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- PDB-7t9x: Saccharomyces cerevisiae Pex12 RING domain -

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Basic information

Entry
Database: PDB / ID: 7t9x
TitleSaccharomyces cerevisiae Pex12 RING domain
ComponentsPeroxisome assembly protein 12
KeywordsLIGASE / Saccharomyces cerevisiae Pex12 RING domain
Function / homology
Function and homology information


protein import into peroxisome matrix, substrate release / peroxisomal importomer complex / protein import into peroxisome matrix, receptor recycling / Class I peroxisomal membrane protein import / protein import into peroxisome matrix / Peroxisomal protein import / : / ubiquitin ligase activator activity / peroxisomal membrane / transmembrane protein transporter activity ...protein import into peroxisome matrix, substrate release / peroxisomal importomer complex / protein import into peroxisome matrix, receptor recycling / Class I peroxisomal membrane protein import / protein import into peroxisome matrix / Peroxisomal protein import / : / ubiquitin ligase activator activity / peroxisomal membrane / transmembrane protein transporter activity / protein monoubiquitination / ubiquitin ligase complex / protein polyubiquitination / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / proteasome-mediated ubiquitin-dependent protein catabolic process / protein ubiquitination / zinc ion binding
Similarity search - Function
Pex, N-terminal / Peroxisome assembly protein 12 / Pex2 / Pex12 amino terminal region / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
Peroxisome assembly protein 12
Similarity search - Component
Biological speciesBacteria (eubacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / AB INITIO PHASING / Resolution: 1.52 Å
AuthorsFeng, P. / Rapoport, T.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2022
Title: A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.
Authors: Peiqiang Feng / Xudong Wu / Satchal K Erramilli / Joao A Paulo / Pawel Knejski / Steven P Gygi / Anthony A Kossiakoff / Tom A Rapoport /
Abstract: Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which ...Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12). Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.
History
DepositionDec 20, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 20, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Feb 28, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Peroxisome assembly protein 12
B: Peroxisome assembly protein 12
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,1474
Polymers16,0172
Non-polymers1312
Water1,33374
1
A: Peroxisome assembly protein 12
hetero molecules


Theoretical massNumber of molelcules
Total (without water)8,0742
Polymers8,0081
Non-polymers651
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Peroxisome assembly protein 12
hetero molecules


Theoretical massNumber of molelcules
Total (without water)8,0742
Polymers8,0081
Non-polymers651
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)27.578, 32.922, 72.488
Angle α, β, γ (deg.)90.000, 100.715, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: PRO / Beg label comp-ID: PRO / End auth comp-ID: ILE / End label comp-ID: ILE / Auth seq-ID: 2 - 74 / Label seq-ID: 1 - 73

Dom-IDSelection detailsAuth asym-IDLabel asym-ID
1chain 'A'AA
2chain 'B'BB

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Components

#1: Protein Peroxisome assembly protein 12 / Peroxin-12


Mass: 8008.321 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteria (eubacteria) / Gene: PEX12, YMR026C, YM9711.16C / Production host: Bacteria (eubacteria) / References: UniProt: Q04370
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 74 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.02 Å3/Da / Density % sol: 39.07 %
Crystal growTemperature: 291.15 K / Method: evaporation
Details: 0.1 M bis-Tris propane, pH 8.5, 0.2 M sodium fluoride, 20% PEG3350

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Data collection

DiffractionMean temperature: 293 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.967 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jun 13, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.967 Å / Relative weight: 1
ReflectionResolution: 1.52→71.22 Å / Num. obs: 34282 / % possible obs: 97.1 % / Redundancy: 9.3 % / Biso Wilson estimate: 13.62 Å2 / Rmerge(I) obs: 0.057 / Net I/σ(I): 36.1
Reflection shellResolution: 1.52→4.8 Å / Rmerge(I) obs: 0.057 / Num. unique obs: 18534 / CC1/2: 0.772 / % possible all: 83.8

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660 / Classification: refinement
RefinementMethod to determine structure: AB INITIO PHASING / Resolution: 1.52→71.22 Å / SU ML: 0.127 / Cross valid method: FREE R-VALUE / σ(F): 1.44 / Phase error: 20.5963
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.197 3441 10.04 %
Rwork0.186 30841 -
obs0.1872 34282 88.6 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 17.8 Å2
Refinement stepCycle: LAST / Resolution: 1.52→71.22 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1114 0 2 74 1190
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00551138
X-RAY DIFFRACTIONf_angle_d0.83821552
X-RAY DIFFRACTIONf_chiral_restr0.0565184
X-RAY DIFFRACTIONf_plane_restr0.005194
X-RAY DIFFRACTIONf_dihedral_angle_d11.4938416
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.52-1.540.2193420.2069384X-RAY DIFFRACTION26.84
1.54-1.560.2022590.2028503X-RAY DIFFRACTION37
1.56-1.580.2082960.1978762X-RAY DIFFRACTION55.14
1.58-1.610.25531230.19151103X-RAY DIFFRACTION79.25
1.61-1.640.20441340.18791218X-RAY DIFFRACTION87.17
1.64-1.660.20321380.1831284X-RAY DIFFRACTION93.25
1.66-1.690.22811450.18821329X-RAY DIFFRACTION95.04
1.69-1.730.2051650.18111375X-RAY DIFFRACTION94.65
1.73-1.760.21541340.17681256X-RAY DIFFRACTION96.86
1.76-1.80.19411420.18991352X-RAY DIFFRACTION94.74
1.8-1.840.23521500.18851353X-RAY DIFFRACTION96.28
1.84-1.890.16421500.18661361X-RAY DIFFRACTION97.23
1.89-1.940.22221540.18911330X-RAY DIFFRACTION94.46
1.94-20.20551420.1911346X-RAY DIFFRACTION98.8
2-2.060.21371500.19721323X-RAY DIFFRACTION93.76
2.06-2.130.22811530.18361360X-RAY DIFFRACTION97.74
2.14-2.220.2151480.18511342X-RAY DIFFRACTION96.01
2.22-2.320.21371410.1891352X-RAY DIFFRACTION98.48
2.32-2.440.2391620.191344X-RAY DIFFRACTION98.05
2.44-2.60.21851500.20171316X-RAY DIFFRACTION94.46
2.6-2.80.21351460.20881381X-RAY DIFFRACTION98.33
2.8-3.080.22561510.20051403X-RAY DIFFRACTION99.3
3.08-3.520.20891570.18011353X-RAY DIFFRACTION99.02
3.52-4.440.13491580.16481359X-RAY DIFFRACTION97.43
4.44-71.220.14681510.17231352X-RAY DIFFRACTION97.22
Refinement TLS params.Method: refined / Origin x: 4.30503115124 Å / Origin y: 9.56719701048 Å / Origin z: 55.2594773437 Å
111213212223313233
T0.115838235184 Å2-0.00470828395339 Å20.00114053231285 Å2-0.119089886296 Å2-0.00705737889077 Å2--0.130973649087 Å2
L0.0420451506354 °2-0.0195586972395 °2-0.000507861499089 °2-0.0893461277377 °2-0.012213767945 °2--0.588892889511 °2
S-0.00372579984893 Å °0.000918111300774 Å °-0.00849623180397 Å °-0.0167299927251 Å °-0.0153050083566 Å °0.0145284730692 Å °0.0311660476216 Å °-0.00332570202764 Å °0.0186144332925 Å °
Refinement TLS groupSelection details: all

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