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- PDB-6gvu: NMR structure of the DNA-bound helix bundle domain from the funct... -

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Basic information

Entry
Database: PDB / ID: 6gvu
TitleNMR structure of the DNA-bound helix bundle domain from the functional pRN1 primase
Components
  • DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')
  • functional pRN1 primase
KeywordsDNA BINDING PROTEIN / primase / single-stranded DNA / replication initiation
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 141160 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

Downloads & links

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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 area1490 Å2
ΔGint-8 kcal/mol
Surface area8920 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: DNA template of the pRN1 primase / 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: helix bundle domain of the functional pRN1 primase / 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 HN(CA)CB
141isotropic23D CBCA(CO)NH
151isotropic33D C(CO)NH
161isotropic33D H(CCO)NH
171isotropic13D 1H-15N NOESY
181isotropic13D 1H-13C NOESY aliphatic
191isotropic13D 1H-13C NOESY aromatic
1102isotropic23D 1H-13C NOESY aromatic
1113isotropic22D 1H-15N HSQC
2123isotropic22D 1H-15N HSQC
3133isotropic22D 1H-15N HSQC
2143isotropic13D 1H-15N NOESY
3153isotropic23D 1H-15N NOESY
1164isotropic22D 1H-1H NOESY
2174isotropic22D 1H-1H NOESY
1184isotropic22D 1H-1H TOCSY
2194isotropic22D 1H-1H TOCSY
1202isotropic22D-F1fF2f-NOESY
1212isotropic22D-F2f-NOESY
1222isotropic23D 13C-aliphatic Hfiltered-Hedited NOESY
1232isotropic23D 13C-aromatic Hfiltered-Hedited NOESY
2242isotropic23D 13C-aliphatic Hfiltered-Hedited NOESY
2252isotropic23D 13C-aromatic Hfiltered-Hedited NOESY

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution11.0 mM [U-99% 13C; U-99% 15N] DNA-bound helix bundle domain, 1.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 90% H2O/10% D2O13C-15N-HBD bound to DNA90% H2O/10% D2O
solution20.7 mM [U-100% 13C] DNA-bound helix bundle domain, 0.7 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 100% D2O13C-HBD-bound to DNA100% D2O
solution31.1 mM [U-99% 15N] DNA-bound helix bundle domain, 1.1 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 90% H2O/10% D2O15N-HBD-bound to DNA90% H2O/10% D2O
solution41.0 mM DNA-bound helix bundle domain, 1.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 90% H2O/10% D2O1H-HBD-bound to DNA90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.0 mMDNA-bound helix bundle domain[U-99% 13C; U-99% 15N]1
1.0 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance1
0.7 mMDNA-bound helix bundle domain[U-100% 13C]2
0.7 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance2
1.1 mMDNA-bound helix bundle domain[U-99% 15N]3
1.1 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance3
1.0 mMDNA-bound helix bundle domainnatural 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)
125mM acetate buffer; 100mM NaCl100mM NaCl mMHigh-temperature5.5 ambient atm323 K
225mM acetate buffer; 100mM NaCl100mM NaCl mMRoom-temperature5.5 ambient atm298 K
325mM phosphate buffer; 50mM NaCl50mM NaCl mMSample-pH77.0 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
SparkyGoddarddata analysis
CANDIDHerrmann, Guntert and Wuthrichpeak picking
SparkyGoddardchemical shift assignment
TopSpinBruker Biospincollection
CYANAGuntert, Mumenthaler and Wuthrichstructure calculation
AmberCase, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, ... and Kollmanrefinement
RefinementMethod: simulated annealing / Software ordinal: 6
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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