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- PDB-8ush: Crystal Structure of Kemp Eliminase HG630 with bound transition s... -

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Basic information

Entry
Database: PDB / ID: 8ush
TitleCrystal Structure of Kemp Eliminase HG630 with bound transition state analogue, 280 K
ComponentsKemp eliminase
KeywordsHYDROLASE / De novo enzyme design / computational protein design / Kemp eliminases / biocatalysis / directed evolution / X-ray crystallography
Function / homology6-NITROBENZOTRIAZOLE
Function and homology information
Biological speciesThermoascus aurantiacus (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.32 Å
AuthorsSeifinoferest, B.
Funding support Canada, France, United States, 4items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canada Foundation for Innovation Canada
Human Frontier Science Program (HFSP) France
Department of Energy (DOE, United States) United States
CitationJournal: J.Am.Chem.Soc. / Year: 2024
Title: Design of Efficient Artificial Enzymes Using Crystallographically Enhanced Conformational Sampling.
Authors: Rakotoharisoa, R.V. / Seifinoferest, B. / Zarifi, N. / Miller, J.D.M. / Rodriguez, J.M. / Thompson, M.C. / Chica, R.A.
History
DepositionOct 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 6, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Kemp eliminase
B: Kemp eliminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,8334
Polymers68,5052
Non-polymers3282
Water9,944552
1
A: Kemp eliminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,4172
Polymers34,2521
Non-polymers1641
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Kemp eliminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,4172
Polymers34,2521
Non-polymers1641
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)51.025, 77.516, 93.912
Angle α, β, γ (deg.)90.000, 105.263, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Kemp eliminase


Mass: 34252.406 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoascus aurantiacus (fungus) / Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-6NT / 6-NITROBENZOTRIAZOLE


Mass: 164.122 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H4N4O2 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 552 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.62 Å3/Da / Density % sol: 52.97 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.8
Details: 10.6 mg/mL protein, 100 mM sodium acetate, pH 4.8, 1.4 M ammonium sulfate

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Data collection

DiffractionMean temperature: 280 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.1 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 17, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 1.32→90.6 Å / Num. obs: 164672 / % possible obs: 99.7 % / Redundancy: 3.3 % / Biso Wilson estimate: 13.06 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.08 / Rpim(I) all: 0.052 / Net I/σ(I): 9.4
Reflection shellResolution: 1.32→1.34 Å / Redundancy: 3.2 % / Rmerge(I) obs: 0.897 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 8177 / CC1/2: 0.477 / Rpim(I) all: 0.585 / % possible all: 98.9

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Processing

Software
NameVersionClassification
xia20.5.492data reduction
DIALSXia2 0.5.492data reduction
AimlessXia2 0.5.492data scaling
Coot0.8.9.236model building
PHASERv1.13.2998phasing
PHENIXv1.13.2998refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.32→90.6 Å / SU ML: 0.1259 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 14.4044
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1476 8245 5.01 %
Rwork0.119 156348 -
obs0.1204 164593 99.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 19.94 Å2
Refinement stepCycle: LAST / Resolution: 1.32→90.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4570 0 24 552 5146
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0075356
X-RAY DIFFRACTIONf_angle_d0.88617403
X-RAY DIFFRACTIONf_chiral_restr0.0731817
X-RAY DIFFRACTIONf_plane_restr0.00681010
X-RAY DIFFRACTIONf_dihedral_angle_d12.30091955
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.32-1.340.28222760.23885115X-RAY DIFFRACTION98.7
1.34-1.350.27242840.23185180X-RAY DIFFRACTION99.38
1.35-1.370.25853010.2265161X-RAY DIFFRACTION99.4
1.37-1.380.23052700.21145252X-RAY DIFFRACTION99.55
1.38-1.40.24012690.20745140X-RAY DIFFRACTION99.36
1.4-1.420.22732950.20275158X-RAY DIFFRACTION99.53
1.42-1.440.22153100.1825135X-RAY DIFFRACTION99.65
1.44-1.460.21392530.17325221X-RAY DIFFRACTION99.53
1.46-1.490.23752390.16115230X-RAY DIFFRACTION99.53
1.49-1.510.20012910.14695206X-RAY DIFFRACTION99.57
1.51-1.540.16752740.13765154X-RAY DIFFRACTION99.45
1.54-1.570.17833240.13085139X-RAY DIFFRACTION99.56
1.57-1.60.17442810.12435222X-RAY DIFFRACTION99.48
1.6-1.630.16952450.12265208X-RAY DIFFRACTION99.47
1.63-1.660.15362610.11535195X-RAY DIFFRACTION99.53
1.66-1.70.17292610.11375223X-RAY DIFFRACTION99.55
1.7-1.740.1572660.10895204X-RAY DIFFRACTION99.64
1.74-1.790.13922890.10295195X-RAY DIFFRACTION99.6
1.79-1.840.13452980.09585210X-RAY DIFFRACTION99.95
1.84-1.90.13672840.09425201X-RAY DIFFRACTION99.96
1.9-1.970.12362970.09135246X-RAY DIFFRACTION99.86
1.97-2.050.12892580.09285211X-RAY DIFFRACTION99.93
2.05-2.140.13522730.09425253X-RAY DIFFRACTION99.89
2.14-2.260.11182820.09065204X-RAY DIFFRACTION99.85
2.26-2.40.11992570.0955244X-RAY DIFFRACTION99.82
2.4-2.580.12552830.10195244X-RAY DIFFRACTION99.93
2.58-2.840.14812550.11565279X-RAY DIFFRACTION99.8
2.84-3.260.14382410.1275260X-RAY DIFFRACTION99.6
3.26-4.10.13112740.11445275X-RAY DIFFRACTION99.5
4.1-90.60.13242540.1255383X-RAY DIFFRACTION99.73

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