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- PDB-8c8w: Priestia megaterium mupirocin hyper-resistant HIGH motif mutant o... -

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Basic information

Entry
Database: PDB / ID: 8c8w
TitlePriestia megaterium mupirocin hyper-resistant HIGH motif mutant of type 2 isoleucyl-tRNA synthetase complexed with an isoleucyl-adenylate analogue
ComponentsIsoleucine--tRNA ligase
KeywordsRNA BINDING PROTEIN / antibiotic / mupirocin-resistant / IleRS2 / RNA-binding protein / C-terminal domain
Function / homology
Function and homology information


isoleucine-tRNA ligase / isoleucine-tRNA ligase activity / isoleucyl-tRNA aminoacylation / aminoacyl-tRNA deacylase activity / tRNA binding / zinc ion binding / ATP binding / cytoplasm
Similarity search - Function
Domain of unknown function (DUF5915) / Isoleucine-tRNA ligase, type 2 / Isoleucyl tRNA synthetase type 2, anticodon-binding domain / Isoleucine-tRNA ligase / Aminoacyl-tRNA synthetase, class Ia / tRNA synthetases class I (I, L, M and V) / Valyl/Leucyl/Isoleucyl-tRNA synthetase, editing domain / Methionyl/Valyl/Leucyl/Isoleucyl-tRNA synthetase, anticodon-binding / Anticodon-binding domain of tRNA ligase / Aminoacyl-tRNA synthetase, class Ia, anticodon-binding ...Domain of unknown function (DUF5915) / Isoleucine-tRNA ligase, type 2 / Isoleucyl tRNA synthetase type 2, anticodon-binding domain / Isoleucine-tRNA ligase / Aminoacyl-tRNA synthetase, class Ia / tRNA synthetases class I (I, L, M and V) / Valyl/Leucyl/Isoleucyl-tRNA synthetase, editing domain / Methionyl/Valyl/Leucyl/Isoleucyl-tRNA synthetase, anticodon-binding / Anticodon-binding domain of tRNA ligase / Aminoacyl-tRNA synthetase, class Ia, anticodon-binding / Aminoacyl-tRNA synthetase, class I, conserved site / Aminoacyl-transfer RNA synthetases class-I signature. / Rossmann-like alpha/beta/alpha sandwich fold
Similarity search - Domain/homology
N-[ISOLEUCINYL]-N'-[ADENOSYL]-DIAMINOSUFONE / D(-)-TARTARIC ACID / Isoleucine--tRNA ligase
Similarity search - Component
Biological speciesPriestia megaterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.29 Å
AuthorsBrkic, A. / Leibundgut, M. / Jablonska, J. / Zanki, V. / Car, Z. / Petrovic Perokovic, V. / Ban, N. / Gruic-Sovulj, I.
Funding support Croatia, Switzerland, European Union, 3items
OrganizationGrant numberCountry
Croatian Science Foundation180567 Croatia
Swiss National Science Foundation180567 Switzerland
European Regional Development FundKK.01.1.1.02.0016European Union
CitationJournal: Nat Commun / Year: 2023
Title: Antibiotic hyper-resistance in a class I aminoacyl-tRNA synthetase with altered active site signature motif.
Authors: Brkic, A. / Leibundgut, M. / Jablonska, J. / Zanki, V. / Car, Z. / Petrovic Perokovic, V. / Marsavelski, A. / Ban, N. / Gruic-Sovulj, I.
History
DepositionJan 21, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Mar 27, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoleucine--tRNA ligase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)119,0548
Polymers117,8641
Non-polymers1,1907
Water4,684260
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1850 Å2
ΔGint0 kcal/mol
Surface area45320 Å2
MethodPISA
Unit cell
Length a, b, c (Å)107.920, 107.920, 239.360
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein Isoleucine--tRNA ligase / Isoleucyl-tRNA synthetase / IleRS


