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- PDB-6coe: AtHNL enantioselectivity mutant At-A9-H7 Apo Y13C,Y121L,P126F,L12... -

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Basic information

Entry
Database: PDB / ID: 6coe
TitleAtHNL enantioselectivity mutant At-A9-H7 Apo Y13C,Y121L,P126F,L128W,C131T,F179L,A209I with benzaldehyde, MANDELIC ACID NITRILE
ComponentsAlpha-hydroxynitrile lyase
KeywordsLYASE / alpha beta hydrolase globular hydroxynitrile lyase
Function / homology
Function and homology information


mandelonitrile lyase activity / (R)-mandelonitrile lyase / glycoside catabolic process / response to wounding
Similarity search - Function
Methylesterase/Alpha-hydroxynitrile lyase / Alpha/beta hydrolase family / alpha/beta hydrolase fold / Alpha/beta hydrolase fold-1 / Alpha/Beta hydrolase fold, catalytic domain / Alpha/Beta hydrolase fold / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
benzaldehyde / (S)-MANDELIC ACID NITRILE / (2R)-hydroxy(phenyl)ethanenitrile / Alpha-hydroxynitrile lyase
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.844 Å
AuthorsJones, B.J. / Kazlauskas, R.J. / Desrouleaux, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM102205 United States
CitationJournal: To be published
Title: AtHNL enantioselectivity mutants
Authors: Jones, B.J. / Desrouleaux, R. / Kazlauskas, R.J.
History
DepositionMar 12, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 20, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 4, 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: Alpha-hydroxynitrile lyase
B: Alpha-hydroxynitrile lyase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,07014
Polymers58,8862
Non-polymers1,18412
Water6,053336
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3320 Å2
ΔGint-13 kcal/mol
Surface area19840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)50.024, 87.231, 123.283
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Alpha-hydroxynitrile lyase / AtHNL / (R)-hydroxynitrile lyase / (R)-oxynitrilase / Methylesterase 5 / AtMES5


Mass: 29442.980 Da / Num. of mol.: 2 / Fragment: AtHNL / Mutation: Y13C,Y121L,P126F,L128W,C131T,F179L,A209I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: HNL, MES5, At5g10300, F18D22_70 / Plasmid: pET21a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q9LFT6, (R)-mandelonitrile lyase

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Non-polymers , 6 types, 348 molecules

#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-HBX / benzaldehyde


Mass: 106.122 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C7H6O / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-MNN / (S)-MANDELIC ACID NITRILE / (S)-HYDROXY(PHENYL)ACETONITRILE


Mass: 133.147 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H7NO / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MXN / (2R)-hydroxy(phenyl)ethanenitrile / (R)-mandelonitrile


Mass: 133.147 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H7NO / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 336 / Source method: isolated from a natural source / Formula: H2O

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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: 2.29 Å3/Da / Density % sol: 46.35 % / Mosaicity: 0.281 ° / Mosaicity esd: 0.004 °
Crystal growTemperature: 292 K / Method: vapor diffusion, sitting drop / pH: 6.4 / Details: 0.1 M bis-tris, 16% PEG 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.0332 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 26, 2016
RadiationMonochromator: Double crystal cryo-cooled Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 1.844→50 Å / Num. obs: 45246 / % possible obs: 95.7 % / Redundancy: 3.5 % / Rmerge(I) obs: 0.072 / Rpim(I) all: 0.042 / Rrim(I) all: 0.083 / Χ2: 0.836 / Net I/σ(I): 7.8
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.844-1.882.80.42221030.7780.2780.5090.56991.6
1.88-1.923.20.36622700.8470.2310.4350.56997.1
1.92-1.953.40.32422450.8830.1970.3810.59496.9
1.95-1.993.50.2922950.9050.1750.340.62597.2
1.99-2.043.50.23122290.940.1390.2710.63397.1
2.04-2.083.50.222970.9440.1210.2350.67797.7
2.08-2.143.60.17522830.9640.1050.2050.70898.1
2.14-2.193.50.14622780.9660.0890.1720.73997.4
2.19-2.263.50.12722940.9740.0780.150.79498.1
2.26-2.333.50.11922550.9790.0730.140.82896.7
2.33-2.413.30.10622770.9770.0670.1260.93496.6
2.41-2.513.20.09922310.9790.0620.1170.98494.7
2.51-2.6330.09421230.9790.060.1130.98190.8
2.63-2.763.40.08822260.9840.0520.1030.98494.6
2.76-2.943.70.08122950.9860.0470.0950.96996.7
2.94-3.163.70.07622980.980.0440.0881.00596.5
3.16-3.483.80.06922800.9880.0390.080.99595.8
3.48-3.993.70.05823080.990.0330.0680.9995.5
3.99-5.023.30.04921530.9940.0280.0571.01288.3
5.02-503.80.04725060.9930.0250.0540.98297.3

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Processing

Software
NameVersionClassification
REFMAC5.8.0155refinement
DENZOdata reduction
HKL-2000data scaling
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: pdbid 3DQZ

3dqz


Resolution: 1.844→50 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.942 / SU B: 5.523 / SU ML: 0.084 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.127 / ESU R Free: 0.124
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.1988 2245 5 %RANDOM
Rwork0.154 ---
obs0.1563 42949 95.47 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 115.89 Å2 / Biso mean: 32.207 Å2 / Biso min: 12.92 Å2
Baniso -1Baniso -2Baniso -3
1-0.07 Å2-0 Å20 Å2
2--0.02 Å2-0 Å2
3----0.08 Å2
Refinement stepCycle: final / Resolution: 1.844→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4112 0 82 336 4530
Biso mean--58.17 37.99 -
Num. residues----517
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0260.0194385
X-RAY DIFFRACTIONr_bond_other_d0.0020.024157
X-RAY DIFFRACTIONr_angle_refined_deg2.2591.9755922
X-RAY DIFFRACTIONr_angle_other_deg1.17539626
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4385537
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.67624.392189
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.57215775
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.1361517
X-RAY DIFFRACTIONr_chiral_restr0.160.2641
X-RAY DIFFRACTIONr_gen_planes_refined0.0130.0214885
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02998
LS refinement shellResolution: 1.844→1.892 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.27 146 -
Rwork0.228 2940 -
all-3086 -
obs--89.79 %
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)
10.61560.09410.00020.10690.1560.7867-0.0890.16730.0462-0.02590.0239-0.0076-0.0558-0.06990.06510.0174-0.0167-0.01030.06970.00390.0215-8.0078-2.71943.0469
20.70430.14320.14630.3378-0.01490.29170.013-0.0816-0.01320.03560.0045-0.0285-0.01130.0134-0.01750.01540.0004-0.01630.0333-0.01640.03019.3664-7.06433.055
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 258
2X-RAY DIFFRACTION2B1 - 259

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