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- PDB-7k4u: Crystal structure of Kemp Eliminase HG3 K50Q in complex with the ... -

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Basic information

Entry
Database: PDB / ID: 7k4u
TitleCrystal structure of Kemp Eliminase HG3 K50Q in complex with the transition state analog 6-nitrobenzotriazole
ComponentsEndo-1,4-beta-xylanaseXylanase
KeywordsHYDROLASE / kemp elimination / directed evolution / transition state analog
Function / homology
Function and homology information


endo-1,4-beta-xylanase activity / endo-1,4-beta-xylanase / xylan catabolic process
Similarity search - Function
Glycosyl hydrolases family 10, active site / Glycosyl hydrolases family 10 (GH10) active site. / Glycoside hydrolase family 10 / Glycosyl hydrolases family 10 (GH10) domain profile. / Glycoside hydrolase family 10 domain / Glycosyl hydrolase family 10 / Glycosyl hydrolase family 10 / Glycoside hydrolase superfamily
Similarity search - Domain/homology
6-NITROBENZOTRIAZOLE / Endo-1,4-beta-xylanase
Similarity search - Component
Biological speciesThermoascus aurantiacus (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.3 Å
AuthorsPadua, R.A.P. / Otten, R. / Bunzel, A. / Nguyen, V. / Pitsawong, W. / Patterson, M. / Sui, S. / Perry, S.L. / Cohen, A.E. / Hilvert, D. / Kern, D.
Funding support United States, Switzerland, 11items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-FG02-05ER15699 United States
Damon Runyon Cancer Research FoundationDRG-2114-12 United States
Department of Energy (DOE, United States)DE-AC02-76SF00515 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103393 United States
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30 GM124169 United States
Department of Energy (DOE, United States)DE-AC02-05CH11231 United States
National Institutes of Health/Office of the DirectorS10OD021832 United States
Swiss National Science Foundation Switzerland
Other government Switzerland
National Science Foundation (NSF, United States)BioXFEL STC (1231306) United States
CitationJournal: Science / Year: 2020
Title: How directed evolution reshapes the energy landscape in an enzyme to boost catalysis.
Authors: Otten, R. / Padua, R.A.P. / Bunzel, H.A. / Nguyen, V. / Pitsawong, W. / Patterson, M. / Sui, S. / Perry, S.L. / Cohen, A.E. / Hilvert, D. / Kern, D.
History
DepositionSep 16, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 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

Downloads & links

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Assembly

Deposited unit
A: Endo-1,4-beta-xylanase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,2352
Polymers33,0711
Non-polymers1641
Water5,008278
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)48.089, 59.829, 80.468
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Endo-1,4-beta-xylanase / Xylanase / Xylanase / 1 / 4-beta-D-xylan xylanohydrolase / TAXI


Mass: 33071.172 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoascus aurantiacus (fungus) / Gene: XYNA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P23360, endo-1,4-beta-xylanase
#2: Chemical ChemComp-6NT / 6-NITROBENZOTRIAZOLE


Mass: 164.122 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H4N4O2
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 278 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 1.74 Å3/Da / Density % sol: 29.45 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / Details: 2 M ammonium sulfate and 5% isopropanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.1 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Sep 19, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.3→33.39 Å / Num. obs: 57674 / % possible obs: 99.57 % / Redundancy: 6.9 % / Biso Wilson estimate: 13.28 Å2 / CC1/2: 0.995 / Net I/σ(I): 8.33
Reflection shellResolution: 1.3→1.346 Å / Num. unique obs: 5663 / CC1/2: 0.222

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
Cootmodel building
Aimlessdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4bs0

