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- PDB-252l: GENERATING LIGAND BINDING SITES IN T4 LYSOZYME USING DEFICIENCY-C... -

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Basic information

Entry
Database: PDB / ID: 252l
TitleGENERATING LIGAND BINDING SITES IN T4 LYSOZYME USING DEFICIENCY-CREATING SUBSTITUTIONS
ComponentsT4 LYSOZYME
KeywordsHYDROLASE / O-GLYCOSYL / T4 LYSOZYME / CAVITY MUTANTS / LIGAND BINDING / PROTEIN ENGINEERING / PROTEIN DESIGN / MULTIPLE CONFORMATIONS
Function / homology
Function and homology information


viral release from host cell by cytolysis / peptidoglycan catabolic process / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / host cell cytoplasm / defense response to bacterium
Similarity search - Function
Lysozyme - #40 / Endolysin T4 type / T4-type lysozyme / Glycoside hydrolase, family 24 / Lysozyme domain superfamily / Phage lysozyme / Lysozyme / Lysozyme-like domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
2-HYDROXYETHYL DISULFIDE / Endolysin
Similarity search - Component
Biological speciesEnterobacteria phage T4 (virus)
MethodX-RAY DIFFRACTION / DIFFERENCE FOURIER / Resolution: 2.1 Å
AuthorsBaldwin, E.P. / Baase, W.A. / Zhang, X.-J. / Feher, V. / Matthews, B.W.
Citation
Journal: J.Mol.Biol. / Year: 1998
Title: Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions.
Authors: Baldwin, E. / Baase, W.A. / Zhang, X. / Feher, V. / Matthews, B.W.
#1: Journal: Nature / Year: 1992
Title: A Cavity-Containing Mutant of T4 Lysozyme is Stabilized by Buried Benzene
Authors: Eriksson, A.E. / Baase, W.A. / Wozniak, J.A. / Matthews, B.W.
#2: Journal: Methods Enzymol. / Year: 1989
Title: Expression and Nitrogen-15 Labeling of Proteins for Proton and Nitrogen-15 Nuclear Magnetic Resonance
Authors: Muchmore, D.C. / Mcintosh, L.P. / Russell, C.B. / Anderson, D.E. / Dahlquist, F.W.
#3: Journal: J.Mol.Biol. / Year: 1987
Title: Structure of Bacteriophage T4 Lysozyme Refined at 1.7 A Resolution
Authors: Weaver, L.H. / Matthews, B.W.
History
DepositionOct 28, 1997Processing site: BNL
Revision 1.0Mar 18, 1998Provider: repository / Type: Initial release
Revision 1.1Mar 24, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Nov 3, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.5Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: T4 LYSOZYME
hetero molecules


Theoretical massNumber of molelcules
Total (without water)18,6983
Polymers18,5081
Non-polymers1902
Water2,450136
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)60.800, 60.800, 97.200
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein T4 LYSOZYME


Mass: 18508.125 Da / Num. of mol.: 1 / Mutation: C54T, C97A, M102A, M106A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T4 (virus) / Genus: T4-like viruses / Species: Enterobacteria phage T4 sensu lato / Cellular location: CYTOPLASM / Gene: GENE E / Plasmid: PHS1403 / Gene (production host): T4 LYSOZYME / Production host: Escherichia coli (E. coli) / Strain (production host): RR1 / References: UniProt: P00720, lysozyme
#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-HED / 2-HYDROXYETHYL DISULFIDE


Mass: 154.251 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O2S2
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 136 / Source method: isolated from a natural source / Formula: H2O
Compound detailsSTRUCTURE OF A T4 LYSOZYME MUTANT THAT CONTAINS TWO DIFFERENT CONFORMATIONS FOR RESIDUES 106 - 114. ...STRUCTURE OF A T4 LYSOZYME MUTANT THAT CONTAINS TWO DIFFERENT CONFORMATIONS FOR RESIDUES 106 - 114. THE MUTANT WAS DESIGNED TO CREATE A DEEP PIT BY TRUNCATING MET 102 AND MET 106 TO ALA. THE STRUCTURE OF SINGLE MUTANT MET102-ALA HAS A BURIED NON-POLAR CAVITY THAT BINDS BENZENE, AND THE MET 106 SIDE CHAIN SEPARATES THE CAVITY FROM THE SURFACE. THE DOUBLE MUTANT FAILED TO BIND ANY LIGANDS DETECTABLY. THE STRUCTURE DISPLAYED TWO DIFFERENT CONFORMATIONS FOR THE REGION 106 - 114, WHICH IS SHORT MOBILE HELIX IN WILDTYPE. THE PREDOMINANT CONFORMER CONTAINED THREE LEFT-HANDED GLYCINES (107, 110, 113) WHILE THE LESSER POPULATED CONFORMER WAS WILDTYPE-LIKE. THE WEIGHTS WERE 0.6 (ALT) AND 0.4 (WILDTYPE). THE SWITCH IN CONFORMATION IS ATTRIBUTED TO CHANGES IN PACKING OF THE HYDROPHOBIC FACE OF THE HELIX.

