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- PDB-4bs0: Crystal Structure of Kemp Eliminase HG3.17 E47N,N300D Complexed w... -

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Basic information

Entry
Database: PDB / ID: 4bs0
TitleCrystal Structure of Kemp Eliminase HG3.17 E47N,N300D Complexed with Transition State Analog 6-Nitrobenzotriazole
ComponentsKEMP ELIMINASE HG3.17
KeywordsLYASE/LYASE INHIBITOR / LYASE-LYASE INHIBITOR COMPLEX / COMPUTATIONAL PROTEIN DESIGN / PROTON TRANSFER / KEMP ELIMINATION / DIRECTED EVOLUTION / TRANSITION STATE TUNING / BOTTOM-UP ENZYME CONSTRUCTION / ELEMENTARY CHEMICAL STEP CATALYSIS
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 / Glycosidases / Glycoside hydrolase superfamily / TIM Barrel ...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 / Glycosidases / Glycoside hydrolase superfamily / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
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: 1.09 Å
AuthorsBlomberg, R. / Kries, H. / Pinkas, D.M. / Mittl, P.R.E. / Gruetter, M.G. / Privett, H.K. / Mayo, S. / Hilvert, D.
CitationJournal: Nature / Year: 2013
Title: Precision is Essential for Efficient Catalysis in an Evolved Kemp Eliminase
Authors: Blomberg, R. / Kries, H. / Pinkas, D.M. / Mittl, P.R.E. / Gruetter, M.G. / Privett, H.K. / Mayo, S.L. / Hilvert, D.
History
DepositionJun 6, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2013Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2013Group: Database references
Revision 1.2Nov 27, 2013Group: Database references
Revision 2.0Dec 20, 2023Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Other / Refinement description
Category: atom_site / chem_comp_atom ...atom_site / chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_site
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "BA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: KEMP ELIMINASE HG3.17
B: KEMP ELIMINASE HG3.17
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,6368
Polymers68,9242
Non-polymers7126
Water14,988832
1
A: KEMP ELIMINASE HG3.17
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,8184
Polymers34,4621
Non-polymers3563
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: KEMP ELIMINASE HG3.17
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,8184
Polymers34,4621
Non-polymers3563
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)76.080, 77.950, 98.280
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein KEMP ELIMINASE HG3.17 / ENDO-1 / 4-BETA-XYLANASE / XYLANASE / 1\ / 4-BETA-D-XYLAN XYLANOHYDROLASE


Mass: 34461.824 Da / Num. of mol.: 2 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: KEMP ELIMINASE HG-2 (PDB ID 3NYD) WITH MUTATION V6I, Q37K, K50Q, G82A, M84C, S89N, Q90F, T105I, A125T, T142N, T208M, S265T, F267M, W275A, R276F, T279S
Source: (gene. exp.) THERMOASCUS AURANTIACUS (fungus) / Description: ENGINEERED ENZYME / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): GOLD / References: UniProt: P23360, Lyases
#2: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-6NT / 6-NITROBENZOTRIAZOLE


Mass: 164.122 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H4N4O2
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 832 / 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: 2.26 Å3/Da / Density % sol: 45.52 % / Description: NONE
Crystal growpH: 5.9
Details: 1.1 M AMMONIUM SULFATE, 100 MM SODIUM ACETATE, PH 5.9, SEEDING

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Feb 25, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.09→28.3 Å / Num. obs: 225765 / % possible obs: 93.1 % / Observed criterion σ(I): -3 / Redundancy: 12.2 % / Biso Wilson estimate: 9.99 Å2 / Rmerge(I) obs: 0.04 / Net I/σ(I): 32.1
Reflection shellResolution: 1.09→1.13 Å / Redundancy: 7.3 % / Rmerge(I) obs: 0.46 / Mean I/σ(I) obs: 4.9 / % possible all: 57

