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- PDB-8bp1: Crystal structure of BHMeHis1.0, an engineered enzyme for the Mor... -

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Basic information

Entry
Database: PDB / ID: 8bp1
TitleCrystal structure of BHMeHis1.0, an engineered enzyme for the Morita-Baylis-Hillman reaction
ComponentsBHMeHis1.0
KeywordsBIOSYNTHETIC PROTEIN / Morita-Baylis-Hillman / non-canonical amino acid / methylhistidine / de novo enzyme
Function / homologyACETATE ION / DI(HYDROXYETHYL)ETHER
Function and homology information
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.72 Å
AuthorsHardy, F.J.
Funding support United Kingdom, European Union, France, 5items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S507040/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M027023 United Kingdom
European Research Council (ERC)757991European Union
Biotechnology and Biological Sciences Research Council (BBSRC)BB/W014483/1 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.J. / 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

Downloads & links

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Assembly

Deposited unit
A: BHMeHis1.0
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,0067
Polymers27,6371
Non-polymers3696
Water3,603200
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)71.013, 71.013, 120.312
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Space group name HallP312"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+1/3
#3: -x+y,-x,z+2/3
#4: x-y,-y,-z+2/3
#5: -x,-x+y,-z+1/3
#6: y,x,-z

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Components

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Protein , 1 types, 1 molecules A

#1: Protein BHMeHis1.0


Mass: 27636.756 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)

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Non-polymers , 5 types, 206 molecules

#2: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#3: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 200 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 3.17 Å3/Da / Density % sol: 61.18 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop
Details: 0.1 M Sodium citrate, 29% w/v PEG 4000, pH 6.5, 0.1 M Magnesium acetate tetrahydrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9762 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Apr 30, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9762 Å / Relative weight: 1
ReflectionResolution: 1.72→61.5 Å / Num. obs: 37997 / % possible obs: 99.94 % / Redundancy: 20.2 % / Biso Wilson estimate: 30.53 Å2 / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.08128 / Rpim(I) all: 0.01838 / Net I/σ(I): 20.45
Reflection shellResolution: 1.72→1.782 Å / Redundancy: 19 % / Rmerge(I) obs: 1.691 / Num. unique obs: 3733 / CC1/2: 0.709 / CC star: 0.911 / % possible all: 99.71

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Processing

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

7o1d


Resolution: 1.72→61.5 Å / SU ML: 0.1634 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 20.7592
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2019 1865 4.91 %
Rwork0.1809 36139 -
obs0.182 37987 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 38.59 Å2
Refinement stepCycle: LAST / Resolution: 1.72→61.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1853 0 22 200 2075
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00422022
X-RAY DIFFRACTIONf_angle_d0.73612749
X-RAY DIFFRACTIONf_chiral_restr0.046299
X-RAY DIFFRACTIONf_plane_restr0.0063355
X-RAY DIFFRACTIONf_dihedral_angle_d5.4503292
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.72-1.770.28951330.25682737X-RAY DIFFRACTION99.41
1.77-1.820.26861200.23442740X-RAY DIFFRACTION100
1.82-1.880.29851280.26782764X-RAY DIFFRACTION99.93
1.88-1.940.26431480.22822722X-RAY DIFFRACTION99.9
1.94-2.020.27611470.20782784X-RAY DIFFRACTION100
2.02-2.110.22421280.17642737X-RAY DIFFRACTION100
2.11-2.220.18391420.18342771X-RAY DIFFRACTION100
2.23-2.360.22171370.19472773X-RAY DIFFRACTION100
2.36-2.550.21241470.18592778X-RAY DIFFRACTION100
2.55-2.80.19351680.18862751X-RAY DIFFRACTION100
2.8-3.210.22851590.18952790X-RAY DIFFRACTION100
3.21-4.040.1961530.15952825X-RAY DIFFRACTION100
4.04-61.50.1571550.16352967X-RAY DIFFRACTION99.84
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)
10.3777765114470.9199961284671.145013880083.054272486163.724741280845.46690639549-0.0802676870569-0.05185932210370.0250134264745-0.2267747241290.0007892567309610.038470905382-0.1432807939610.02234389986920.1059860821740.246206927708-0.04721356961720.03009970192490.23701148652-0.02116261224210.213722606961-23.358393086533.5168751533-7.67623430767
20.948078003331-0.3964376342790.1405044636528.506368574123.472800648212.378869431030.01801201336310.329407771839-0.369361530872-0.8996893118950.461448042108-0.590194477007-0.2254125797490.231727369135-0.3973541888320.281963682604-0.09011392242740.05365013830270.42027585992-0.1543717628450.433165491772-13.115304291133.693150111-0.18914666555
30.4053308338450.7439719647840.9718774440718.2888758285.35208747093.961239788730.00216261839123-0.0205609181984-0.07388651233760.3262249227640.30778100079-0.04686255661740.4514587680580.142479878539-0.3651337003930.321786233491-0.0579999989869-0.0007306022913290.278986326357-0.01716726115380.314915267632-16.409713554126.26491569595.31740727031
43.96618015071.901267377662.356212255351.328347438560.9722980191582.93269485185-0.0133527959658-0.283471936213-0.0808552080145-0.0591729757557-0.09014466266180.04634552936330.149304543016-0.6903043587020.1267651176280.297967406909-0.04669336179590.07056813257670.357628432154-0.06082542756310.28999256104-29.151260125432.54703739824.12713346687
51.19700985922-0.989254163170.7597972173384.25347783107-0.7100363288482.83690423811-0.074240941168-0.05638505986290.006787285596530.03107888119660.08970555400970.116687261158-0.147920786461-0.240506188664-0.01071899380350.238582353989-0.01475186067550.03621520051770.197261178262-0.005773759336770.211521542615-25.737393333520.1068010408-15.2467479498
62.320592159371.26948848767-0.1059795053934.623009647310.2164629979141.58138793366-0.0403723945003-0.08176357941270.0561462842051-0.2675008606020.103370893024-0.593274407614-0.2696360919850.289053576535-0.06518738414890.396388767129-0.08035360559130.09744154462020.285670358773-0.02639425591860.274103790064-14.690685380824.3599424349-24.7663231654
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 1 through 37 )1 - 371 - 37
22chain 'A' and (resid 38 through 56 )38 - 5638 - 56
33chain 'A' and (resid 57 through 79 )57 - 7957 - 79
44chain 'A' and (resid 80 through 98 )80 - 9880 - 98
55chain 'A' and (resid 99 through 168 )99 - 16899 - 168
66chain 'A' and (resid 169 through 231 )169 - 231169 - 231

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