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- PDB-6vt9: Naegleria gruberi RNA ligase E227A mutant with ATP and Mn -

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Basic information

Entry
Database: PDB / ID: 6vt9
TitleNaegleria gruberi RNA ligase E227A mutant with ATP and Mn
ComponentsRNA ligase
KeywordsLIGASE / RNA repair / adenylyltransferase
Function / homologyRNA ligase, DRB0094 / PHA02142 OB-fold domain / RNA ligase domain, REL/Rln2 / RNA ligase / ATP binding / metal ion binding / ADENOSINE-5'-TRIPHOSPHATE / : / Predicted protein
Function and homology information
Biological speciesNaegleria gruberi (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.49 Å
AuthorsUnciuleac, M.C. / Goldgur, Y. / Shuman, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM126945 United States
CitationJournal: Nucleic Acids Res. / Year: 2020
Title: Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation.
Authors: Unciuleac, M.C. / Goldgur, Y. / Shuman, S.
History
DepositionFeb 12, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 8, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 10, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
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: RNA ligase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,1663
Polymers38,6041
Non-polymers5622
Water27015
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)88.432, 88.432, 105.347
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-512-

HOH

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Components

#1: Protein RNA ligase


Mass: 38603.797 Da / Num. of mol.: 1 / Mutation: E227A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Naegleria gruberi (eukaryote) / Gene: NAEGRDRAFT_82186 / Production host: Escherichia coli (E. coli) / References: UniProt: D2W2Z5
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.67 Å3/Da / Density % sol: 53.89 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: NgrRnlE227A (12 mg/ml) was adjusted to 2 mM ATP and 5 mM MnCl2 and incubated for 15 min on ice before aliquots of the protein solution (1 ul) were mixed on a coverslip with an equal volume ...Details: NgrRnlE227A (12 mg/ml) was adjusted to 2 mM ATP and 5 mM MnCl2 and incubated for 15 min on ice before aliquots of the protein solution (1 ul) were mixed on a coverslip with an equal volume of precipitant solution containing 0.1 M HEPES pH 6.5, 30% PEG6000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.9793 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Aug 3, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 2.49→30 Å / Num. obs: 27618 / % possible obs: 99.9 % / Redundancy: 14.7 % / Rmerge(I) obs: 0.144 / Rpim(I) all: 0.041 / Rrim(I) all: 0.15 / Χ2: 3.084 / Net I/σ(I): 8.9
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.49-2.5416.62.0067490.6450.5162.0721.341100
2.54-2.5916.71.6427180.6990.4211.6961.434100
2.59-2.6416.51.4337480.7890.3681.481.511100
2.64-2.6916.61.1647390.7540.2991.2021.62100
2.69-2.7516.60.9617350.8780.2460.9931.759100
2.75-2.8216.30.7197260.9390.1860.7431.972100
2.82-2.8916.20.6447390.9430.1670.6652.116100
2.89-2.9616.10.5197500.960.1350.5362.41100
2.96-3.0515.80.3997390.9760.1050.4132.669100
3.05-3.1515.40.3257420.9840.0870.3372.95100
3.15-3.2614.90.277460.9890.0740.283.169100
3.26-3.3914.30.2077440.9920.0580.2153.533100
3.39-3.5513.90.1777500.9890.050.1844.153100
3.55-3.7312.70.1777580.9920.0530.1855.176100
3.73-3.97130.1387450.9940.0410.1444.928100
3.97-4.2712.70.1117660.9960.0330.1164.925100
4.27-4.712.30.0927710.9960.0280.0965.165100
4.7-5.3812.50.0887650.9970.0270.0925.008100
5.38-6.7613.30.0797940.9980.0230.0824.34100
6.76-30120.068570.9960.0190.0644.3198.8

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Processing

Software
NameVersionClassification
SCALEPACKdata scaling
PHENIX1.17.1_3660refinement
PDB_EXTRACT3.25data extraction
HKL-2000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5COT

5cot


Resolution: 2.49→27.5 Å / SU ML: 0.48 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 36.31
RfactorNum. reflection% reflection
Rfree0.3055 2785 10.08 %
Rwork0.2186 --
obs0.2272 27618 98.73 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 169.29 Å2 / Biso mean: 80.5114 Å2 / Biso min: 33 Å2
Refinement stepCycle: final / Resolution: 2.49→27.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2677 0 32 15 2724
Biso mean--82.9 67.92 -
Num. residues----335
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.49-2.530.46451080.3752972108078
2.53-2.580.43061400.361712681408100
2.58-2.630.4191410.348512381379100
2.63-2.680.47441410.341712661407100
2.68-2.740.44771390.346112771416100
2.74-2.80.40721380.31312541392100
2.8-2.870.36111360.301712651401100
2.87-2.950.42411420.287112671409100
2.95-3.040.30691400.264112421382100
3.04-3.140.35041430.259312561399100
3.14-3.250.35641410.271612601401100
3.25-3.380.30641420.253912471389100
3.38-3.530.34641470.254712721419100
3.53-3.720.44361390.25411233137299
3.72-3.950.31461410.23761252139399
3.95-4.250.241410.18912471388100
4.25-4.680.25891420.155212741416100
4.68-5.350.22181400.159612431383100
5.36-6.720.25671440.179212571401100
6.73-27.50.24551400.16361243138398
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
16.12360.27792.30934.0691-0.53415.8051-0.1545-0.12030.0323-0.29010.27180.63290.0714-0.7904-0.0580.4631-0.1011-0.08590.44790.09470.5508-3.082428.122424.7
23.5289-3.1014-1.01883.79072.18121.79590.12970.00440.08170.09130.1897-0.61020.39190.4197-0.34150.770.0929-0.07230.5754-0.0540.707422.519112.955320.9115
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 4 through 117 )A4 - 117
2X-RAY DIFFRACTION2chain 'A' and (resid 118 through 338 )A118 - 338

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