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- PDB-6y8p: Crystal structure of SNAP-tag labeled with a benzyl-tetramethylrh... -

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Basic information

Entry
Database: PDB / ID: 6y8p
TitleCrystal structure of SNAP-tag labeled with a benzyl-tetramethylrhodamine fluorophore
ComponentsO6-alkylguanine-DNA alkyltransferase mutant
KeywordsTRANSFERASE / SNAP-tag / self-labeling protein / tetramethylrhodamine / synthetic fluorophore
Function / homology
Function and homology information


methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / DNA modification / methylation / DNA repair / DNA binding / metal ion binding / nucleus
Similarity search - Function
Methylguanine DNA methyltransferase, ribonuclease-like domain / 6-O-methylguanine DNA methyltransferase, ribonuclease-like domain / Methylated DNA-protein cysteine methyltransferase domain superfamily / Methylated-DNA-[protein]-cysteine S-methyltransferase, active site / Methylated-DNA--protein-cysteine methyltransferase active site. / Methylated-DNA-[protein]-cysteine S-methyltransferase, DNA binding / Methylated DNA-protein cysteine methyltransferase, DNA binding domain / 6-O-methylguanine DNA methyltransferase, DNA binding domain / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Chem-OGQ / Methylated-DNA--protein-cysteine methyltransferase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsGotthard, G. / Tanzer, T. / Johnsson, K. / Hiblot, J.
Citation
Journal: Biochemistry / Year: 2021
Title: Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag.
Authors: Wilhelm, J. / Kuhn, S. / Tarnawski, M. / Gotthard, G. / Tunnermann, J. / Tanzer, T. / Karpenko, J. / Mertes, N. / Xue, L. / Uhrig, U. / Reinstein, J. / Hiblot, J. / Johnsson, K.
#1: Journal: Biorxiv / Year: 2021
Title: Kinetic and structural characterization of the self-labeling protein tags HaloTag7, SNAP-tag and CLIP-tag
Authors: Wilhelm, J. / Kuhn, S. / Tarnawski, M. / Gotthard, G. / Tunnermann, J. / Tanzer, T. / Karpenko, J. / Mertes, N. / Xue, L. / Uhrig, U. / Reinstein, J. / Hiblot, J. / Johnsson, K.
History
DepositionMar 5, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 31, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 27, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / pdbx_database_proc
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Nov 10, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / pdbx_database_proc
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / pdbx_initial_refinement_model
Item: _citation.journal_id_ISSN

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: O6-alkylguanine-DNA alkyltransferase mutant
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,6144
Polymers18,9521
Non-polymers6623
Water90150
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area230 Å2
ΔGint3 kcal/mol
Surface area8290 Å2
MethodPISA
Unit cell
Length a, b, c (Å)65.515, 65.515, 97.067
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein O6-alkylguanine-DNA alkyltransferase mutant


Mass: 18951.752 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pET51b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): -pLysS / References: UniProt: E5BBQ0
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-OGQ / [9-[2-carboxy-5-[(4-methylphenyl)methylcarbamoyl]phenyl]-6-(dimethylamino)xanthen-3-ylidene]-dimethyl-azanium


Mass: 534.625 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C33H32N3O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 50 / 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: 3.48 Å3/Da / Density % sol: 61.24 % / Description: Red hexaedral crystals
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 100 mM Na HEPES pH 7.5 25% PEG 8000 10 mg/mL protein 100 nl:100 nL

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.976 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Nov 3, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 2.3→48.98 Å / Num. obs: 11154 / % possible obs: 99.94 % / Redundancy: 10.7 % / CC1/2: 0.999 / Net I/σ(I): 15
Reflection shellResolution: 2.3→2.382 Å / Num. unique obs: 1087 / CC1/2: 0.193

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3l00

3l00


Resolution: 2.3→48.98 Å / SU ML: 0.43 / Cross valid method: THROUGHOUT / σ(F): 1.38 / Phase error: 37.4
RfactorNum. reflection% reflection
Rfree0.2767 557 4.99 %
Rwork0.2356 --
obs0.2374 11154 99.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 143.77 Å2 / Biso mean: 73.1096 Å2 / Biso min: 31.06 Å2
Refinement stepCycle: final / Resolution: 2.3→48.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1213 0 45 50 1308
Biso mean--60.68 62.41 -
Num. residues----158
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 4 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
2.3-2.530.42221350.399425992734
2.53-2.90.36861370.333825952732
2.9-3.650.31491400.256626512791
3.65-48.980.22241450.181427522897
Refinement TLS params.Method: refined / Origin x: 16.4644 Å / Origin y: -5.4937 Å / Origin z: 2.3287 Å
111213212223313233
T0.337 Å20.0286 Å20.0696 Å2-0.4242 Å2-0.122 Å2--0.607 Å2
L3.8415 °2-2.3714 °20.1666 °2-4.1022 °20.239 °2--3.0876 °2
S0.2629 Å °0.3946 Å °-0.401 Å °-0.2289 Å °-0.1815 Å °-0.708 Å °0.3591 Å °0.4522 Å °-0.0575 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA4 - 179
2X-RAY DIFFRACTION1allA180 - 301
3X-RAY DIFFRACTION1allW1 - 58

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