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- PDB-7k4w: Crystal structure of Kemp Eliminase HG3.17 in the inactive state -

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Basic information

Entry
Database: PDB / ID: 7k4w
TitleCrystal structure of Kemp Eliminase HG3.17 in the inactive state
ComponentsEndo-1,4-beta-xylanaseXylanase
KeywordsHYDROLASE / kemp elimination / directed evolution / inactive state
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
Endo-1,4-beta-xylanase
Similarity search - Component
Biological speciesThermoascus aurantiacus (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 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)33,2312
Polymers33,1901
Non-polymers401
Water2,810156
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)61.390, 67.390, 74.700
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: 33190.457 Da / Num. of mol.: 1 / Mutation: E47N, N300D
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-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 156 / 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: 2.32 Å3/Da / Density % sol: 47.07 %
Crystal growTemperature: 310 K / Method: vapor diffusion, sitting drop
Details: The protein was dialyzed in 380 mM Bis-Tris propane, pH 10, and 200 mM calcium chloride. Crystals were obtained in 2.1 M malic acid, pH 7.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 0.9774 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Nov 29, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9774 Å / Relative weight: 1
ReflectionResolution: 1.9→50.04 Å / Num. obs: 25033 / % possible obs: 99.82 % / Redundancy: 11.9 % / Biso Wilson estimate: 20.6 Å2 / CC1/2: 0.979 / Net I/σ(I): 4.74
Reflection shellResolution: 1.9→1.968 Å / Num. unique obs: 2465 / CC1/2: 0.251

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Processing

Software
NameVersionClassification
PHENIXdev_3965refinement
Cootmodel building
iMOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4bs0

4bs0


Resolution: 1.9→50.04 Å / SU ML: 0.2854 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 25.3854
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2546 3761 8 %
Rwork0.2101 43239 -
obs0.2136 25011 99.63 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 25.04 Å2
Refinement stepCycle: LAST / Resolution: 1.9→50.04 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2171 0 1 156 2328
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00812318
X-RAY DIFFRACTIONf_angle_d0.92063177
X-RAY DIFFRACTIONf_chiral_restr0.05353
X-RAY DIFFRACTIONf_plane_restr0.0081422
X-RAY DIFFRACTIONf_dihedral_angle_d6.1054322
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.9-1.920.36521340.3571560X-RAY DIFFRACTION97.13
1.92-1.950.35481370.33411567X-RAY DIFFRACTION97.59
1.95-1.980.37511380.34911594X-RAY DIFFRACTION98.07
1.98-20.36641390.32511569X-RAY DIFFRACTION99.24
2-2.030.36191400.311614X-RAY DIFFRACTION99.55
2.03-2.070.35321410.30611605X-RAY DIFFRACTION99.89
2.07-2.10.32171390.28791600X-RAY DIFFRACTION99.89
2.1-2.140.29471410.28821590X-RAY DIFFRACTION99.6
2.14-2.170.23431390.2771615X-RAY DIFFRACTION99.94
2.18-2.220.30671400.26021597X-RAY DIFFRACTION100
2.22-2.260.26921350.25961619X-RAY DIFFRACTION99.89
2.26-2.310.26621440.26141617X-RAY DIFFRACTION99.94
2.31-2.360.3281400.25051584X-RAY DIFFRACTION99.88
2.37-2.420.30281420.23711587X-RAY DIFFRACTION99.94
2.42-2.490.27531430.22511627X-RAY DIFFRACTION100
2.49-2.560.26741390.22711618X-RAY DIFFRACTION99.94
2.56-2.650.27531410.21191582X-RAY DIFFRACTION100
2.65-2.740.25861430.18821610X-RAY DIFFRACTION100
2.74-2.850.23761450.20661606X-RAY DIFFRACTION100
2.85-2.980.2251350.18821596X-RAY DIFFRACTION100
2.98-3.140.22651370.18151629X-RAY DIFFRACTION100
3.14-3.330.19851390.15751608X-RAY DIFFRACTION100
3.33-3.590.20821400.14911630X-RAY DIFFRACTION99.94
3.59-3.950.20221360.13931592X-RAY DIFFRACTION99.94
3.95-4.520.21021340.12981606X-RAY DIFFRACTION100
4.52-5.690.18521480.15931610X-RAY DIFFRACTION100
5.7-50.040.2731320.22191607X-RAY DIFFRACTION99.94
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)
10.987282169945-0.7115813408870.1257634188231.478260940510.07610879101230.3718075689240.056994585328-0.00562984425689-0.0748547957625-0.0495408297148-0.04087565948180.06736823668790.0360187797448-0.0215841423021-0.01951668132630.2033220561-0.0091663106650.003116320425650.1624628438730.002374355366730.1709028797794.89786349599-8.9140899284721.7545324718
20.513745386469-0.20967998150.350600536020.5988959968241.059670185363.20548627586-0.0193801084664-0.03976818760540.02845637071870.09872850456590.0596103233504-0.04904124984680.0431898435060.07598454554430.01900726511290.160905890329-0.01741410592650.008740132414460.1129579132890.001509205196750.16669352624115.45093967811.4034150129729.8417476244
32.40379689613-1.21125083980.8545940995483.665797283540.9581042773555.78430546792-0.143406487475-0.449718838049-0.1121498725410.6246096939250.2578104005840.2618218883010.16162528908-0.6659873711010.02774812529610.1938963133970.02164288520740.003268604568530.2236217690580.002456332467340.1816323168746.837513784297.9838502998839.9569449116
46.40765473046-0.0334280383996-4.34097271172.72384612437-0.2434860100077.01258615766-0.3637287414-0.561637903568-0.1088234631210.370846459460.2169760142560.323604570540.133072066941-0.4131661139360.1515268032890.3083037606840.134810338888-0.02079070421920.3742245511830.05150492947770.182638520866-1.9029945702510.226418885440.9012306381
55.373806721480.216135661656-0.5193161556545.33764329130.9646617074954.452715733740.00269262241847-0.153868497812-0.09390545134720.449584194166-0.09173075354560.4292363661070.111040613609-0.3864900689390.09988723474380.1744302557840.05600319104560.0124333147810.181757170836-0.00272974366820.162982194924-6.465426755417.8444672262432.634455196
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth asym-ID: A / Label asym-ID: A

IDRefine TLS-IDSelection detailsAuth seq-IDLabel seq-ID
11chain 'A' and (resid 46 through 149 )46 - 14944 - 147
22chain 'A' and (resid 150 through 228 )150 - 228148 - 226
33chain 'A' and (resid 229 through 257 )229 - 257227 - 255
44chain 'A' and (resid 258 through 302 )258 - 302256 - 286
55chain 'A' and (resid 3 through 45 )3 - 451 - 43

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