Mass: 117863.844 Da / Num. of mol.: 1 / Mutation: H54G, G56H
Source method: isolated from a genetically manipulated source
Details: Gram-positive bacterium / Source: (gene. exp.) Priestia megaterium (bacteria) / Strain: DSM-32 / Gene: ileS, BG04_5198 / Plasmid: pET28b(+) / Details (production host): EMD Biosciences, Cat: 69865-3 / Cell (production host): bacterial / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): B / Variant (production host): BL21(DE3) / References: UniProt: A0A0B6AVD3, isoleucine-tRNA ligase
#2: Chemical ChemComp-ILA / N-[ISOLEUCINYL]-N'-[ADENOSYL]-DIAMINOSUFONE


Mass: 458.493 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H26N8O6S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-TAR / D(-)-TARTARIC ACID


Mass: 150.087 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H6O6
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 260 / 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: 2.96 Å3/Da / Density % sol: 58.4 %
Description: Single rhombohedral crystals suspended in the solution. Mechanically robust but prone to cracking during temperature change.
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 5 mM Ile-AMS and 16.5 mg/ml mut-GVHH-PmIleRS2 were mixed in 1:1 ratio with the well solution containing: 0.2-0.5 M Potassium tartrate, 10-20 % (w/v) PEG3350, 25 mM Hepes-KOH pH=7,5 at 20 oC, ...Details: 5 mM Ile-AMS and 16.5 mg/ml mut-GVHH-PmIleRS2 were mixed in 1:1 ratio with the well solution containing: 0.2-0.5 M Potassium tartrate, 10-20 % (w/v) PEG3350, 25 mM Hepes-KOH pH=7,5 at 20 oC, 50 mM NaCl. The drop volume of 2 microliters, was equilibrated towards 300 microliters of the well solution at 4 degrees.
PH range: 6.8-8.2
Temp details: Crystal suscteptible to breaking upon temperature change.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1.0000356682334 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Sep 28, 2020 / Details: 2.9 T superbend magnet
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0000356682334 Å / Relative weight: 1
ReflectionResolution: 2.29→47.31 Å / Num. obs: 64159 / % possible obs: 92.59 % / Redundancy: 13 % / Biso Wilson estimate: 46.79 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.1209 / Rpim(I) all: 0.03477 / Rrim(I) all: 0.1259 / Net I/σ(I): 16.65
Reflection shellResolution: 2.29→2.372 Å / Rmerge(I) obs: 2.125 / Mean I/σ(I) obs: 1.2 / Num. unique obs: 4825 / CC1/2: 0.454 / CC star: 0.79 / Rpim(I) all: 0.6216 / Rrim(I) all: 2.217 / % possible all: 76.38

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDS20190806data reduction
XSCALE20190806data scaling
PHASERCCP4Interface 7.0.077phasing
Coot0.8.9.2model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.29→47.31 Å / SU ML: 0.3141 / Cross valid method: FREE R-VALUE / σ(F): 0.01 / Phase error: 24.8943
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2314 1645 2.76 %
Rwork0.1986 58036 -
obs0.1995 59681 92.52 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 64.49 Å2
Refinement stepCycle: LAST / Resolution: 2.29→47.31 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8313 0 73 260 8646
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00258615
X-RAY DIFFRACTIONf_angle_d0.509811690
X-RAY DIFFRACTIONf_chiral_restr0.04021274
X-RAY DIFFRACTIONf_plane_restr0.00391506
X-RAY DIFFRACTIONf_dihedral_angle_d12.67053160
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.29-2.360.34861110.33273790X-RAY DIFFRACTION74.06
2.36-2.430.32631250.28784267X-RAY DIFFRACTION83.36
2.43-2.520.28691210.26064525X-RAY DIFFRACTION87.38
2.52-2.620.30911280.24044544X-RAY DIFFRACTION88.17
2.62-2.740.26641350.24284742X-RAY DIFFRACTION91.81
2.74-2.880.32891390.24134864X-RAY DIFFRACTION94.08
2.88-3.070.27321420.22774974X-RAY DIFFRACTION95.72
3.07-3.30.29881430.23315073X-RAY DIFFRACTION97.55
3.3-3.630.23141470.21375163X-RAY DIFFRACTION98.7
3.63-4.160.20251460.17455223X-RAY DIFFRACTION98.99
4.16-5.240.17851510.15735300X-RAY DIFFRACTION99.62
5.24-47.310.19661570.16995571X-RAY DIFFRACTION99.57

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