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- PDB-4cla: ALTERNATIVE BINDING MODES FOR CHLORAMPHENICOL AND 1-SUBSTITUTED C... -

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Entry
Database: PDB / ID: 4cla
TitleALTERNATIVE BINDING MODES FOR CHLORAMPHENICOL AND 1-SUBSTITUTED CHLORAMPHENICOL ANALOGUES REVEALED BY SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY OF CHLORAMPHENICOL ACETYLTRANSFERASE
ComponentsTYPE III CHLORAMPHENICOL ACETYLTRANSFERASE
KeywordsTRANSFERASE (ACYLTRANSFERASE)
Function / homology
Function and homology information


chloramphenicol O-acetyltransferase activity / chloramphenicol O-acetyltransferase / response to antibiotic
Similarity search - Function
Chloramphenicol acetyltransferase, active site / Chloramphenicol acetyltransferase active site. / Chloramphenicol acetyltransferase / Chloramphenicol acetyltransferase / Chloramphenicol acetyltransferase / Chloramphenicol Acetyltransferase / Chloramphenicol acetyltransferase-like domain / Chloramphenicol acetyltransferase-like domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
CHLORAMPHENICOL / : / Chloramphenicol acetyltransferase 3
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / Resolution: 2 Å
AuthorsLeslie, A.G.W.
Citation
Journal: Biochemistry / Year: 1991
Title: Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogues revealed by site-directed mutagenesis and X-ray crystallography of chloramphenicol acetyltransferase.
Authors: Murray, I.A. / Lewendon, A. / Williams, J.A. / Cullis, P.M. / Shaw, W.V.
#1: Journal: Annu.Rev.Biophys.Biophys.Chem. / Year: 1991
Title: Chloramphenicol Acetyltransferase
Authors: Shaw, W.V. / Leslie, A.G.W.
#2: Journal: J.Mol.Biol. / Year: 1990
Title: Refined Crystal Structure of Type III Chloramphenicol Acetyltransferase at 1.75 Angstroms Resolution
Authors: Leslie, A.G.W.
#3: Journal: Biochemistry / Year: 1990
Title: Crystal Structure of the Asp-199-Asn Mutant of Chloramphenicol Acetyltransferase to 2.35 Angstroms Resolution. Structural Consequences of Disruption of a Buried Salt-Bridge.
Authors: Gibbs, M.R. / Moody, P.C.E. / Leslie, A.G.W.
#4: Journal: Biochemistry / Year: 1990
Title: Evidence for Transition-State Stabilization by Serine-148 in the Catalytic Mechanism of Chloramphenicol Acetyltransferase
Authors: Lewendon, A. / Murray, I.A. / Shaw, W.V. / Gibbs, M.R. / Leslie, A.G.W.
#5: Journal: Biochemistry / Year: 1988
Title: Substitutions in the Active Site of Chloramphenicol Acetyltransferase. Role of a Conserved Aspartate
Authors: Lewendon, A. / Murray, I.A. / Kleanthous, C. / Cullis, P.M. / Shaw, W.V.
#6: Journal: Proc.Natl.Acad.Sci.USA / Year: 1988
Title: Structure of Chloramphenicol Acetyltransferase at 1.75-Angstroms Resolution
Authors: Leslie, A.G.W. / Moody, P.C.E. / Shaw, W.V.
#7: Journal: J.Mol.Biol. / Year: 1986
Title: Crystallization of a Type III Chloramphenicol Acetyl Transferase
Authors: Leslie, A.G.W. / Liddell, J.M. / Shaw, W.V.
History
DepositionOct 23, 1990Processing site: BNL
Revision 1.0Jan 15, 1992Provider: repository / Type: Initial release
Revision 1.1Mar 25, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Feb 28, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_struct_conn_angle / struct_conn / 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 / _pdbx_database_status.process_site / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr1_symmetry / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 700SHEET SHEET 1 ACTUALLY HAS SEVEN STRANDS. RESIDUES 157 - 162 FORM AN EXTENSION TO THE SIX STRANDED ...SHEET SHEET 1 ACTUALLY HAS SEVEN STRANDS. RESIDUES 157 - 162 FORM AN EXTENSION TO THE SIX STRANDED BETA- SHEET OF AN ADJACENT SUBUNIT OF THE TRIMER, RESULTING IN A SEVEN-STRANDED SHEET WHICH SPANS THE SUBUNIT INTERFACE. THE FINAL STRAND, SER 157 - VAL 162, IS IN AN ADJACENT (THREE-FOLD RELATED) SUBUNIT OF THE TRIMER. N ASN 159 IS HYDROGEN BONDED TO O SEH 34. THERE IS A WIDE BETA-BULGE INVOLVING RESIDUES LYS 177, TYR 178, AND LEU 187.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TYPE III CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,4974
Polymers25,0561
Non-polymers4413
Water3,657203
1
A: TYPE III CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules

