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- PDB-8c49: Crystal structure of pyrrolysyl-tRNA synthetase from Methanomethy... -

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Basic information

Entry
Database: PDB / ID: 8c49
TitleCrystal structure of pyrrolysyl-tRNA synthetase from Methanomethylophilus alvus engineered for 3-Methyl-L-histidine, bound to AMPPNP
ComponentsPyrrolysyl-tRNA synthetasePyrrolysine—tRNAPyl ligase
KeywordsLIGASE / pyrrolysyl-tRNA synthetase / genetic code expansion / engineered translation components / Methanomethylophilus alvus
Function / homology
Function and homology information


pyrrolysine-tRNAPyl ligase / pyrrolysyl-tRNA synthetase activity
Similarity search - Function
Phenylalanyl tRNA synthetase beta chain, core domain / Phenylalanyl tRNA synthetase beta chain CLM domain / Pyrrolysyl-tRNA ligase, C-terminal / Aminoacyl-tRNA synthetase, class II / Aminoacyl-transfer RNA synthetases class-II family profile. / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL)
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DI(HYDROXYETHYL)ETHER / TRIETHYLENE GLYCOL / Pyrrolysyl-tRNA synthetase
Similarity search - Component
Biological speciesCandidatus Methanomethylophilus alvus (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.82 Å
AuthorsHardy, F.J. / Levy, C.W.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MR/T041722/1 United Kingdom
CitationJournal: Protein Sci. / Year: 2023
Title: Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.
Authors: Taylor, C.J. / Hardy, F.J. / Burke, A.J. / Bednar, R.M. / Mehl, R.A. / Green, A.P. / Lovelock, S.L.
History
DepositionJan 3, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 19, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / diffrn_source / pdbx_initial_refinement_model
Item: _diffrn_source.pdbx_synchrotron_site / _pdbx_initial_refinement_model.accession_code

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pyrrolysyl-tRNA synthetase
B: Pyrrolysyl-tRNA synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,44029
Polymers63,7182
Non-polymers2,72127
Water4,594255
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11490 Å2
ΔGint-69 kcal/mol
Surface area22800 Å2
MethodPISA
Unit cell
Length a, b, c (Å)59.932, 59.932, 263.231
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number169
Space group name H-MP61
Space group name HallP61
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/6
#3: y,-x+y,z+5/6
#4: -y,x-y,z+1/3
#5: -x+y,-x,z+2/3
#6: -x,-y,z+1/2

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Pyrrolysyl-tRNA synthetase / Pyrrolysine—tRNAPyl ligase


Mass: 31859.135 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Methanomethylophilus alvus (archaea)
Gene: MMALV_11280 / Production host: Escherichia coli (E. coli) / References: UniProt: M9SC49, pyrrolysine-tRNAPyl ligase

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Non-polymers , 6 types, 282 molecules

#2: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#3: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL / Polyethylene glycol


Mass: 150.173 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H14O4
#5: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 255 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.14 Å3/Da / Density % sol: 42.57 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop
Details: 0.2 M Magnesium chloride hexahydrate, 0.1 M tris, pH = 8, 25% (v/v) PEG Smear High, 10% glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Jul 30, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.82→51.9 Å / Num. obs: 47516 / % possible obs: 99.74 % / Redundancy: 21.1 % / Biso Wilson estimate: 23.43 Å2 / CC1/2: 0.997 / CC star: 0.999 / Rmerge(I) obs: 0.2091 / Net I/σ(I): 9.02
Reflection shellResolution: 1.82→1.885 Å / Mean I/σ(I) obs: 1.13 / Num. unique obs: 4638 / CC1/2: 0.759 / CC star: 0.929

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
DIALSdata reduction
DIALSdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6jp2

6jp2


Resolution: 1.82→51.9 Å / SU ML: 0.2155 / Cross valid method: FREE R-VALUE / σ(F): 1.42 / Phase error: 21.9964
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2094 1998 4.21 %
Rwork0.1945 45486 -
obs0.1952 47484 99.74 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 28.86 Å2
Refinement stepCycle: LAST / Resolution: 1.82→51.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4294 0 168 255 4717
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00244553
X-RAY DIFFRACTIONf_angle_d0.56436120
X-RAY DIFFRACTIONf_chiral_restr0.04665
X-RAY DIFFRACTIONf_plane_restr0.0035781
X-RAY DIFFRACTIONf_dihedral_angle_d15.297659
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.82-1.870.3151380.2973144X-RAY DIFFRACTION97.24
1.87-1.920.29681400.28643219X-RAY DIFFRACTION99.88
1.92-1.970.27831420.25653260X-RAY DIFFRACTION99.91
1.97-2.040.23361510.22733260X-RAY DIFFRACTION99.88
2.04-2.110.24721450.23163231X-RAY DIFFRACTION99.91
2.11-2.190.20611440.20483261X-RAY DIFFRACTION100
2.19-2.290.24191430.19733279X-RAY DIFFRACTION99.91
2.29-2.410.20371350.19493252X-RAY DIFFRACTION99.88
2.41-2.570.18051440.19243231X-RAY DIFFRACTION99.97
2.57-2.760.25281430.20123261X-RAY DIFFRACTION99.91
2.76-3.040.20541460.18643287X-RAY DIFFRACTION100
3.04-3.480.20531450.17673248X-RAY DIFFRACTION99.97
3.48-4.380.19991370.16133266X-RAY DIFFRACTION100
4.39-51.90.20361450.18343287X-RAY DIFFRACTION99.91

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