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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: 2.8 Å3/Da / Density % sol: 56.1 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7
Details: CRYSTALS GROWN IN HANGING DROPS AT 4 DEGREES C. PROTEIN 10-20 MG/ML IN A BUFFER CONTAINING 0.5 M NACL 0.1 M NAPO4 PH 6.5 WAS DILUTED 1/2 WITH A WELL SOLUTION CONTAINING MIXED K/NAPO4 PH 6.3- ...Details: CRYSTALS GROWN IN HANGING DROPS AT 4 DEGREES C. PROTEIN 10-20 MG/ML IN A BUFFER CONTAINING 0.5 M NACL 0.1 M NAPO4 PH 6.5 WAS DILUTED 1/2 WITH A WELL SOLUTION CONTAINING MIXED K/NAPO4 PH 6.3-7.1,1.8-2.2 MOLAR., pH 7.0, vapor diffusion - hanging drop, temperature 277K
PH range: 6.3-7.1
Crystal grow
*PLUS
Method: batch method / Details: Remington, S.J., (1978). J. Mol. Biol., 118, 81. / pH: 6.7
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
120 mg/mlprotein11
20.55 M11NaCl
314 mMmercaptoethanol11
41 mM11MgCl2
50.01 Msodium phospahte11
62.2 M11NaH2PO4
71.8 M11K2HPO4

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Data collection

DiffractionMean temperature: 298 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RUH2R / Wavelength: 1.5418
DetectorType: XUONG-HAMLIN MULTIWIRE / Detector: AREA DETECTOR / Date: Mar 25, 1993 / Details: GRAPHITE MONOCHROMATOR
RadiationMonochromator: GRAPHITE(002) / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.1→30 Å / Num. obs: 11176 / % possible obs: 85 % / Rmerge(I) obs: 0.049
Reflection shellHighest resolution: 2.1 Å / Rmerge(I) obs: 0.2 / Mean I/σ(I) obs: 2

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Processing

Software
NameVersionClassification
TNTrefinement
SDMSDETECTOR SYSTEM (NIELSEN)data reduction
SDMSDETECTOR SYSTEM (NIELSEN)data scaling
TNTphasing
RefinementMethod to determine structure: DIFFERENCE FOURIER
Starting model: CYS-FREE WILD TYPE LYSOZYME WITH RESIDUES 106 - 114 DELETED

Resolution: 2.1→30 Å / σ(F): 0 / Stereochemistry target values: TNT PROTGEO
Details: THE STARTING MODEL WAS CYS-FREE WILDTYPE WITH RESIDUES 106 - 115 DELETED AND 102 CONVERTED TO ALA. RESIDUES 106 - 114 WERE FIRST FIT IN A NON-WILDTYPE CONFORMATION. AFTER REFINEMENT THE ...Details: THE STARTING MODEL WAS CYS-FREE WILDTYPE WITH RESIDUES 106 - 115 DELETED AND 102 CONVERTED TO ALA. RESIDUES 106 - 114 WERE FIRST FIT IN A NON-WILDTYPE CONFORMATION. AFTER REFINEMENT THE WILDTYPE CONFORMATION WAS VISIBLE IN FO-FC DIFFERENCE MAPS, AND WAS ADDED TO THE MODEL. RELATIVE OCCUPANCIES WERE ESTIMATED BASED ON REFINED GROUP OCCUPANCIES (INITIALLY AT 0.5) AND EITHER FIXING OR REFINING THE B VALUES AND POSITIONS. THE WEIGHTS OF EACH CONFORMER ARE 0.6 (ALT) AND 0.4 (WT). IN MOST T4 MUTANT STRUCTURES, RESIDUES 163 AND 164 ARE DISORDERED AND NOT OBSERVED IN ELECTRON DENSITY MAPS. IN THIS MUTANT, THESE RESIDUES WERE RESOLVED IN REFINED FO-FC DIFFERENCE MAPS AND THE RESIDUES ADDED TO THE MODEL.
RfactorNum. reflection% reflection
Rwork0.163 --
all-11176 -
obs-11176 85 %
Solvent computationSolvent model: BABINET SCALING / Bsol: 630 Å2 / ksol: 1.1 e/Å3
Refinement stepCycle: LAST / Resolution: 2.1→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1360 0 9 136 1505
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONt_bond_d0.015
X-RAY DIFFRACTIONt_angle_deg2.1
X-RAY DIFFRACTIONt_dihedral_angle_d0
X-RAY DIFFRACTIONt_incorr_chiral_ct0
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes0.009
X-RAY DIFFRACTIONt_gen_planes0.014
X-RAY DIFFRACTIONt_it6.1
X-RAY DIFFRACTIONt_nbd0.044
Software
*PLUS
Name: TNT / Version: 5F / Classification: refinement
Refinement
*PLUS
Rfactor all: 0.163
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d
X-RAY DIFFRACTIONt_dihedral_angle_deg0
X-RAY DIFFRACTIONt_planar_d0.009
X-RAY DIFFRACTIONt_plane_restr0.014

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