A: TYPE III CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules

A: TYPE III CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,49012
Polymers75,1673
Non-polymers1,3239
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area8970 Å2
ΔGint-85 kcal/mol
Surface area25140 Å2
MethodPISA, PQS
2
A: TYPE III CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)152,97924
Polymers150,3336
Non-polymers2,64618
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
crystal symmetry operation4_555y,x,-z1
crystal symmetry operation5_555x-y,-y,-z1
crystal symmetry operation6_555-x,-x+y,-z1
Buried area20020 Å2
ΔGint-225 kcal/mol
Surface area48570 Å2
MethodPISA
Unit cell
Length a, b, c (Å)107.750, 107.750, 123.920
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number155
Space group name H-MH32
Atom site foot note1: THERE IS A WIDE BETA-BULGE INVOLVING RESIDUES LYS 177, TYR 178, AND LEU 187.
2: RESIDUES 157 - 162 FORM AN EXTENSION TO THE SIX STRANDED BETA-SHEET OF AN ADJACENT SUBUNIT OF THE TRIMER, RESULTING IN A SEVEN-STRANDED SHEET WHICH SPANS THE SUBUNIT INTERFACE.
Components on special symmetry positions
IDModelComponents
11A-222-

CO

21A-223-

CO

31A-309-

HOH

41A-310-

HOH

DetailsCAT IS A TRIMER OF IDENTICAL SUBUNITS EACH CONTAINING 213 AMINO ACIDS (SUBUNIT MOLECULAR WEIGHT 25000). THE THREE-FOLD AXIS OF THE TRIMER IS COINCIDENT WITH THE CRYSTALLOGRAPHIC THREE-FOLD AXIS. THE COORDINATES PRESENTED IN THIS ENTRY ARE FOR ONE SUBUNIT OF THE TRIMER. THE COORDINATES FOR THE OTHER TWO SUBUNITS CAN BE DERIVED BY APPLYING ROTATIONS OF 120 DEGREES AND 240 DEGREES ABOUT THE Z AXIS. TO GENERATE THESE SYMMETRY RELATED SUBUNITS APPLY THE FOLLOWING MATRICES TO THE COORDINATES GIVEN BELOW: 1. -0.50000 -0.86603 0.00000 0.86603 -0.50000 0.00000 0.00000 0.00000 1.00000 2. -0.50000 0.86603 0.00000 -0.86603 -0.50000 0.00000 0.00000 0.00000 1.00000

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Components

#1: Protein TYPE III CHLORAMPHENICOL ACETYLTRANSFERASE


Mass: 25055.510 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
References: UniProt: P00484, chloramphenicol O-acetyltransferase
#2: Chemical ChemComp-CO / COBALT (II) ION


Mass: 58.933 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Co
#3: Chemical ChemComp-CLM / CHLORAMPHENICOL / Chloramphenicol


