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- PDB-7kw9: NMR Structure of a tRNA 2'-phosphotransferase from Runella slithy... -

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Basic information

Entry
Database: PDB / ID: 7kw9
TitleNMR Structure of a tRNA 2'-phosphotransferase from Runella slithyformis in complex with NAD+
ComponentstRNA 2'-phosphotransferase
KeywordsTRANSFERASE / tRNA 2'-phosphotransferase NAD+
Function / homologyNICOTINAMIDE-ADENINE-DINUCLEOTIDE
Function and homology information
Biological speciesRunella slithyformis (bacteria)
MethodSOLUTION NMR / simulated annealing
AuthorsAlphonse, S. / Dantuluri, S. / Banerjee, A. / Shuman, S. / Ghose, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)NSF MCB 1412007 United States
CitationJournal: Nucleic Acids Res. / Year: 2021
Title: NMR solution structures of Runella slithyformis RNA 2'-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity.
Authors: Alphonse, S. / Banerjee, A. / Dantuluri, S. / Shuman, S. / Ghose, R.
History
DepositionNov 30, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 14, 2023Group: Other / Category: pdbx_database_status / Item: _pdbx_database_status.status_code_nmr_data
Revision 1.2May 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: tRNA 2'-phosphotransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,5092
Polymers19,8461
Non-polymers6631
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area970 Å2
ΔGint-7 kcal/mol
Surface area10190 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with acceptable covalent geometry
RepresentativeModel #1lowest energy

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Components

#1: Protein tRNA 2'-phosphotransferase / Tpt1


Mass: 19846.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Runella slithyformis (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: 2'-phosphotransferase
#2: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Nicotinamide adenine dinucleotide


Mass: 663.425 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic13D HNCO
121isotropic13D HN(CA)CO
132isotropic43D HNCA
141isotropic23D HN(CO)CA
151isotropic23D HN(CA)CB
162isotropic43D CBCA(CO)NH
171isotropic53D HBHA(CO)NH
181isotropic13D C(CO)NH
191isotropic13D H(CCO)NH
2103isotropic43D CCH-TOCSY
2113isotropic43D (H)CCH-TOCSY
2124isotropic32D 1H-1H NOESY
1135isotropic63D 1H-15N NOESY
1141isotropic43D 1H-15N NOESY
1151isotropic43D 1H-13C NOESY aromatic
2163isotropic43D 1H-13C NOESY aromatic
1171isotropic23D 1H-13C NOESY aliphatic
2183isotropic43D 1H-13C NOESY aliphatic
2194isotropic23D 1H-13C NOESY methyl
2206isotropic44D 1H-13C-13C-1H NOESY methyl

