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- PDB-6nxk: Ubiquitin binding variants -

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Basic information

Entry
Database: PDB / ID: 6nxk
TitleUbiquitin binding variants
Components
  • Anaphase-promoting complex subunit 2
  • Polyubiquitin-C
KeywordsLIGASE / APC / WHB / Ubiquitin / Diubiquitin
Function / homology
Function and homology information


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 / anaphase-promoting complex / anaphase-promoting complex-dependent catabolic process / regulation of meiotic cell cycle / protein branched polyubiquitination / Aberrant regulation of mitotic exit in cancer due to RB1 defects / metaphase/anaphase transition of mitotic cell cycle ...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 / anaphase-promoting complex / anaphase-promoting complex-dependent catabolic process / regulation of meiotic cell cycle / protein branched polyubiquitination / Aberrant regulation of mitotic exit in cancer due to RB1 defects / metaphase/anaphase transition of mitotic cell cycle / Phosphorylation of the APC/C / positive regulation of synaptic plasticity / positive regulation of dendrite morphogenesis / protein K11-linked ubiquitination / Regulation of APC/C activators between G1/S and early anaphase / Transcriptional Regulation by VENTX / positive regulation of axon extension / protein K48-linked ubiquitination / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / regulation of mitotic cell cycle / FLT3 signaling by CBL mutants / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Prevention of phagosomal-lysosomal fusion / Endosomal Sorting Complex Required For Transport (ESCRT) / Membrane binding and targetting of GAG proteins / Negative regulation of FLT3 / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Constitutive Signaling by NOTCH1 HD Domain Mutants / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / APC-Cdc20 mediated degradation of Nek2A / Regulation of FZD by ubiquitination / p75NTR recruits signalling complexes / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / NF-kB is activated and signals survival / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / Pexophagy / Downregulation of ERBB2:ERBB3 signaling / Regulation of innate immune responses to cytosolic DNA / NRIF signals cell death from the nucleus / Regulation of PTEN localization / VLDLR internalisation and degradation / Activated NOTCH1 Transmits Signal to the Nucleus / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by REV1 / TICAM1, RIP1-mediated IKK complex recruitment / Regulation of BACH1 activity / Translesion synthesis by POLK / InlB-mediated entry of Listeria monocytogenes into host cell / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Downregulation of TGF-beta receptor signaling / Translesion synthesis by POLI / Josephin domain DUBs / Gap-filling DNA repair synthesis and ligation in GG-NER / IKK complex recruitment mediated by RIP1 / PINK1-PRKN Mediated Mitophagy / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / Regulation of activated PAK-2p34 by proteasome mediated degradation / TNFR1-induced NF-kappa-B signaling pathway / TCF dependent signaling in response to WNT / Regulation of NF-kappa B signaling / activated TAK1 mediates p38 MAPK activation / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Asymmetric localization of PCP proteins / NOTCH3 Activation and Transmission of Signal to the Nucleus / Regulation of signaling by CBL / Negative regulators of DDX58/IFIH1 signaling / Ubiquitin-dependent degradation of Cyclin D / Fanconi Anemia Pathway / Negative regulation of FGFR3 signaling / Peroxisomal protein import / SCF-beta-TrCP mediated degradation of Emi1 / Deactivation of the beta-catenin transactivating complex / NIK-->noncanonical NF-kB signaling / AUF1 (hnRNP D0) binds and destabilizes mRNA / TNFR2 non-canonical NF-kB pathway / Stabilization of p53 / Negative regulation of FGFR2 signaling / Negative regulation of FGFR4 signaling / Assembly of the pre-replicative complex / Vpu mediated degradation of CD4
Similarity search - Function
Anaphase-promoting complex subunit 2, C-terminal / Anaphase-promoting complex subunit 2 / : / Anaphase promoting complex (APC) subunit 2 / ANAPC2-like, TPR repeats / Anaphase promoting complex (APC) subunit 2 / Cullin / : / Cullin alpha+beta domain / Cullin homology domain ...Anaphase-promoting complex subunit 2, C-terminal / Anaphase-promoting complex subunit 2 / : / Anaphase promoting complex (APC) subunit 2 / ANAPC2-like, TPR repeats / Anaphase promoting complex (APC) subunit 2 / Cullin / : / Cullin alpha+beta domain / Cullin homology domain / Cullin homology domain superfamily / Cullin family profile. / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Arc Repressor Mutant, subunit A / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Polyubiquitin-C / Anaphase-promoting complex subunit 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsMiller, D.J. / Watson, E.R.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site.
Authors: Edmond R Watson / Christy R R Grace / Wei Zhang / Darcie J Miller / Iain F Davidson / J Rajan Prabu / Shanshan Yu / Derek L Bolhuis / Elizaveta T Kulko / Ronnald Vollrath / David Haselbach / ...Authors: Edmond R Watson / Christy R R Grace / Wei Zhang / Darcie J Miller / Iain F Davidson / J Rajan Prabu / Shanshan Yu / Derek L Bolhuis / Elizaveta T Kulko / Ronnald Vollrath / David Haselbach / Holger Stark / Jan-Michael Peters / Nicholas G Brown / Sachdev S Sidhu / Brenda A Schulman /
Abstract: Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential ...Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub's C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at "exosites." However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.
History
DepositionFeb 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polyubiquitin-C
B: Polyubiquitin-C
C: Anaphase-promoting complex subunit 2
D: Anaphase-promoting complex subunit 2


