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- PDB-6gvt: Hybrid structure of the pRN1 helix bundle domain in complex with ... -

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Basic information

Entry
Database: PDB / ID: 6gvt
TitleHybrid structure of the pRN1 helix bundle domain in complex with DNA and 2 ATP molecules
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 / 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 ...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 / Methyltransferase, Methionine Synthase (B12-binding Domains); Chain A, domain 1 / Bacteriophage/plasmid primase, P4, C-terminal / D5 N terminal like / 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
ADENOSINE-5'-TRIPHOSPHATE / DNA / SF3 helicase domain-containing protein
Similarity search - Component
Biological speciesSulfolobus islandicus (acidophilic)
MethodSOLUTION NMR / SOLID-STATE NMR / simulated annealing
AuthorsBoudet, J. / Wiegand, T. / Meier, B.H. / 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 1.3Aug 21, 2019Group: Data collection / Category: pdbx_nmr_software / Item: _pdbx_nmr_software.name
Revision 1.4Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)17,2986
Polymers16,2362
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2680 Å2
ΔGint-31 kcal/mol
Surface area8380 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 / 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: The first stage of the hybrid structure determination includes structural data from the helix bundle domain quaternary structure
Source: (gene. exp.) Sulfolobus islandicus (acidophilic) / Plasmid: pET28b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q54324
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has protein modificationY

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Experimental details

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Experiment

Experiment
Method
SOLUTION NMR
SOLID-STATE NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
114isotropic23D HNCA
124isotropic23D HN(CO)CA
134isotropic23D HN(CA)CB
144isotropic23D CBCA(CO)NH
154isotropic23D HNCO
164isotropic23D C(CO)NH
174isotropic23D H(CCO)NH
184isotropic13D 1H-15N NOESY
194isotropic13D 1H-13C NOESY aliphatic
1104isotropic13D 1H-13C NOESY aromatic
1115isotropic13D 1H-13C NOESY aromatic
2126isotropic13D 1H-15N NOESY
1139isotropic22D 1H-1H NOESY
1149isotropic22D 1H-1H TOCSY
2157isotropic22D 1H-1H NOESY
2166isotropic22D 1H-1H TOCSY
1175isotropic32D 1H-13C HSQC aromatic
1185isotropic32D 1H-13C HSQC aliphatic
1195isotropic22D-F1fF2f-NOESY
1205isotropic22D-F2f-NOESY
1215isotropic23D-13C-aliphatic-HfilteredHedited-NOESY
1225isotropic23D-13C-aliphatic-HfilteredHedited-NOESY
22310isotropic23D-13C-aromatic-HfilteredHedited-NOESY
22410isotropic23D-13C-aromatic-HfilteredHedited-NOESY
3257isotropic22D-imino-NOESY
1267isotropic32D-natural-abundance-CHSQC
4278isotropic4DARR
4288isotropic4NCX(R)
4298isotropic4NCX(W)
4308isotropic5CHHP
4318isotropic5NHHP
4328isotropic531P CPMAS
4338isotropic531P-31P-DARR
4348isotropic431P CPMAS
1354isotropic12D 1H-15N HSQC
2364isotropic12D 1H-15N HSQC

