMBINANT
Molecular weightValue: 400 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.5
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_2919: / Classification: refinement
EM softwareName: RELION / Version: 2.1-beta-1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 354157 / Symmetry type: POINT

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