Theoretical massNumber of molelcules
Total (without water)37,6734
Polymers37,6734
Non-polymers00
Water2,090116
1
A: Polyubiquitin-C
C: Anaphase-promoting complex subunit 2


Theoretical massNumber of molelcules
Total (without water)18,8362
Polymers18,8362
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Polyubiquitin-C
D: Anaphase-promoting complex subunit 2


Theoretical massNumber of molelcules
Total (without water)18,8362
Polymers18,8362
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)58.893, 63.368, 100.135
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Polyubiquitin-C


Mass: 8576.831 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
Strain (production host): BL21(DE3)-RIL / References: UniProt: P0CG48
#2: Protein Anaphase-promoting complex subunit 2 / APC2 / Cyclosome subunit 2


Mass: 10259.662 Da / Num. of mol.: 2 / Fragment: WHB domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC2, APC2, KIAA1406
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
Strain (production host): BL21(DE3)-RIL / References: UniProt: Q9UJX6
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 116 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.5 Å3/Da / Density % sol: 50.78 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop
Details: 0.1M HEPES pH 7.7, 27.5% PEG3350, 2.5mM APC2 WHB (735-C) + 2.08mM Ubiquitin protein co-mixed

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Aug 3, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.2→53.55 Å / Num. obs: 17815 / % possible obs: 96.5 % / Observed criterion σ(I): -3 / Redundancy: 4.4 % / Rmerge(I) obs: 0.069 / Rpim(I) all: 0.036 / Rsym value: 0.069 / Net I/σ(I): 12
Reflection shellResolution: 2.2→2.27 Å / Redundancy: 4.4 % / Rmerge(I) obs: 0.583 / Rpim(I) all: 0.303 / Rsym value: 0.583 / % possible all: 99

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Processing

Software
NameVersionClassification
REFMAC5.8.0135refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4s1z, 4YII chain A

4s1z

4yii


Resolution: 2.2→53.55 Å / Cor.coef. Fo:Fc: 0.95 / Cor.coef. Fo:Fc free: 0.918 / SU B: 8.963 / SU ML: 0.215 / Cross valid method: THROUGHOUT / ESU R: 0.286 / ESU R Free: 0.228 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2662 980 5.2 %RANDOM
Rwork0.2205 ---
obs0.22287 17815 95.64 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 48.829 Å2
Baniso -1Baniso -2Baniso -3
1--3.75 Å2-0 Å2-0 Å2
2--1.17 Å20 Å2
3---2.59 Å2
Refinement stepCycle: 1 / Resolution: 2.2→53.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2395 0 0 116 2511
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.022436
X-RAY DIFFRACTIONr_bond_other_d0.0020.022445
X-RAY DIFFRACTIONr_angle_refined_deg1.341.9943289
X-RAY DIFFRACTIONr_angle_other_deg0.89835622
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.9245294
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.84225.263114
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.45715480
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.7411515
X-RAY DIFFRACTIONr_chiral_restr0.0740.2387
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.022690
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02521
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.7084.591182
X-RAY DIFFRACTIONr_mcbond_other2.7084.5871181
X-RAY DIFFRACTIONr_mcangle_it3.9986.8691471
X-RAY DIFFRACTIONr_mcangle_other3.9976.8711472
X-RAY DIFFRACTIONr_scbond_it3.7575.1341254
X-RAY DIFFRACTIONr_scbond_other3.7565.1361255
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.1097.4961817
X-RAY DIFFRACTIONr_long_range_B_refined7.58535.6672688
X-RAY DIFFRACTIONr_long_range_B_other7.56135.5472663
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.308 76 -
Rwork0.331 1335 -
obs--98.67 %

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