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution41.0 mM [U-99% 13C; U-99% 15N] pRN1 helix bundle domain in complex with DNA and ATP, 1.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 4.0 mM ADENOSINE-5'-TRIPHOSPHATE, 10 mM magnesium, 90% H2O/10% D2O13C-15N-hbd-hybrid-H2O-pH790% H2O/10% D2O
solution50.7 mM [U-100% 13C] pRN1 helix bundle domain in complex with DNA and ATP, 0.7 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 4.0 mM ADENOSINE-5'-TRIPHOSPHATE, 10.0 mM magnesium, 100% D2O13C-hbd-hybrid-D2O-pH7100% D2O
solution61.0 mM [U-99% 15N] pRN1 helix bundle domain in complex with DNA and ATP, 1.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 4.0 mM ADENOSINE-5'-TRIPHOSPHATE, 10.0 mM magnesium, 90% H2O/10% D2O15N-hbd-hybrid-H2O-pH590% H2O/10% D2O
solution71.0 mM pRN1 the helix bundle domain in complex with DNA and ATP, 1.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 4.0 mM ADENOSINE-5'-TRIPHOSPHATE, 10.0 mM magnesium, 90% H2O/10% D2O1H-hbd-hybrid-H2O-pH590% H2O/10% D2O
solution91.0 mM pRN1 helix bundle domain in complex with DNA and ATP, 1.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 4.0 mM ADENOSINE-5'-TRIPHOSPHATE, 10.0 mM magnesium, 90% H2O/10% D2O1H-hbd-hybrid-H2O-pH790% H2O/10% D2O
solid84.0 mM [U-99% 13C; U-99% 15N] functional-pRN1-primase, 4.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 10.0 mM ADENOSINE-5'-TRIPHOSPHATE, 10.0 mM magnesium, 90% H2O/10% D2O13C-15N-pRN1-primase-solid-state90% H2O/10% D2O
solution101.0 mM [U-100% 13C] pRN1 helix bundle domain in complex with DNA and ATP, 1.0 mM DNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3'), 4.0 mM ADENOSINE-5'-TRIPHOSPHATE, 10 mM magnesium, 100% D2O13C-hbd-hybrid-D2O-pH5100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.0 mMpRN1 helix bundle domain in complex with DNA and ATP[U-99% 13C; U-99% 15N]4
1.0 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance4
4.0 mMADENOSINE-5'-TRIPHOSPHATEnatural abundance4
10 mMmagnesiumnatural abundance4
0.7 mMpRN1 helix bundle domain in complex with DNA and ATP[U-100% 13C]5
0.7 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance5
4.0 mMADENOSINE-5'-TRIPHOSPHATEnatural abundance5
10.0 mMmagnesiumnatural abundance5
1.0 mMpRN1 helix bundle domain in complex with DNA and ATP[U-99% 15N]6
1.0 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance6
4.0 mMADENOSINE-5'-TRIPHOSPHATEnatural abundance6
10.0 mMmagnesiumnatural abundance6
1.0 mMpRN1 the helix bundle domain in complex with DNA and ATPnatural abundance7
1.0 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance7
4.0 mMADENOSINE-5'-TRIPHOSPHATEnatural abundance7
10.0 mMmagnesiumnatural abundance7
1.0 mMpRN1 helix bundle domain in complex with DNA and ATPnatural abundance9
1.0 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance9
4.0 mMADENOSINE-5'-TRIPHOSPHATEnatural abundance9
10.0 mMmagnesiumnatural abundance9
4.0 mMfunctional-pRN1-primase[U-99% 13C; U-99% 15N]8
4.0 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance8
10.0 mMADENOSINE-5'-TRIPHOSPHATEnatural abundance8
10.0 mMmagnesiumnatural abundance8
1.0 mMpRN1 helix bundle domain in complex with DNA and ATP[U-100% 13C]10
1.0 mMDNA (5'-D(*CP*TP*GP*TP*GP*CP*TP*CP*A)-3')natural abundance10
4.0 mMADENOSINE-5'-TRIPHOSPHATEnatural abundance10
10 mMmagnesiumnatural abundance10
Sample conditions
Conditions-IDDetailsIonic strengthLabelpHPressure (kPa)Temperature (K)
125mM phosphate buffer, NaCl 50mM50 mM NaCl 10mM MgCl2 mMNMR-solution-pH77ambient atm298 K
225mM acetate buffer, NaCl 100mM100 mM NaCl 10mM MgCl2 mMNMR-solution-pH5-25C5ambient atm298 K
3100 mM NaCl 10mM MgCl2 mMNMR-solution-pH5-5C5ambient atm278 K
450 mM NaCl 10mM MgCl2 mMNMR-solid-state7ambient atm278 K

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Data collection

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE III HDBrukerAVANCE III HD9001
Bruker AVANCE IIIBrukerAVANCE III7002
Bruker AVANCE IIIBrukerAVANCE III6003
Bruker AVANCE IIIBrukerAVANCE III8504
Bruker AVANCE IIIBrukerAVANCE III5005

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Processing

NMR software
NameDeveloperClassification
CYANAGuntert P.structure calculation
SparkyGoddarddata analysis
MARSJung and Zweckstetterchemical shift assignment
CANDIDHerrmann, Guntert and Wuthrichpeak picking
AmberCase, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, ... and Kollmanrefinement
TopSpinBruker Biospinprocessing
SparkyGoddardchemical shift assignment
CcpNmr AnalysisCCPNdata analysis
TopSpinBruker Biospincollection
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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