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- PDB-7k4s: Crystal structure of Kemp Eliminase HG3.7 -

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Basic information

Entry
Database: PDB / ID: 7k4s
TitleCrystal structure of Kemp Eliminase HG3.7
ComponentsEndo-1,4-beta-xylanase
KeywordsHYDROLASE / kemp elimination / directed evolution
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. / Glycosyl hydrolases family 10 (GH10) domain profile. / Glycoside hydrolase family 10 / 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: 2 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
Revision 1.3Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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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)33,6945
Polymers33,3091
Non-polymers3844
Water5,206289
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)50.470, 68.220, 73.210
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 / 1 / 4-beta-D-xylan xylanohydrolase / TAXI


Mass: 33309.480 Da / Num. of mol.: 1 / Mutation: K50Q
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-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 289 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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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: 1.89 Å3/Da / Density % sol: 34.98 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / Details: 100 mM Bis-Tris, pH 5.5, and 2 M ammonium sulfate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.1 / Wavelength: 0.9998 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Jul 21, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9998 Å / Relative weight: 1
ReflectionResolution: 1.8→41.55 Å / Num. obs: 24041 / % possible obs: 97.85 % / Redundancy: 5.4 % / Biso Wilson estimate: 19.84 Å2 / CC1/2: 0.959 / Net I/σ(I): 3.52
Reflection shellResolution: 1.8→1.864 Å / Num. unique obs: 2351 / CC1/2: 0.0142

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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: 2→41.55 Å / SU ML: 0.2792 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.9371
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2463 1461 8.28 %
Rwork0.1864 16178 -
obs0.1913 17639 99.86 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 22.59 Å2
Refinement stepCycle: LAST / Resolution: 2→41.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2296 0 20 289 2605
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0022524
X-RAY DIFFRACTIONf_angle_d0.4573465
X-RAY DIFFRACTIONf_chiral_restr0.0406383
X-RAY DIFFRACTIONf_plane_restr0.003452
X-RAY DIFFRACTIONf_dihedral_angle_d5.6272348
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.070.38781440.30381586X-RAY DIFFRACTION99.65
2.07-2.150.34551390.2681591X-RAY DIFFRACTION99.88
2.15-2.250.30191440.23341565X-RAY DIFFRACTION99.71
2.25-2.370.29091410.22471615X-RAY DIFFRACTION99.83
2.37-2.520.27521520.21141593X-RAY DIFFRACTION99.89
2.52-2.710.24681480.20011608X-RAY DIFFRACTION99.94
2.71-2.990.26451430.17921602X-RAY DIFFRACTION99.89
2.99-3.420.22171430.15851632X-RAY DIFFRACTION100
3.42-4.310.20141530.13921654X-RAY DIFFRACTION99.83
4.31-41.550.18241540.15711732X-RAY DIFFRACTION99.95
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)
12.66633363109-0.915654108302-1.961428839410.8767502230980.1844314579059.189664248410.0136089033160.102614838359-0.00238110634448-0.0849472747810.0517259485513-0.09467888780620.07248283563220.151270838598-0.02370305759360.130158737722-0.0323485190359-0.0121910306660.105335554133-0.02532586696290.17686771915725.02260075953.976802471949.5709298492
27.956814317061.50254038532-2.146613981261.24740817335-0.3689223990552.271999913260.0691843459294-0.184040601971-0.1436658257090.153375952881-0.0450476132322-0.0174403144243-0.161657936219-0.02315642287610.007314614231010.162289215726-0.00397787070945-0.02118086369380.133758442170.04508194879420.08711501265488.7231870250910.979520124420.0532132104
32.030336396470.807537665625-0.5420298409281.947211771561.126365494092.130464794540.0443701172061-0.184595762236-0.01143372925260.0551167567257-0.1121872471630.107732658658-0.0356785624335-0.2043547955250.06757859846330.1127910369920.030324955123-0.01766378347350.1321947710120.0325087273890.0921450596293-0.088689865553411.884193175112.7353603519
41.19606355149-0.551217717833-1.120308047225.652902448031.867036117232.08215887924-0.01138304243390.0966428758557-0.04747845919390.127725583406-0.08809077992710.113562684377-0.0408979027154-0.1049043841890.07186149672310.0957441464872-0.00350634333965-0.03110328972370.135995735760.02431262707390.08453266958672.8040831757710.12455946254.82499860597
52.01632502052-1.427970600850.3922084093469.19726637968-4.463348273562.82695021735-0.17534515640.175334712204-0.24780941728-0.4792286838640.268045039197-0.08070774663140.284838549887-0.490750842404-0.1402639990420.1296720496250.0136189571674-0.01233731583560.241053985665-0.03948486720010.123599885489-2.805951793535.27464532607-2.96123102311
61.91203297305-1.925594129820.7643921737432.00542952857-0.5301148498680.846385749111-0.0410791368415-0.083671010825-0.0031037976112-0.002212558395560.05837599563540.0007657345778360.08031449185590.0584387987369-0.02313400876320.12237937785-0.02064263043480.01887064821920.1927770288550.004250398242170.15577816060212.6654151855-0.6719869336140.334246990422
74.754598213731.611155325951.262432839565.98251110802-1.436522191314.59365154717-0.0635636021103-0.4872294927180.1266369337170.3867659226930.06883462950530.08183114578780.353608084028-0.13202732266-0.09167712062910.135531753850.0388405130091-0.02597964406540.22706894938-0.02924146620380.16538571349924.0022954582-8.189453545979.16336421221
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 3 through 45 )3 - 451 - 43
22chain 'A' and (resid 46 through 100 )46 - 10044 - 98
33chain 'A' and (resid 101 through 149 )101 - 14999 - 147
44chain 'A' and (resid 150 through 181 )150 - 181148 - 179
55chain 'A' and (resid 182 through 196 )182 - 196180 - 194
66chain 'A' and (resid 197 through 270 )197 - 270195 - 268
77chain 'A' and (resid 271 through 303 )271 - 303269 - 301

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