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- PDB-6gvq: DNA-bound pRN1 helix bundle domain with ATP and magnesium in the ... -

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Basic information

Entry
Database: PDB / ID: 6gvq
TitleDNA-bound pRN1 helix bundle domain with ATP and magnesium in the interaction buffer
Components
  • DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')
  • functional pRN1 primase
KeywordsDNA BINDING PROTEIN / pRN1 primase / dinucleotide formation / quaternary structure of the helix bundle domain / synergistic effect
Function / homology
Function and homology information


hydrolase activity / ATP binding / metal ion binding
Similarity search - Function
Methyltransferase, Methionine Synthase (B12-binding Domains); Chain A, domain 1 - #50 / DNA primase/polymerase, bifunctional, N-terminal / Primase helical domain / Bifunctional DNA primase/polymerase, N-terminal / Primase helical domain / Bifunctional DNA primase/polymerase, N-terminal / DNA primase, phage/plasmid / D5 N terminal like / Domain of unknown function DUF5906 / Family of unknown function (DUF5906) ...Methyltransferase, Methionine Synthase (B12-binding Domains); Chain A, domain 1 - #50 / DNA primase/polymerase, bifunctional, N-terminal / Primase helical domain / Bifunctional DNA primase/polymerase, N-terminal / Primase helical domain / Bifunctional DNA primase/polymerase, N-terminal / DNA primase, phage/plasmid / D5 N terminal like / Domain of unknown function DUF5906 / Family of unknown function (DUF5906) / DNA primase/nucleoside triphosphatase, C-terminal / Poxvirus D5 protein-like / Bacteriophage/plasmid primase, P4, C-terminal / D5 N terminal like / Methyltransferase, Methionine Synthase (B12-binding Domains); Chain A, domain 1 / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / Winged helix DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA / SF3 helicase domain-containing protein
Similarity search - Component
Biological speciesSulfolobus islandicus (acidophilic)
MethodSOLUTION NMR / simulated annealing
AuthorsBoudet, J. / Lipps, G. / Allain, F.H.-T.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_163345 Switzerland
CitationJournal: Cell / Year: 2019
Title: A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template.
Authors: Boudet, J. / Devillier, J.C. / Wiegand, T. / Salmon, L. / Meier, B.H. / Lipps, G. / Allain, F.H.
History
DepositionJun 21, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 26, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 9, 2019Group: Data collection / 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Jan 23, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3May 8, 2019Group: Data collection / Category: pdbx_nmr_software / pdbx_seq_map_depositor_info
Item: _pdbx_nmr_software.name / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.4Jul 3, 2024Group: Data collection / Database references / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')
B: functional pRN1 primase


Theoretical massNumber of molelcules
Total (without water)16,2362
Polymers16,2362
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1400 Å2
ΔGint-6 kcal/mol
Surface area8410 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 200structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: DNA chain DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')


Mass: 2706.785 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Sequence of the DNA template from the Sulfolobus islandicus pRN1 plasmid
Source: (synth.) Sulfolobus islandicus (acidophilic)
#2: Protein functional pRN1 primase


Mass: 13528.738 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: DNA-bound pRN1 helix bundle domain with ATP and magnesium in interaction buffer at pH 7.0
Source: (gene. exp.) Sulfolobus islandicus (acidophilic) / Plasmid: pET28b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q54324

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic23D HNCA
121isotropic23D HN(CO)CA
131isotropic23D CBCA(CO)NH
141isotropic23D HN(CA)CB
151isotropic23D HNCO
161isotropic2HN(CA)CO
171isotropic23D C(CO)NH
181isotropic23D H(CCO)NH
191isotropic13D 1H-15N NOESY
1101isotropic13D 1H-13C NOESY aliphatic
1111isotropic13D 1H-13C NOESY aromatic
1123isotropic13D 1H-13C NOESY aromatic
1134isotropic22D 1H-1H NOESY
1144isotropic22D 1H-1H TOCSY
1151isotropic12D 1H-15N HSQC
1161isotropic22D 1H-15N HSQC
3171isotropic22D 1H-15N HSQC
2181isotropic22D 1H-15N HSQC
1191isotropic22D 1H-13C HSQC aliphatic
1201isotropic22D 1H-13C HSQC aromatic
1213isotropic22D 1H-13C HSQC aliphatic
1223isotropic22D 1H-13C HSQC aromatic
1233isotropic22D-F1fF2f-NOESY
1243isotropic22D-F2f-NOESY
1253isotropic23D 13Caliphatic Hfiltered-Hedited NOESY
1263isotropic23D 13Caromatic Hfiltered-Hedited NOESY
1274isotropic22D imino NOESY
2284isotropic22D imino NOESY
1294isotropic32D natural abundance CHSQC

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution11.1 mM [U-99% 13C; U-99% 15N] DNA-bound pRN1 helix bundle domain with ATP, 1.1 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 90% H2O/10% D2O13C-15N-labeled-hbd bound to DNA with 10mM ATP and magnesium in interaction buffer13C-15N-hbd-quaternary90% H2O/10% D2O
solution20.9 mM [U-99% 13C; U-99% 15N] DNA-bound pRN1 helix bundle domain with ATP, 0.9 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 100% D2O13C-15N-labeled-hbd bound to DNA with 10mM ATP and magnesium in interaction buffer (100% D2O)13C-15N-hbd-quaternary-D2O100% D2O
solution30.7 mM [U-100% 13C] DNA-bound pRN1 helix bundle domain with ATP, 0.7 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 100% D2O13C-labeled-hbd bound to DNA with 10mM ATP and magnesium in interaction buffer (100% D2O)13C-hbd-quaternary-D2O100% D2O
solution41.0 mM DNA-bound pRN1 helix bundle domain with ATP, 1.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 90% H2O/10% D2O1H-hbd bound to DNA with 10mM ATP and magnesium in interaction buffer1H-hbd-quaternary90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.1 mMDNA-bound pRN1 helix bundle domain with ATP[U-99% 13C; U-99% 15N]1
1.1 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance1
0.9 mMDNA-bound pRN1 helix bundle domain with ATP[U-99% 13C; U-99% 15N]2
0.9 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance2
0.7 mMDNA-bound pRN1 helix bundle domain with ATP[U-100% 13C]3
0.7 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance3
1.0 mMDNA-bound pRN1 helix bundle domain with ATPnatural abundance4
1.0 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance4
Sample conditions
Conditions-IDDetailsIonic strengthLabelpHPressure (kPa)Temperature (K)
1Experimental conditions for quaternary arrangement assignment and structure determination 25mM phosphate buffer; 50mM NaCl50mM NaCl mMhbd-assignment-structure7.0 ambient atm298 K
2Complementary experimental conditions for quaternary structure determination 25mM phosphate buffer; 50mM NaCl50mM NaCl mMhbd-structure-complement-temp7.0 ambient atm278 K
3Complementary experimental conditions for quaternary arrangement assignment 25mM acetate buffer; 100mM NaCl100mM NaCl mMhbd-complement-pH5.0 ambient atm298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE III HDBrukerAVANCE III HD9001
Bruker AVANCE IIIBrukerAVANCE III7002
Bruker AVANCE IIIBrukerAVANCE III6003

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Processing

NMR software
NameDeveloperClassification
CYANAGuntert P.structure calculation
TopSpinBruker Biospinprocessing
SparkyGoddarddata analysis
CANDIDHerrmann, Guntert and Wuthrichpeak picking
AmberCase, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, ... and Kollmanrefinement
TopSpinBruker Biospincollection
MARSJung and Zweckstetterchemical shift assignment
RefinementMethod: simulated annealing / Software ordinal: 5
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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