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- PDB-7kw8: NMR Structure of a tRNA 2'-phosphotransferase from Runella slithy... -

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Basic information

Entry
Database: PDB / ID: 7kw8
TitleNMR Structure of a tRNA 2'-phosphotransferase from Runella slithyformis
ComponentstRNA 2'-phosphotransferase
KeywordsTRANSFERASE / tRNA 2'-phosphotransferase
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


Theoretical massNumber of molelcules
Total (without water)19,8461
Polymers19,8461
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
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

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic13D 1H-15N NOESY
122isotropic13D 1H-13C NOESY aliphatic
133isotropic23D 1H-13C NOESY aromatic
144isotropic13D 1H-13C NOESY aromatic
255isotropic33D 1H-13C Methyl NOESY
266isotropic41H-13C-13C-1H 4D NOESY
377isotropic13D HNCA
388isotropic43D HNCO
398isotropic43D HN(CA)CO
3108isotropic43D CBCA(CO)NH
3118isotropic43D HN(CA)CB
3128isotropic43D C(CO)NH
3138isotropic13D H(CCO)NH
4149isotropic43D CCH-TOCSY
4159isotropic43D (H)CCH-TOCSY

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent systemDetails
solution1300 uM [U-13C; U-15N] Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 0.5 mM AEBSF protease inhibitor, 0.04 % v/v sodium azide, 50 uM DSS, 95% H2O/5% D2O15N-13C_sample195% H2O/5% D2O
solution2360 uM [U-13C; U-15N] Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 0.5 mM AEBSF protease inhibitor, 0.04 % v/v sodium azide, 50 uM DSS, 95% H2O/5% D2O15N-13C_sample295% H2O/5% D2O
solution3300 uM [U-13C; U-15N] Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 1 mM AEBSF protease inhibitor, 0.05 % w/v sodium azide, 50 uM DSS, 95% H2O/5% D2O15N-13C_sample395% H2O/5% D2O
solution4400 uM [U-13C; U-15N] Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 0.5 mM AEBSF protease inhibitor, 0.05 % w/v sodium azide, 50 mM DSS, 100% D2O15N-13C_sample4_d2o100% D2O
solution7420 uM [U-13C; U-15N] Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 0.5 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 mM DSS, 95% H2O/5% D2O15N-13C_sample795% H2O/5% D2O
solution8380 uM [U-13C; U-15N] Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 0.5 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 mM DSS, 95% H2O/5% D2O15N-13C_sample895% H2O/5% D2O
solution9400 uM [U-13C; U-15N] Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 0.5 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 mM DSS, 100% D2O15N-13C_sample9_d2o100% D2O
solution5400 uM [U-2H; U-15N];[13CD1,1H3]ILE;[13CD1,1H3;13CD2,1H3]LEU;[13CG1,1H3;13CG2,1H3]VAL;[13CE1,1H3]MET;[13CG2,1H3]THR Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 0.5 mM AEBSF protease inhibitor, 0.05 % w/v sodium azide, 50 uM DSS, 100% D2O2H-15N_ILVMT_sample5_d2O100% D2OUniformly [2H-15N] sample with [13C-1H] labeling on methyl groups for ILE [d1 only] VAL (g1 and g2), Leu (d1 and d2) and THR (g2).
solution6350 uM [U-2H; U-15N];[13CD1,1H3]ILE;[13CD1,1H3;13CD2,1H3]LEU;[13CG1,1H3;13CG2,1H3]VAL;[13CE1,1H3]MET;[13CG2,1H3]THR Tpt1, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-100% 2H] EDTA, 5 % v/v [U-100% 2H] glycerol, 0.5 mM AEBSF protease inhibitor, 0.05 % w/v sodium azide, 50 uM DSS, 100% D2O2H-15N_ILVMT_sample6_d2O100% D2OUniformly [2H-15N] sample with [13C-1H] labeling on methyl groups for ILE [d1 only] VAL (g1 and g2), Leu (d1 and d2) and THR (g2).
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
300 uMTpt1[U-13C; U-15N]1
20 mMHEPESnatural abundance1
200 mMsodium chloridenatural abundance1
2 mMDTTnatural abundance1
2 mMEDTA[U-100% 2H]1
5 % v/vglycerol[U-100% 2H]1
