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- PDB-6x94: An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical am... -

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Basic information

Entry
Database: PDB / ID: 6x94
TitleAn orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in Escherichia coli
ComponentsSerine--tRNA ligase
KeywordsLIGASE / Serine tRNA-ligase / Protein translation / Serine aminoacylation / Non-hydrolyzable Serine-AMP
Function / homology
Function and homology information


: / selenocysteine biosynthetic process / serine-tRNA ligase / serine-tRNA ligase activity / seryl-tRNA aminoacylation / ATP binding / cytoplasm
Similarity search - Function
Serine-tRNA synthetase, type1, N-terminal / Seryl-tRNA synthetase N-terminal domain / Serine-tRNA ligase, type1 / Serine-tRNA ligase catalytic core domain / Serine-tRNA synthetase, type1, N-terminal domain superfamily / Class I and II aminoacyl-tRNA synthetase, tRNA-binding arm / Aminoacyl-tRNA synthetase, class II (G/ P/ S/T) / tRNA synthetase class II core domain (G, H, P, S and T) / Aminoacyl-tRNA synthetase, class II / Aminoacyl-transfer RNA synthetases class-II family profile.
Similarity search - Domain/homology
: / DI(HYDROXYETHYL)ETHER / THIOCYANATE ION / 5'-O-(N-(L-SERYL)-SULFAMOYL)ADENOSINE / Serine--tRNA ligase
Similarity search - Component
Biological speciesMethanosarcina mazei (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.45 Å
AuthorsZambaldo, C. / Koh, M. / Nasertorabi, F. / Han, G.W. / Chatterjee, A. / Stevens, R.C. / Schultz, P.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 - GM062159 United States
CitationJournal: Bioorg.Med.Chem. / Year: 2020
Title: An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in Escherichia coli.
Authors: Zambaldo, C. / Koh, M. / Nasertorabi, F. / Han, G.W. / Chatterjee, A. / Stevens, R.C. / Schultz, P.G.
History
DepositionJun 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 4, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 18, 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

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Assembly

Deposited unit
A: Serine--tRNA ligase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,85413
Polymers48,6591
Non-polymers1,19512
Water12,304683
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Size Exclusion Chromatography
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2910 Å2
ΔGint-8 kcal/mol
Surface area20000 Å2
Unit cell
Length a, b, c (Å)72.782, 159.415, 84.004
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11A-923-

HOH

21A-1018-

HOH

31A-1161-

HOH

41A-1210-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Serine--tRNA ligase / Seryl-tRNA synthetase / SerRS / Seryl-tRNA(Ser/Sec) synthetase


Mass: 48659.363 Da / Num. of mol.: 1 / Mutation: E402V E416V K419P V420E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88) (archaea)
Strain: ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Gene: serS, MM_0865 / Plasmid: pET22b
Details (production host): T7 RNA polimerase (IPTG indicuble expression system), Ampicillin resistant
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8PYJ6, serine-tRNA ligase

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Non-polymers , 7 types, 695 molecules

#2: Chemical ChemComp-SSA / 5'-O-(N-(L-SERYL)-SULFAMOYL)ADENOSINE


Mass: 433.397 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H19N7O8S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE / Dimethyl sulfoxide


Mass: 78.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#5: Chemical
ChemComp-SCN / THIOCYANATE ION / Thiocyanate


Mass: 58.082 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: CNS
#6: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
#7: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 683 / 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: 2.71 Å3/Da / Density % sol: 54.6 % / Description: Large, rectangular 20 x 30 x (50-300) um
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: 18% PEG3350, 200 mM thiocyanate / PH range: 6.5 - 7.5

