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- PDB-5vf0: Solution NMR structure of human RAD18 (198-240) in complex with u... -

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Basic information

Entry
Database: PDB / ID: 5vf0
TitleSolution NMR structure of human RAD18 (198-240) in complex with ubiquitin
Components
  • E3 ubiquitin-protein ligase RAD18
  • Polyubiquitin-B
KeywordsTRANSFERASE / Ubiquitin ligase / Ubiquitin / Nucleosome / DNA damage response
Function / homology
Function and homology information


Rad6-Rad18 complex / positive regulation of chromosome segregation / Y-form DNA binding / nuclear inclusion body / postreplication repair / symbiont entry into host cell via disruption of host cell glycocalyx / symbiont entry into host cell via disruption of host cell envelope / virus tail / polyubiquitin modification-dependent protein binding / protein monoubiquitination ...Rad6-Rad18 complex / positive regulation of chromosome segregation / Y-form DNA binding / nuclear inclusion body / postreplication repair / symbiont entry into host cell via disruption of host cell glycocalyx / symbiont entry into host cell via disruption of host cell envelope / virus tail / polyubiquitin modification-dependent protein binding / protein monoubiquitination / protein autoubiquitination / replication fork / Recognition of DNA damage by PCNA-containing replication complex / RING-type E3 ubiquitin transferase / ubiquitin protein ligase activity / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / single-stranded DNA binding / damaged DNA binding / nuclear body / DNA repair / ubiquitin protein ligase binding / centrosome / DNA damage response / protein-containing complex binding / zinc ion binding / nucleoplasm / identical protein binding / nucleus / cytoplasm
Similarity search - Function
E3 ubiquitin-protein ligase Rad18 / Rad18-like CCHC zinc finger / Zinc finger, C3HC4 type (RING finger) / SAP domain superfamily / SAP motif profile. / SAP domain / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / Pectate lyase superfamily protein / Rad18, zinc finger UBZ4-type ...E3 ubiquitin-protein ligase Rad18 / Rad18-like CCHC zinc finger / Zinc finger, C3HC4 type (RING finger) / SAP domain superfamily / SAP motif profile. / SAP domain / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / Pectate lyase superfamily protein / Rad18, zinc finger UBZ4-type / Zinc finger UBZ4-type profile. / Rhamnogalacturonase A/epimerase, pectate lyase-like / Pectin lyase fold / Pectin lyase fold/virulence factor / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Ubiquitin family / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
Tail fiber / E3 ubiquitin-protein ligase RAD18
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsHu, Q. / Botuyan, M.V. / Mer, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM116829 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA132878 United States
CitationJournal: Mol. Cell / Year: 2017
Title: Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18.
Authors: Hu, Q. / Botuyan, M.V. / Cui, G. / Zhao, D. / Mer, G.
History
DepositionApr 6, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 17, 2017Provider: repository / Type: Initial release
Revision 1.1May 31, 2017Group: Database references
Revision 1.2Sep 20, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 4, 2019Group: Author supporting evidence / Data collection
Category: pdbx_audit_support / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.4Jun 14, 2023Group: Database references / Other / Category: database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data
Revision 1.5May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polyubiquitin-B
B: E3 ubiquitin-protein ligase RAD18
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,8673
Polymers13,8022
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area880 Å2
ΔGint-7 kcal/mol
Surface area8270 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 200structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Polyubiquitin-B


Mass: 8576.831 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG47
#2: Protein/peptide E3 ubiquitin-protein ligase RAD18 / Postreplication repair protein RAD18 / hRAD18 / RING finger protein 73 / RING-type E3 ubiquitin ...Postreplication repair protein RAD18 / hRAD18 / RING finger protein 73 / RING-type E3 ubiquitin transferase RAD18


Mass: 5225.018 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD18, RNF73 / Plasmid: pTEV / Production host: Escherichia coli (E. coli)
References: UniProt: Q9NS91, RING-type E3 ubiquitin transferase
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
115isotropic12D 1H-15N HSQC
126isotropic12D 1H-15N HSQC
136isotropic12D 1H-13C HSQC
143isotropic12D 1H-13C HSQC
154isotropic12D 1H-13C HSQC
163isotropic12D 1H-15N HSQC
174isotropic12D 1H-15N HSQC
186isotropic13D CBCA(CO)NH
196isotropic13D HN(CA)CB
1106isotropic13D HBHA(CO)NH
1116isotropic13D (H)CCH-TOCSY
1133isotropic13D CBCA(CO)NH
1123isotropic13D HN(CA)CB
1143isotropic13D HBHA(CO)NH
1153isotropic13D (H)CCH-TOCSY
1164isotropic13D CBCA(CO)NH
1174isotropic13D HN(CA)CB
1184isotropic13D HBHA(CO)NH
1194isotropic13D (H)CCH-TOCSY
1205isotropic13D 1H-15N NOESY
1213isotropic13D 1H-15N NOESY
1224isotropic13D 1H-15N NOESY
1236isotropic13D 1H-13C NOESY
1243isotropic13D 1H-13C NOESY
1254isotropic13D 1H-13C NOESY
1293isotropic13D 15N/13C-FILTERED EDITED NOESY
1304isotropic13D 15N/13C-FILTERED EDITED NOESY
1317anisotropic12D 1H-15N IPAP HSQC
1328anisotropic12D 1H-15N IPAP HSQC