Mass: 323.129 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H12Cl2N2O5 / Comment: antibiotic*YM
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 203 / Source method: isolated from a natural source / Formula: H2O
Compound detailsTHE MUTATED RESIDUE FORMS PART OF THE CHLORAMPHENICOL BINDING POCKET. THE CONFORMATION OF THE ...THE MUTATED RESIDUE FORMS PART OF THE CHLORAMPHENICOL BINDING POCKET. THE CONFORMATION OF THE ENZYME IS ESSENTIALLY IDENTICAL TO THE WILD TYPE. HOWEVER, CHLORAMPHENICOL BINDS IN A DIFFERENT ORIENTATION TO THAT OBSERVED IN THE WILD TYPE BINARY COMPLEX, WITH SHIFTS OF UP TO 2 ANGSTROMS IN ATOMIC POSITIONS. THE OBSERVED MODE OF BINDING IN THE CRYSTAL STRUCTURE IS ALMOST CERTAINLY NON-PRODUCTIVE. IN SPITE OF THIS OBSERVATION, THE MUTANT ENZYME IS STILL ACTIVE WITH A 60 FOLD DROP IN CATALYTIC ACTIVITY.
Nonpolymer detailsSOLVENT ATOMS (COBALT OR WATER) LYING ON CRYSTALLOGRAPHIC SYMMETRY AXES HAVE BEEN GIVEN OCCUPANCIES ...SOLVENT ATOMS (COBALT OR WATER) LYING ON CRYSTALLOGRAPHIC SYMMETRY AXES HAVE BEEN GIVEN OCCUPANCIES EQUAL TO 1.0/(ROTATIONAL SYMMETRY OF THAT AXIS). ATOMS OF THE SUBSTRATE CHLORAMPHENICOL HAVE BEEN ASSIGNED AN OCCUPANCY OF 0.66 BECAUSE OF UNCERTAINTY ABOUT WHETHER THE SITE IS FULLY OCCUPIED. THE LOW VALUES OF THE REFINED TEMPERATURE FACTORS SUGGEST THAT THE OCCUPANCY IS SOMEWHAT HIGHER THAN 0.66 AND IS PROBABLY CLOSE TO 1.0. IT SHOULD BE NOTED THAT THE TEMPERATURE FACTORS QUOTED IN THE JRNL REFERENCE RELATE TO AN INPUT OCCUPANCY OF 0.66, NOT AN OCCUPANCY OF 1.0. THE COBALT IONS PLAY A CRUCIAL ROLE IN STABILIZING THE CRYSTAL LATTICE.
Sequence detailsTHE NUMBERING SCHEME ADOPTED IS BASED ON THE ALIGNMENT OF A NUMBER OF CAT SEQUENCES. FOR THE TYPE ...THE NUMBERING SCHEME ADOPTED IS BASED ON THE ALIGNMENT OF A NUMBER OF CAT SEQUENCES. FOR THE TYPE III ENZYME WHOSE COORDINATES ARE PRESENTED IN THIS ENTRY, MET 6 IS THE N-TERMINAL RESIDUE AND THERE IS NO RESIDUE NUMBER 79.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.76 Å3/Da / Density % sol: 55.46 %
Crystal grow
*PLUS
Temperature: 4 ℃ / Method: microdialysis / pH: 6.3
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
150 mg/mlprotein1buttom
210 mMMES1buttom
31 mMchloramphenicol1reservoir
45 %MPD1reservoir
510 mMMES1reservoir
60.5 mMhexammine cobalt(III) chloride1reservoir

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Data collection

Reflection
*PLUS
Highest resolution: 2 Å

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Processing

SoftwareName: DERIV / Classification: refinement
RefinementResolution: 2→6 Å / Rfactor Rwork: 0.157
Details: ATOMS OF SIDECHAINS SHOWING VERY WEAK (LESS THAN 0.2 $E/A**3) OR UNINTERPRETABLE ELECTRON DENSITY HAVE BEEN OMITTED FROM THE MODEL.
Refinement stepCycle: LAST / Resolution: 2→6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1711 0 22 203 1936
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONo_bond_d0.021
X-RAY DIFFRACTIONo_bond_d_na
X-RAY DIFFRACTIONo_bond_d_prot
X-RAY DIFFRACTIONo_angle_d
X-RAY DIFFRACTIONo_angle_d_na
X-RAY DIFFRACTIONo_angle_d_prot
X-RAY DIFFRACTIONo_angle_deg3
X-RAY DIFFRACTIONo_angle_deg_na
X-RAY DIFFRACTIONo_angle_deg_prot
X-RAY DIFFRACTIONo_dihedral_angle_d
X-RAY DIFFRACTIONo_dihedral_angle_d_na
X-RAY DIFFRACTIONo_dihedral_angle_d_prot
X-RAY DIFFRACTIONo_improper_angle_d
X-RAY DIFFRACTIONo_improper_angle_d_na
X-RAY DIFFRACTIONo_improper_angle_d_prot
X-RAY DIFFRACTIONo_mcbond_it
X-RAY DIFFRACTIONo_mcangle_it
X-RAY DIFFRACTIONo_scbond_it
X-RAY DIFFRACTIONo_scangle_it
Software
*PLUS
Name: PROLSQ / Classification: refinement
Refinement
*PLUS
Highest resolution: 2 Å / Lowest resolution: 6 Å / Num. reflection all: 17970 / Rfactor obs: 0.147
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeWeightDev ideal
X-RAY DIFFRACTIONp_bond_d0.02
X-RAY DIFFRACTIONp_angle_d3
X-RAY DIFFRACTIONp_dihedral_angle_d0.20.16
X-RAY DIFFRACTIONp_improper_angle_d
X-RAY DIFFRACTIONp_planar_d0.050.062
X-RAY DIFFRACTIONp_plane_restr0.030.02
X-RAY DIFFRACTIONp_chiral_restr0.150.22
X-RAY DIFFRACTIONp_mcbond_it
X-RAY DIFFRACTIONp_scbond_it
X-RAY DIFFRACTIONp_mcangle_it
X-RAY DIFFRACTIONp_scangle_it

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