0.5 mMAEBSF protease inhibitornatural abundance1
0.04 % v/vsodium azidenatural abundance1
50 uMDSSnatural abundance1
360 uMTpt1[U-13C; U-15N]2
20 mMHEPESnatural abundance2
200 mMsodium chloridenatural abundance2
2 mMDTTnatural abundance2
2 mMEDTA[U-100% 2H]2
5 % v/vglycerol[U-100% 2H]2
0.5 mMAEBSF protease inhibitornatural abundance2
0.04 % v/vsodium azidenatural abundance2
50 uMDSSnatural abundance2
300 uMTpt1[U-13C; U-15N]3
20 mMHEPESnatural abundance3
200 mMsodium chloridenatural abundance3
2 mMDTTnatural abundance3
2 mMEDTA[U-100% 2H]3
5 % v/vglycerol[U-100% 2H]3
1 mMAEBSF protease inhibitornatural abundance3
0.05 % w/vsodium azidenatural abundance3
50 uMDSSnatural abundance3
400 uMTpt1[U-13C; U-15N]4
20 mMHEPESnatural abundance4
200 mMsodium chloridenatural abundance4
2 mMDTTnatural abundance4
2 mMEDTA[U-100% 2H]4
5 % v/vglycerol[U-100% 2H]4
0.5 mMAEBSF protease inhibitornatural abundance4
0.05 % w/vsodium azidenatural abundance4
50 mMDSSnatural abundance4
420 uMTpt1[U-13C; U-15N]7
20 mMHEPESnatural abundance7
200 mMsodium chloridenatural abundance7
2 mMDTTnatural abundance7
2 mMEDTA[U-100% 2H]7
5 % v/vglycerol[U-100% 2H]7
0.5 mMAEBSF protease inhibitornatural abundance7
0.04 % w/vsodium azidenatural abundance7
50 mMDSSnatural abundance7
380 uMTpt1[U-13C; U-15N]8
20 mMHEPESnatural abundance8
200 mMsodium chloridenatural abundance8
2 mMDTTnatural abundance8
2 mMEDTA[U-100% 2H]8
5 % v/vglycerol[U-100% 2H]8
0.5 mMAEBSF protease inhibitornatural abundance8
0.04 % w/vsodium azidenatural abundance8
50 mMDSSnatural abundance8
400 uMTpt1[U-13C; U-15N]9
20 mMHEPESnatural abundance9
200 mMsodium chloridenatural abundance9
2 mMDTTnatural abundance9
2 mMEDTA[U-100% 2H]9
5 % v/vglycerol[U-100% 2H]9
0.5 mMAEBSF protease inhibitornatural abundance9
0.04 % w/vsodium azidenatural abundance9
50 mMDSSnatural abundance9
400 uMTpt1[U-2H; U-15N];[13CD1,1H3]ILE;[13CD1,1H3;13CD2,1H3]LEU;[13CG1,1H3;13CG2,1H3]VAL;[13CE1,1H3]MET;[13CG2,1H3]THR5
20 mMHEPESnatural abundance5
200 mMsodium chloridenatural abundance5
2 mMDTTnatural abundance5
2 mMEDTA[U-100% 2H]5
5 % v/vglycerol[U-100% 2H]5
0.5 mMAEBSF protease inhibitornatural abundance5
0.05 % w/vsodium azidenatural abundance5
50 uMDSSnatural abundance5
350 uMTpt1[U-2H; U-15N];[13CD1,1H3]ILE;[13CD1,1H3;13CD2,1H3]LEU;[13CG1,1H3;13CG2,1H3]VAL;[13CE1,1H3]MET;[13CG2,1H3]THR6
20 mMHEPESnatural abundance6
200 mMsodium chloridenatural abundance6
2 mMDTTnatural abundance6
2 mMEDTA[U-100% 2H]6
5 % v/vglycerol[U-100% 2H]6
0.5 mMAEBSF protease inhibitornatural abundance6
0.05 % w/vsodium azidenatural abundance6
50 uMDSSnatural abundance6
Sample conditions
Conditions-IDDetailsIonic strengthLabelpHPressure (kPa)Temperature (K)
1Used 4mm shigemi tubes0 Not definedcondition_171 atm290.15 K
3Used 5mm shigemi tubes0 Not definedcondition_371 atm290.15 K
2Sample in 100% D2O, pH of the solution was corrected to match the pH of other {95% H2O-5% D2O} samples. Used 4mm shigemi tubes0 Not definedcondition_27 pH*1 atm290.15 K
4Sample in 100% D2O, pH of the solution was corrected to match the pH of other {95% H2O-5% D2O} samples. Used 5mm shigemi tubes0 Not definedcondition_47 pH*1 atm290.15 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE III HDBrukerAVANCE III HD8001cryogenically-cooled probe
Bruker AVANCE III HDBrukerAVANCE III HD7002cryogenically-cooled probe
Bruker AVANCE IIIBrukerAVANCE III6003cryogenically-cooled probe
Bruker AVANCE III HDBrukerAVANCE III HD8004cryogenically-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 2997 restraints, 2587 are NOE-derived distance constraints, 278 dihedral angle restraints, 132 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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