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution1600 uM [U-13C; U-15N] Tpt1, 2.4 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 95% H2O/5% D2O4-fold excess of NAD+ was used to assure full saturation of Tpt1CN_sample195% H2O/5% D2O
solution2280 uM [U-13C; U-15N] Tpt1, 1.12 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 95% H2O/5% D2O4-fold excess of NAD+ was used to assure full saturation of Tpt1CN_sample295% H2O/5% D2O
solution3325 uM [U-13C; U-15N] Tpt1, 1.3 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 100% D2O4-fold excess of NAD+ was used to assure full saturation of Tpt1CN_sample3100% D2O
solution4300 uM [U-13C; U-15N] Tpt1, 1.2 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 100% D2OTpt1 was uniformly [2H-15N] label with specific [13C-1H] labeling on methyl groups for ILE [d1 only] VAL (g1 and g2), Leu (d1 and d2). 4-fold excess of NAD+ was used to assure full saturation of Tpt12H-15N_ILVM_sample4_d2O100% D2O
solution5500 uM [U-13C; U-15N; U-2H] Tpt1, 2 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 95% H2O/5% D2OTpt1 was uniformly [2H-15N] label with specific [13C-1H] labeling on methyl groups for ILE [d1 only] VAL (g1 and g2), Leu (d1 and d2). 4-fold excess of NAD+ was used to assure full saturation of Tpt1DCN_sample595% H2O/5% D2O
solution6400 uM [U-13C; U-15N] Tpt1, 1.6 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 100% D2OTpt1 was uniformly [2H-15N] label with specific [13C-1H] labeling on methyl groups for ILE [d1 only] VAL (g1 and g2), Leu (d1 and d2). 4-fold excess of NAD+ was used to assure full saturation of Tpt12H-15N_ILVM_sample5_d2O100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
600 uMTpt1[U-13C; U-15N]1
2.4 mMNICOTINAMIDE-ADENINE-DINUCLEOTIDEnatural abundance1
20 mMHEPESnatural abundance1
200 mMsodium chloridenatural abundance1
2 mMDTTnatural abundance1
2 mMEDTA[U-2H]1
5 % v/vglycerol[U-2H]1
1 mMAEBSF protease inhibitornatural abundance1
0.04 % w/vsodium azidenatural abundance1
50 uMDSSnatural abundance1
280 uMTpt1[U-13C; U-15N]2
1.12 mMNICOTINAMIDE-ADENINE-DINUCLEOTIDEnatural abundance2
20 mMHEPESnatural abundance2
200 mMsodium chloridenatural abundance2
2 mMDTTnatural abundance2
2 mMEDTA[U-2H]2
5 % v/vglycerol[U-2H]2
1 mMAEBSF protease inhibitornatural abundance2
0.04 % w/vsodium azidenatural abundance2
50 uMDSSnatural abundance2
325 uMTpt1[U-13C; U-15N]3
1.3 mMNICOTINAMIDE-ADENINE-DINUCLEOTIDEnatural abundance3
20 mMHEPESnatural abundance3
200 mMsodium chloridenatural abundance3
2 mMDTTnatural abundance3
2 mMEDTA[U-2H]3
5 % v/vglycerol[U-2H]3
1 mMAEBSF protease inhibitornatural abundance3
0.04 % w/vsodium azidenatural abundance3
50 uMDSSnatural abundance3
300 uMTpt1[U-13C; U-15N]4
1.2 mMNICOTINAMIDE-ADENINE-DINUCLEOTIDEnatural abundance4
20 mMHEPESnatural abundance4
200 mMsodium chloridenatural abundance4
2 mMDTTnatural abundance4
2 mMEDTA[U-2H]4
5 % v/vglycerol[U-2H]4
1 mMAEBSF protease inhibitornatural abundance4
0.04 % w/vsodium azidenatural abundance4
50 uMDSSnatural abundance4
500 uMTpt1[U-13C; U-15N; U-2H]5
2 mMNICOTINAMIDE-ADENINE-DINUCLEOTIDEnatural abundance5
20 mMHEPESnatural abundance5
200 mMsodium chloridenatural abundance5
2 mMDTTnatural abundance5
2 mMEDTA[U-2H]5
5 % v/vglycerol[U-2H]5
1 mMAEBSF protease inhibitornatural abundance5
0.04 % w/vsodium azidenatural abundance5
50 uMDSSnatural abundance5
400 uMTpt1[U-13C; U-15N]6
1.6 mMNICOTINAMIDE-ADENINE-DINUCLEOTIDEnatural abundance6
20 mMHEPESnatural abundance6
200 mMsodium chloridenatural abundance6
2 mMDTTnatural abundance6
2 mMEDTA[U-2H]6
5 % v/vglycerol[U-2H]6
1 mMAEBSF protease inhibitornatural abundance6
0.04 % w/vsodium azidenatural abundance6
50 uMDSSnatural abundance6
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)Details
10 Not definedcondition_17 1 atm290.15 K
20 Not definedcondition_27 pH*1 atm290.15 KSample in 100% D2O, pH of the solution was corrected to match the pH of other {95% H2O-5% D2O} samples.

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE III HDBrukerAVANCE III HD6001cryogenically-cooled probe
Bruker AVANCE III HDBrukerAVANCE III HD7002cryogenically-cooled probe
Bruker AVANCE III HDBrukerAVANCE III HD8003cryogenically-cooled probe
Bruker AVANCE IIIBrukerAVANCE III7005cryogenically-cooled probe
Bruker AVANCE III HDBrukerAVANCE III HD8004cryogenically-cooled probe
Bruker AVANCE IIBrukerAVANCE II9006cryogenically-cooled probe

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Processing

NMR software
NameVersionDeveloperClassification
NMRPipe10.3 Revision 2019.220.13.40Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
TopSpin3.5p15Bruker Biospincollection
NMRViewJohnson, One Moon Scientificchemical shift assignment
ARIA2.3.2Linge, O'Donoghue and Nilgesstructure calculation
X-PLOR NIH2.52Schwieters, Kuszewski, Tjandra and Clorerefinement
RefinementMethod: simulated annealing / Software ordinal: 5
Details: the structures are based on a total of 3773 restraints, 3368 are NOE-derived distance constraints, 269 dihedral angle restraints, 136 from hydrogen bonds.
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with acceptable covalent geometry
Conformers calculated total number: 100 / Conformers submitted total number: 20

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