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: Cryo / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 6, 2018
RadiationMonochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 1.45→79.707 Å / Num. obs: 86540 / % possible obs: 99.9 % / Redundancy: 9.9 % / CC1/2: 1 / Rmerge(I) obs: 0.06 / Rpim(I) all: 0.02 / Rrim(I) all: 0.063 / Rsym value: 0.06 / Net I/av σ(I): 8.8 / Net I/σ(I): 18.1
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allRsym value% possible all
1.45-1.53101.1540.7125510.7250.3831.2171.154100
1.53-1.629.60.6911.1118260.2320.730.69199.8
1.62-1.7310.20.4221.9111750.1380.4440.422100
1.73-1.879.80.2433.2103750.0810.2570.24399.9
1.87-2.0510.10.1415.596040.0460.1480.141100
2.05-2.2910.10.0878.687060.0290.0920.087100
2.29-2.659.70.05912.477040.020.0620.05999.9
2.65-3.249.70.04215.565540.0140.0450.042100
3.24-4.599.30.03120.651280.010.0320.03199.9
4.59-29.2049.20.02524.329170.0080.0260.02599.5

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
SCALA3.3.22data scaling
PHASERphasing
REFMAC5.8.0238refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2DQ3

2dq3


Resolution: 1.45→29.22 Å / Cor.coef. Fo:Fc: 0.98 / Cor.coef. Fo:Fc free: 0.969 / SU B: 2.051 / SU ML: 0.04 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.054 / ESU R Free: 0.056 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.1685 4206 4.9 %RANDOM
Rwork0.1442 ---
obs0.1454 82304 99.87 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 98.21 Å2 / Biso mean: 22.611 Å2 / Biso min: 12.56 Å2
Baniso -1Baniso -2Baniso -3
1-0.26 Å20 Å20 Å2
2---0.75 Å20 Å2
3---0.5 Å2
Refinement stepCycle: final / Resolution: 1.45→29.22 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3279 0 71 694 4044
Biso mean--27.6 38.34 -
Num. residues----413
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0133607
X-RAY DIFFRACTIONr_bond_other_d0.0020.0173409
X-RAY DIFFRACTIONr_angle_refined_deg1.9371.6654903
X-RAY DIFFRACTIONr_angle_other_deg1.5251.587938
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.9455463
X-RAY DIFFRACTIONr_dihedral_angle_2_deg29.21221.9200
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.18815652
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.3071530
X-RAY DIFFRACTIONr_chiral_restr0.1130.2473
X-RAY DIFFRACTIONr_gen_planes_refined0.0120.024033
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02761
LS refinement shellResolution: 1.45→1.488 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.256 321 -
Rwork0.25 6042 -
all-6363 -
obs--99.97 %
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)
10.3568-0.0180.54310.103-0.04810.87010.0281-0.08770.01240.0149-0.04530.03120.0792-0.14910.01720.0393-0.04020.00980.0692-0.00760.0348-56.5991-31.5887-17.6438
22.45440.56920.87261.37450.93330.8580.18-0.1032-0.25970.2157-0.1137-0.04090.2628-0.0947-0.06630.117-0.0278-0.00630.0230.02070.0419-27.6237-31.275110.1313
30.4575-0.12360.20910.23050.03960.1430.0053-0.06490.04440.0727-0.0224-0.00360.0439-0.04780.01710.0513-0.0008-0.00990.06530.00780.0283-20.7904-1.84420.3293
40.1556-0.14960.00850.70960.45460.38440.0083-0.00840.00050.01510.0071-0.04450.01320.0024-0.01540.03280.0024-0.00350.04590.00780.0654-12.9491-5.623.7011
50.00530.03750.02140.31830.18160.1055-0.0040.00240.0026-0.0031-0.02560.03750.0023-0.01010.02960.0328-0.00210.01530.0526-0.00370.057-32.676-11.8709-6.6739
60.12730.05320.09530.09720.08420.16240.0151-0.014-0.01340.0196-0.0244-0.03480.0375-0.02560.00930.0443-0.00210.00330.03750.00380.0511-23.6537-19.25182.7712
70.22630.24030.07940.34640.13280.7762-0.0097-0.006-0.02140.03840.0224-0.09770.00610.0443-0.01260.02670.0063-0.0420.0653-0.00340.0722-7.9375-2.506416.7999
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 105
2X-RAY DIFFRACTION2A106 - 126
3X-RAY DIFFRACTION3A127 - 168
4X-RAY DIFFRACTION4A169 - 188
5X-RAY DIFFRACTION5A189 - 247
6X-RAY DIFFRACTION6A248 - 390
7X-RAY DIFFRACTION7A391 - 420

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