4bs0


Resolution: 1.3→33.39 Å / SU ML: 0.2414 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 26.7029
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2113 3737 3.47 %
Rwork0.1852 104090 -
obs0.1861 57538 97.78 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 18.99 Å2
Refinement stepCycle: LAST / Resolution: 1.3→33.39 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2329 0 0 278 2607
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0062595
X-RAY DIFFRACTIONf_angle_d0.93575
X-RAY DIFFRACTIONf_chiral_restr0.0678392
X-RAY DIFFRACTIONf_plane_restr0.0064484
X-RAY DIFFRACTIONf_dihedral_angle_d14.4088372
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.3-1.320.4051190.38933258X-RAY DIFFRACTION85
1.32-1.330.39931170.38833259X-RAY DIFFRACTION81.55
1.33-1.350.35421280.38223471X-RAY DIFFRACTION88.67
1.35-1.370.44421390.38563814X-RAY DIFFRACTION95.83
1.37-1.390.36631350.36483831X-RAY DIFFRACTION97.85
1.39-1.410.35421400.35893864X-RAY DIFFRACTION98.09
1.41-1.440.38131430.35353924X-RAY DIFFRACTION99.41
1.44-1.460.44061400.37383988X-RAY DIFFRACTION99.35
1.46-1.490.39331420.33353885X-RAY DIFFRACTION99.68
1.49-1.520.30871400.30513916X-RAY DIFFRACTION99.73
1.52-1.550.30641410.28043969X-RAY DIFFRACTION99.83
1.55-1.580.31731410.263908X-RAY DIFFRACTION99.85
1.58-1.620.26711440.24683974X-RAY DIFFRACTION99.98
1.62-1.660.26791440.23173944X-RAY DIFFRACTION99.95
1.66-1.70.24951390.22323930X-RAY DIFFRACTION99.9
1.7-1.750.25371410.20563935X-RAY DIFFRACTION99.98
1.75-1.810.25021380.18993930X-RAY DIFFRACTION99.95
1.81-1.870.2241420.17313985X-RAY DIFFRACTION99.93
1.87-1.950.20611380.16023926X-RAY DIFFRACTION99.9
1.95-2.040.17951400.15413922X-RAY DIFFRACTION100
2.04-2.150.20941420.14433957X-RAY DIFFRACTION99.81
2.15-2.280.18381410.13853949X-RAY DIFFRACTION99.83
2.28-2.460.15171360.13923917X-RAY DIFFRACTION99.83
2.46-2.70.14331430.13483964X-RAY DIFFRACTION99.71
2.7-3.090.16731460.1393904X-RAY DIFFRACTION99.53
3.09-3.90.14181400.13013871X-RAY DIFFRACTION98.31
3.9-33.390.14481380.14083895X-RAY DIFFRACTION98.56
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)
12.021318404930.00650644286421-0.3777706224191.64334241599-0.3179060426313.1677563034-0.0174274781269-0.0873958172825-0.02817748209830.102426994222-0.00662164581573-0.1650779875830.03619646744620.128302943572-0.006137728354070.112115267239-0.0110863751356-0.01224802707770.07303788973270.01290665562890.089642451457422.737110666-0.06353950351629.6599435056
21.768248994950.0932536270934-0.465795686310.3551206575670.05458554511590.1331805394690.0444383851613-0.5733150288860.03014534130530.125383115454-0.05180583562540.04432440749170.0173703463080.03731018490610.008469043856350.139807738949-0.02041770223080.01409701699740.2309872045080.002348356318410.1030499309284.245807397786.7579745633719.3361095784
32.187833644270.400800776612-0.3836445557220.9573806031090.2627023490970.7672359817570.0234482456479-0.2234107381570.1770610171530.0415871269601-0.05462495367250.145476670523-0.01553035002780.0003349448008780.01714313655120.1094643174210.003746264754390.005172260867840.09199621207910.004196001745280.0856312447149-3.346853064519.5859424816711.7183714291
41.46555613297-0.275307776345-0.2755577221242.649338987780.9401024888321.4181569556-0.01411623510370.0420400770002-0.0518555387379-0.0531328081991-0.01412699989010.01490036870730.04170847872490.0009229029725180.02334413611930.1015379551380.00196950172691-0.003382955352870.08141903209720.02737565024210.0863684524385-0.3474482488226.679537183413.17021954758
50.974456435383-0.762183751912-0.06915860877541.9664099704-1.010301076661.59365272571-0.02978870957810.161326937239-0.111045704772-0.3105847712590.05338383471370.2641586209370.0373487809731-0.13416462994-0.01611890878350.138227350835-0.00410930579538-0.040856602210.09001446342860.00110012248040.125954179068-4.848964103293.74675164823-5.29058052687
61.42199747591-0.736761959540.2653765061571.16561263728-0.3715672922610.6348974300150.02641776651820.0218915484324-0.124391721752-0.0642765046049-0.02436347757160.01212418656750.03821459575750.0334357962348-0.004727485389840.113147824143-0.011520592155-0.004863591028020.07076099937910.009342778213150.0853650860613.4270734328-5.454450374191.62700533636
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid -2 through 45 )
2X-RAY DIFFRACTION2chain 'A' and (resid 46 through 100 )
3X-RAY DIFFRACTION3chain 'A' and (resid 101 through 149 )
4X-RAY DIFFRACTION4chain 'A' and (resid 150 through 181 )
5X-RAY DIFFRACTION5chain 'A' and (resid 182 through 196 )
6X-RAY DIFFRACTION6chain 'A' and (resid 197 through 302 )

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