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- PDB-8r1x: Solution structure and chemical shift assignments for HMG-D Y12F ... -

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Basic information

Entry
Database: PDB / ID: 8r1x
TitleSolution structure and chemical shift assignments for HMG-D Y12F mutant complexed to a 14:12 dA2 bulge DNA
Components
  • DNA (5'-D(*CP*GP*AP*TP*AP*TP*TP*AP*AP*GP*AP*GP*CP*C)-3')
  • DNA (5'-D(*GP*GP*CP*TP*CP*AP*AP*TP*AP*TP*CP*G)-3')
  • High mobility group protein D
KeywordsDNA BINDING PROTEIN / HMG protein / DNA-binding protein / protein-DNA complex
Function / homology
Function and homology information


Mitochondrial transcription initiation / Mitochondrial protein degradation / DNA binding, bending / minor groove of adenine-thymine-rich DNA binding / chromatin organization / regulation of transcription by RNA polymerase II / chromatin / DNA binding / nucleus
Similarity search - Function
: / HMG (high mobility group) box / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain / High mobility group box domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / High mobility group protein D
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
synthetic construct (others)
MethodSOLUTION NMR / molecular dynamics
AuthorsYang, J.C. / Hill, G.R. / Neuhaus, D.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)U105178934 United Kingdom
CitationJournal: Chembiochem / Year: 2024
Title: A Single Interfacial Point Mutation Rescues Solution Structure Determination of the Complex of HMG-D with a DNA Bulge.
Authors: Hill, G.R. / Yang, J.C. / Easton, L.E. / Cerdan, R. / McLaughlin, S.H. / Stott, K. / Travers, A.A. / Neuhaus, D.
History
DepositionNov 2, 2023Deposition site: PDBE / Processing site: PDBE
SupersessionSep 11, 2024ID: 1E7J
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details
Item: _citation.journal_volume / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: High mobility group protein D
B: DNA (5'-D(*CP*GP*AP*TP*AP*TP*TP*AP*AP*GP*AP*GP*CP*C)-3')
C: DNA (5'-D(*GP*GP*CP*TP*CP*AP*AP*TP*AP*TP*CP*G)-3')


Theoretical massNumber of molelcules
Total (without water)20,3783
Polymers20,3783
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: NMR Distance Restraints, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3490 Å2
ΔGint-8 kcal/mol
Surface area9100 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 60structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein High mobility group protein D


Mass: 12426.941 Da / Num. of mol.: 1 / Mutation: Y12F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: HmgD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q05783
#2: DNA chain DNA (5'-D(*CP*GP*AP*TP*AP*TP*TP*AP*AP*GP*AP*GP*CP*C)-3')


Mass: 4288.817 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The DNA ligand (chains B and C) was designed to be a structural mimic of bent DNA; it is not based on any specific natural sequence.
Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*GP*GP*CP*TP*CP*AP*AP*TP*AP*TP*CP*G)-3')


Mass: 3662.404 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The DNA ligand (chains B and C) was designed to be a structural mimic of bent DNA; it is not based on any specific natural sequence.
Source: (synth.) synthetic construct (others)
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic22D 1H-15N HSQC
121isotropic22D 1H-13C HSQC
131isotropic22D 1H-1H NOESY (mixing time = 60 ms)
141isotropic22D 1H-1H NOESY (mixing time = 150 ms)
151isotropic13D HNCA
161isotropic13D HN(CO)CA
171isotropic13D HN(CA)CB
181isotropic13D HBHANH
191isotropic13D HBHA(CO)NH
1101isotropic13D (H)CCH-COSY
1111isotropic13D (H)CCH-TOCSY
1121isotropic23D 1H-15N NOESY (mixing time = 150 ms)
1131isotropic23D 1H-13C NOESY aliphatic (mixing time = 150 ms)
1141isotropic23D 1H-13C NOESY aromatic (mixing time = 150 ms)
1152isotropic22D [13C,15N]-filtered [1H-1H] NOESY (mixing time = 100 ms, with filter elements set to reject 13C and 15N coupled signals in F1 and to accept only 13C coupled signals in F2)
1162isotropic22D [13C,15N]-filtered [1H-1H] NOESY (mixing time = 200 ms, with filter elements set to reject 13C and 15N coupled signals in F1 and to accept only 13C coupled signals in F2)
1172isotropic213C-filtered 13C NOESY-HSQC (mixing time = 150 ms, with filter elements set to reject 13C- and 15N-coupled (protein) signals in F1)

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution11.8 mM [U-13C; U-15N] HMG-D Y12F, 1.8 mM DNA (5'-D(*CP*GP*AP*TP*AP*TP*TP*AP*AP*GP*AP*GP*CP*C)-3'), 1.8 mM DNA (5'-D(*GP*GP*CP*TP*CP*AP*AP*TP*AP*TP*CP*G)-3'), 10 mM sodium phosphate, 20 mM sodium chloride, 3 mM sodium azide, 95% H2O/5% D2O13C,15N labelled protein complexed to natural abundance DNA bulge ligand in H2O13C,15N protein + DNA in H2O95% H2O/5% D2O
solution21.8 mM [U-13C; U-15N] HMG-D Y12F, 1.8 mM DNA (5'-D(*CP*GP*AP*TP*AP*TP*TP*AP*AP*GP*AP*GP*CP*C)-3'), 1.8 mM DNA (5'-D(*GP*GP*CP*TP*CP*AP*AP*TP*AP*TP*CP*G)-3'), 10 mM sodium phosphate, 20 mM sodium chloride, 3 mM sodium azide, 100% D2O13C,15N labelled protein complexed to natural abundance DNA bulge ligand in D2O13C,15N protein + DNA in D2O100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.8 mMHMG-D Y12F[U-13C; U-15N]1
1.8 mMDNA (5'-D(*CP*GP*AP*TP*AP*TP*TP*AP*AP*GP*AP*GP*CP*C)-3')natural abundance1
1.8 mMDNA (5'-D(*GP*GP*CP*TP*CP*AP*AP*TP*AP*TP*CP*G)-3')natural abundance1
10 mMsodium phosphatenatural abundance1
20 mMsodium chloridenatural abundance1
3 mMsodium azidenatural abundance1
1.8 mMHMG-D Y12F[U-13C; U-15N]2
1.8 mMDNA (5'-D(*CP*GP*AP*TP*AP*TP*TP*AP*AP*GP*AP*GP*CP*C)-3')natural abundance2
1.8 mMDNA (5'-D(*GP*GP*CP*TP*CP*AP*AP*TP*AP*TP*CP*G)-3')natural abundance2
10 mMsodium phosphatenatural abundance2
20 mMsodium chloridenatural abundance2
3 mMsodium azidenatural abundance2
Sample conditionsIonic strength: 83 mM / Label: conditions_1 / pH: 6 / Pressure: 1 atm / Temperature: 300 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCEBrukerAVANCE5001
Bruker AVANCEBrukerAVANCE8002

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin3.2 and 3.5Bruker Biospincollection
Sparky3.115Goddardchemical shift assignment
X-PLOR NIH2.28Schwieters, Kuszewski, Tjandra and Clorestructure calculation
Amber11Case, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, ... and Kollmanrefinement
RefinementMethod: molecular dynamics / Software ordinal: 4
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 60 / Conformers submitted total number: 20

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