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- PDB-7oo4: HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with... -

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Basic information

Entry
Database: PDB / ID: 7oo4
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. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 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 26, 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
Revision 1.3Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.4Oct 9, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Haloalkane dehalogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,6093
Polymers34,2161
Non-polymers3932
Water66737
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area130 Å2
ΔGint-13 kcal/mol
Surface area11660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.319, 68.986, 86.020
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Haloalkane dehalogenase


Mass: 34216.027 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodococcus sp. (bacteria) / Gene: dhaA / Plasmid: pET30 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A3G3, haloalkane dehalogenase
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#3: 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.: 1 / Source method: obtained synthetically / Formula: C22H30ClN2 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 37 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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: 2.27 Å3/Da / Density % sol: 45.78 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.6
Details: 0.1 M Na3 Cit 5.6 pH 20 %v/v 2-PropOH 20 %w/v PEG 4K

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1.000031 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: 1.000031 Å / Relative weight: 1
ReflectionResolution: 2.1→43.05 Å / Num. obs: 18774 / % possible obs: 99.9 % / Redundancy: 12.6 % / CC1/2: 0.999 / Rmerge(I) obs: 0.1213 / Rpim(I) all: 0.03554 / Rrim(I) all: 0.1265 / Net I/σ(I): 15.5
Reflection shellResolution: 2.1→2.175 Å / Rmerge(I) obs: 0.9583 / Mean I/σ(I) obs: 2.75 / Num. unique obs: 1842 / CC1/2: 0.88 / Rpim(I) all: 0.2778 / Rrim(I) all: 0.9984

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5y2x

5y2x


Resolution: 2.1→43.05 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.92 / SU B: 8.329 / SU ML: 0.202 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.242 / ESU R Free: 0.207 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2632 914 4.9 %RANDOM
Rwork0.2097 ---
obs0.2123 17847 99.91 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 90.05 Å2 / Biso mean: 37.939 Å2 / Biso min: 23.02 Å2
Baniso -1Baniso -2Baniso -3
1--0.21 Å20 Å2-0 Å2
2---4.49 Å20 Å2
3---4.71 Å2
Refinement stepCycle: final / Resolution: 2.1→43.05 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2342 0 29 37 2408
Biso mean--43.75 34.98 -
Num. residues----292
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0132451
X-RAY DIFFRACTIONr_bond_other_d0.0010.0172247
X-RAY DIFFRACTIONr_angle_refined_deg1.6731.6593346
X-RAY DIFFRACTIONr_angle_other_deg1.3091.575187
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.2735291
X-RAY DIFFRACTIONr_dihedral_angle_2_deg29.08421.667132
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.29215369
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.2131517
X-RAY DIFFRACTIONr_chiral_restr0.0820.2300
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.022748
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02558
LS refinement shellResolution: 2.1→2.155 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.361 69 -
Rwork0.353 1304 -
all-1373 -
obs--100 %

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