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3NYD

3nyd


Resolution: 1.09→28.339 Å / SU ML: 0.08 / σ(F): 1.92 / Phase error: 13.99 / Stereochemistry target values: LS_WUNIT_K1
Details: THE MAXIMUM-LIKELIHOOD TARGET FUNCTION WAS USED WITH OPTIMIZED STEREOCHEMICAL AND ATOMIC-DISPLACEMENT PARAMETER RESTRAINTS FOR THE MAJORITY OF THE REFINEMENT. IN THE FINAL STAGE, THE LIGAND ...Details: THE MAXIMUM-LIKELIHOOD TARGET FUNCTION WAS USED WITH OPTIMIZED STEREOCHEMICAL AND ATOMIC-DISPLACEMENT PARAMETER RESTRAINTS FOR THE MAJORITY OF THE REFINEMENT. IN THE FINAL STAGE, THE LIGAND AS WELL AS THE CATALYTIC RESIDUES ASP127 AND GLN50 FOR THE A-CHAIN WERE REFINED UNRESTRAINED, USING THE LEAST SQUARES TARGET FUNCTION.
RfactorNum. reflection% reflection
Rfree0.1452 11399 5.1 %
Rwork0.1241 --
obs0.1252 225746 93.09 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: PHENIX V1.8.2-1309
Displacement parametersBiso mean: 14.13 Å2
Refinement stepCycle: LAST / Resolution: 1.09→28.339 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4556 0 44 832 5432
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0157340
X-RAY DIFFRACTIONf_angle_d1.30810051
X-RAY DIFFRACTIONf_dihedral_angle_d12.4422624
X-RAY DIFFRACTIONf_chiral_restr0.0741107
X-RAY DIFFRACTIONf_plane_restr0.0081335
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.09-1.10240.20221930.18883729X-RAY DIFFRACTION49
1.1024-1.11540.21682330.18564272X-RAY DIFFRACTION56
1.1154-1.1290.22132630.17594930X-RAY DIFFRACTION65
1.129-1.14330.20033000.16615462X-RAY DIFFRACTION72
1.1433-1.15830.2073260.15846147X-RAY DIFFRACTION80
1.1583-1.17420.18323370.15856678X-RAY DIFFRACTION88
1.1742-1.19090.19724040.15777081X-RAY DIFFRACTION94
1.1909-1.20870.18223770.15297498X-RAY DIFFRACTION98
1.2087-1.22760.18153650.14737548X-RAY DIFFRACTION98
1.2276-1.24770.17293870.1417568X-RAY DIFFRACTION99
1.2477-1.26930.17624140.14237518X-RAY DIFFRACTION99
1.2693-1.29230.16863710.13247584X-RAY DIFFRACTION99
1.2923-1.31720.17664030.12777562X-RAY DIFFRACTION99
1.3172-1.34410.15544450.12627558X-RAY DIFFRACTION99
1.3441-1.37330.15824100.12577566X-RAY DIFFRACTION99
1.3733-1.40520.15323840.1237645X-RAY DIFFRACTION100
1.4052-1.44040.15264080.11797632X-RAY DIFFRACTION100
1.4404-1.47930.14144150.11377579X-RAY DIFFRACTION99
1.4793-1.52290.1443790.10627671X-RAY DIFFRACTION100
1.5229-1.5720.13244250.10467617X-RAY DIFFRACTION100
1.572-1.62820.14314410.10327668X-RAY DIFFRACTION100
1.6282-1.69340.13854080.10487665X-RAY DIFFRACTION100
1.6934-1.77040.14623940.10437685X-RAY DIFFRACTION100
1.7704-1.86380.12534220.10467665X-RAY DIFFRACTION100
1.8638-1.98050.12824120.10637689X-RAY DIFFRACTION100
1.9805-2.13340.13844180.10817729X-RAY DIFFRACTION100
2.1334-2.3480.12864020.10887751X-RAY DIFFRACTION100
2.348-2.68750.14114020.11857752X-RAY DIFFRACTION100
2.6875-3.38510.13864310.1297830X-RAY DIFFRACTION100
3.3851-28.34840.13344300.13938068X-RAY DIFFRACTION99

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