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- PDB-8bp0: Crystal structure of BHMeHis1.8, an engineered enzyme for the Mor... -

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Basic information

Entry
Database: PDB / ID: 8bp0
TitleCrystal structure of BHMeHis1.8, an engineered enzyme for the Morita-Baylis-Hillman reaction
ComponentsBHMeHis1.8
KeywordsBIOSYNTHETIC PROTEIN / Morita-Baylis-Hillmanase / non-canonical amino acid / methylhistidine / de novo enzyme
Function / homologyTRIETHYLENE GLYCOL
Function and homology information
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.621 Å
AuthorsHardy, F.J.
Funding support United Kingdom, European Union, France, 5items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M027023/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S507040/1 United Kingdom
European Research Council (ERC)75991European Union
Biotechnology and Biological Sciences Research Council (BBSRC)BB/W104483/1 United Kingdom
Human Frontier Science Program (HFSP)RGP0004/2022 France
Citation
Journal: Nat Commun / Year: 2024
Title: A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.
Authors: Hutton, A.E. / Foster, J. / Crawshaw, R. / Hardy, F.J. / Johannissen, L.O. / Lister, T.M. / Gerard, E.F. / Birch-Price, Z. / Obexer, R. / Hay, S. / Green, A.P.
#1: Journal: Res Sq / Year: 2023
Title: A Non-Canonical Nucleophile Unlocks a New Mechanistic Pathway in a Designed Enzyme
Authors: Crossley, A. / Crawshaw, R. / Hardy, F. / Johannissen, L. / Lister, T. / Gerard, E. / Birch-Price, Z. / Obexer, R. / Hay, S. / Green, A.
History
DepositionNov 15, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation / citation_author
Revision 1.2Mar 13, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: BHMeHis1.8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,8334
Polymers27,5591
Non-polymers2743
Water1,38777
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)35.457, 42.431, 152.583
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 BHMeHis1.8


Mass: 27558.570 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#3: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 77 / 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: 2.08 Å3/Da / Density % sol: 40.93 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop
Details: 0.2 M magnesium chloride hexahydrate, 0.1 M Bis-Tris, pH = 6.5, 25% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Jan 28, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.621→38.15 Å / Num. obs: 7422 / % possible obs: 99.81 % / Redundancy: 26.1 % / Biso Wilson estimate: 33.11 Å2 / CC1/2: 0.996 / CC star: 0.999 / Rmerge(I) obs: 0.2535 / Rpim(I) all: 0.04998 / Net I/σ(I): 9.87
Reflection shellResolution: 2.621→2.714 Å / Redundancy: 25.3 % / Mean I/σ(I) obs: 2.25 / Num. unique obs: 715 / CC1/2: 0.964 / % possible all: 99.58

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
DIALSdata reduction
DIALSdata scaling
PHENIX1.20.1_4487phasing
PDB-REDOrefinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7O1D

7o1d


Resolution: 2.621→38.15 Å / SU ML: 0.3148 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.4079
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2419 400 5.4 %
Rwork0.1902 7012 -
obs0.1933 7412 99.84 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 36.87 Å2
Refinement stepCycle: LAST / Resolution: 2.621→38.15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1845 0 18 77 1940
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00231925
X-RAY DIFFRACTIONf_angle_d0.44852588
X-RAY DIFFRACTIONf_chiral_restr0.0386274
X-RAY DIFFRACTIONf_plane_restr0.0043331
X-RAY DIFFRACTIONf_dihedral_angle_d6.787269
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.621-30.30881340.23392267X-RAY DIFFRACTION99.71
3-3.780.23591350.19092294X-RAY DIFFRACTION99.84
3.78-38.150.21721310.1742451X-RAY DIFFRACTION99.96

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