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution30.6 mM [U-100% 13C; U-100% 15N] RAD18, 3 mM Ubiquitin, 25 mM MES-Bis-TRIS, 50 mM KCl, 10 uM ZnCl2, 90% H2O/10% D2O15N13C_RAD18-Ub90% H2O/10% D2O
solution43 mM RAD18, 0.6 mM [U-100% 13C; U-100% 15N] Ubiquitin, 25 mM MES-Bis-TRIS, 50 mM KCl, 10 uM ZnCl2, 90% H2O/10% D2O15N13C_Ub-RAD1890% H2O/10% D2O
solution50.9 mM [U-15N] RAD18, 25 mM MES-Bis-TRIS, 50 mM KCl, 10 uM ZnCl2, 90% H2O/10% D2O15N_RAD1890% H2O/10% D2O
solution60.9 mM [U-100% 13C; U-100% 15N] RAD18, 25 mM MES-Bis-TRIS, 50 mM KCl, 10 uM ZnCl2, 90% H2O/10% D2O15N13C_RAD1890% H2O/10% D2O
gel solution70.2 mM [U-100% 15N] RAD18, 1.0 mM Ubiquitin, 25 mM MES-Bis-TRIS, 50 mM KCl, 10 uM ZnCl2, 5 % Alkyl-polyethylene glycol (C12E5)/n-hexanol mixture, 90% H2O/10% D2O15N_RAD18-Ub_RDCs90% H2O/10% D2O
gel solution81.0 mM RAD18, 0.2 mM [U-100% 15N] Ubiquitin, 25 mM MES-Bis-TRIS, 50 mM KCl, 10 uM ZnCl2, 5 % Alkyl-polyethylene glycol (C12E5)/n-hexanol mixture, 90% H2O/10% D2O15N_Ub-RAD18_RDCs90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.6 mMRAD18[U-100% 13C; U-100% 15N]3
3 mMUbiquitinnatural abundance3
25 mMMES-Bis-TRISnatural abundance3
50 mMKClnatural abundance3
10 uMZnCl2natural abundance3
3 mMRAD18natural abundance4
0.6 mMUbiquitin[U-100% 13C; U-100% 15N]4
25 mMMES-Bis-TRISnatural abundance4
50 mMKClnatural abundance4
10 uMZnCl2natural abundance4
0.9 mMRAD18[U-15N]5
25 mMMES-Bis-TRISnatural abundance5
50 mMKClnatural abundance5
10 uMZnCl2natural abundance5
0.9 mMRAD18[U-100% 13C; U-100% 15N]6
25 mMMES-Bis-TRISnatural abundance6
50 mMKClnatural abundance6
10 uMZnCl2natural abundance6
0.2 mMRAD18[U-100% 15N]7
1.0 mMUbiquitinnatural abundance7
25 mMMES-Bis-TRISnatural abundance7
50 mMKClnatural abundance7
10 uMZnCl2natural abundance7
5 %Alkyl-polyethylene glycol (C12E5)/n-hexanol mixturenatural abundance7
1.0 mMRAD18natural abundance8
0.2 mMUbiquitin[U-100% 15N]8
25 mMMES-Bis-TRISnatural abundance8
50 mMKClnatural abundance8
10 uMZnCl2natural abundance8
5 %Alkyl-polyethylene glycol (C12E5)/n-hexanol mixturenatural abundance8
Sample conditionsIonic strength: 50 mM KCl mM / Label: Conditions_1 / pH: 6 / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE III / Manufacturer: Bruker / Model: AVANCE III / Field strength: 700 MHz

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Processing

NMR software
NameDeveloperClassification
TopSpinBruker Biospincollection
SparkyGoddarddata analysis
SparkyGoddardpeak picking
SparkyGoddardchemical shift assignment
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorestructure calculation
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorerefinement
RefinementMethod: simulated annealing / Software ordinal: 4
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 200 / Conformers submitted total number: 20

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