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- PDB-7ond: HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with... -

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Basic information

Entry
Database: PDB / ID: 7ond
TitleHaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridine Dye
ComponentsHaloalkane dehalogenase
KeywordsFLUORESCENT PROTEIN / HaloTag / Fluorescence / Imaging / mammalian cells / fluorescent reporter / channel dye / directed evolution
Function / homology
Function and homology information


haloalkane dehalogenase / haloalkane dehalogenase activity / response to toxic substance
Similarity search - Function
Haloalkane dehalogenase, subfamily 2 / Epoxide hydrolase-like / alpha/beta hydrolase fold / Alpha/beta hydrolase fold-1 / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Chem-VF2 / Haloalkane dehalogenase
Similarity search - Component
Biological speciesRhodococcus sp. (unknown)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.45 Å
AuthorsStein, A. / Liang, A.D.
CitationJournal: Chembiochem / Year: 2021
Title: HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye.
Authors: Miro-Vinyals, C. / Stein, A. / Fischer, S. / Ward, T.R. / Deliz Liang, A.
History
DepositionMay 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 21, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 20, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Dec 22, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Haloalkane dehalogenase
B: Haloalkane dehalogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,6447
Polymers68,8322
Non-polymers8115
Water11,115617
1
A: Haloalkane dehalogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,8103
Polymers34,4161
Non-polymers3932
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Haloalkane dehalogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,8344
Polymers34,4161
Non-polymers4183
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)47.586, 68.558, 81.179
Angle α, β, γ (deg.)90.000, 98.570, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Haloalkane dehalogenase /


Mass: 34416.242 Da / Num. of mol.: 2 / Mutation: R133C, E143M, F144H, M175Y, V245A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodococcus sp. (unknown) / Gene: dhaA / Plasmid: pET30 / Production host: Escherichia coli BL21(DE3) (unknown) / References: UniProt: P0A3G3, haloalkane dehalogenase
#2: Chemical ChemComp-VF2 / 4-[(E)-2-[1-(7-chloranylheptyl)pyridin-1-ium-4-yl]ethenyl]-N,N-dimethyl-aniline


Mass: 357.940 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H30ClN2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 617 / 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: 1.9 Å3/Da / Density % sol: 35.34 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.9
Details: 0.2 M Magnesium Chloride hexahydrate, 20 % w/v PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1.0000314 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Mar 20, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0000314 Å / Relative weight: 1
ReflectionResolution: 1.45→47.1 Å / Num. obs: 90750 / % possible obs: 99.24 % / Redundancy: 6.7 % / CC1/2: 0.999 / Rmerge(I) obs: 0.06203 / Net I/σ(I): 18.68
Reflection shellResolution: 1.45→1.502 Å / Rmerge(I) obs: 0.3734 / Num. unique obs: 8968 / CC1/2: 0.965 / Rpim(I) all: 0.1506 / Rrim(I) all: 0.4031

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
PHASERphasing
Aimlessdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5y2x
Resolution: 1.45→47.1 Å / Cor.coef. Fo:Fc: 0.973 / Cor.coef. Fo:Fc free: 0.965 / SU B: 1.106 / SU ML: 0.043 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.07 / ESU R Free: 0.069 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1859 4538 5 %RANDOM
Rwork0.1625 ---
obs0.1637 86202 99.29 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 52.28 Å2 / Biso mean: 11 Å2 / Biso min: 4.48 Å2
Baniso -1Baniso -2Baniso -3
1--0.38 Å2-0 Å2-0.07 Å2
2---0.02 Å20 Å2
3---0.4 Å2
Refinement stepCycle: final / Resolution: 1.45→47.1 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4738 0 51 617 5406
Biso mean--18.77 20.79 -
Num. residues----592
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0134988
X-RAY DIFFRACTIONr_bond_other_d0.0010.0174534
X-RAY DIFFRACTIONr_angle_refined_deg1.8231.6546818
X-RAY DIFFRACTIONr_angle_other_deg1.5011.5710477
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.0265596
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.51321.798267
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.97815743
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.0061532
X-RAY DIFFRACTIONr_chiral_restr0.0960.2613
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.025620
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021132
LS refinement shellResolution: 1.45→1.488 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.25 343 -
Rwork0.197 6328 -
all-6671 -
obs--98.51 %

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