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- PDB-1aj6: NOVOBIOCIN-RESISTANT MUTANT (R136H) OF THE N-TERMINAL 24 KDA FRAG... -

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Entry
Database: PDB / ID: 1aj6
TitleNOVOBIOCIN-RESISTANT MUTANT (R136H) OF THE N-TERMINAL 24 KDA FRAGMENT OF DNA GYRASE B COMPLEXED WITH NOVOBIOCIN AT 2.3 ANGSTROMS RESOLUTION
ComponentsGYRASEDNA gyrase
KeywordsTOPOISOMERASE / GYRASE / NOVOBIOCIN / ANTIBIOTIC / RESISTANT MUTANT
Function / homology
Function and homology information


DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / DNA negative supercoiling activity / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / ATP-dependent activity, acting on DNA / DNA-templated DNA replication / chromosome / response to xenobiotic stimulus / response to antibiotic ...DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / DNA negative supercoiling activity / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / ATP-dependent activity, acting on DNA / DNA-templated DNA replication / chromosome / response to xenobiotic stimulus / response to antibiotic / DNA-templated transcription / DNA binding / ATP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
: / GyrB, hook / DNA gyrase subunit B insert domain / DNA gyrase B subunit insert domain / DNA gyrase subunit B, TOPRIM domain / DNA gyrase, subunit B / DNA topoisomerase, type IIA, subunit B / DNA gyrase B subunit, C-terminal / DNA gyrase B subunit, carboxyl terminus / DNA topoisomerase, type IIA, subunit B, domain 2 ...: / GyrB, hook / DNA gyrase subunit B insert domain / DNA gyrase B subunit insert domain / DNA gyrase subunit B, TOPRIM domain / DNA gyrase, subunit B / DNA topoisomerase, type IIA, subunit B / DNA gyrase B subunit, C-terminal / DNA gyrase B subunit, carboxyl terminus / DNA topoisomerase, type IIA, subunit B, domain 2 / DNA gyrase B / DNA topoisomerase, type IIA / DNA topoisomerase, type IIA, conserved site / DNA topoisomerase II signature. / TopoisomeraseII / DNA topoisomerase, type IIA, subunit B, C-terminal / Toprim domain / DNA topoisomerase, type IIA-like domain superfamily / Toprim domain profile. / TOPRIM domain / Histidine kinase-like ATPase, C-terminal domain / Heat Shock Protein 90 / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
NOVOBIOCIN / DNA gyrase subunit B / DNA gyrase subunit B
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / DIFFERENCE FOURIER / Resolution: 2.3 Å
AuthorsWeston, S.A. / Tunnicliffe, A. / Pauptit, R.A.
Citation
Journal: Biochemistry / Year: 1997
Title: The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic study.
Authors: Holdgate, G.A. / Tunnicliffe, A. / Ward, W.H. / Weston, S.A. / Rosenbrock, G. / Barth, P.T. / Taylor, I.W. / Pauptit, R.A. / Timms, D.
#1: Journal: To be Published
Title: Antibacterial Design Based on the Structures of Gyrase-Inhibitor Complexes
Authors: Pauptit, R.A. / Weston, S.A. / Breeze, A.L. / Derbyshire, D.J. / Tucker, A.D. / Hales, N. / Hollinshead, D. / Timms, D.
#2: Journal: Proteins / Year: 1997
Title: The High-Resolution Crystal Structure of a 24-kDa Gyrase B Fragment from E. Coli Complexed with One of the Most Potent Coumarin Inhibitors, Clorobiocin
Authors: Tsai, F.T. / Singh, O.M. / Skarzynski, T. / Wonacott, A.J. / Weston, S. / Tucker, A. / Pauptit, R.A. / Breeze, A.L. / Poyser, J.P. / O'Brien, R. / Ladbury, J.E. / Wigley, D.B.
#3: Journal: Embo J. / Year: 1996
Title: The Nature of Inhibition of DNA Gyrase by the Coumarins and the Cyclothialidines Revealed by X-Ray Crystallography
Authors: Lewis, R.J. / Singh, O.M. / Smith, C.V. / Skarzynski, T. / Maxwell, A. / Wonacott, A.J. / Wigley, D.B.
History
DepositionMay 15, 1997Processing site: BNL
Revision 1.0May 20, 1998Provider: repository / Type: Initial release
Revision 1.1Mar 3, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.3Nov 3, 2021Group: Database references / Derived calculations / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / 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 / _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 7, 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: GYRASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,6542
Polymers24,0411
Non-polymers6131
Water1,60389
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)39.500, 47.720, 114.410
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein GYRASE / DNA gyrase


Mass: 24040.936 Da / Num. of mol.: 1 / Fragment: N-TERMINAL 24 KDA / Mutation: R136H
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Description: PLASMID PTB382 ENCODING MUTANT DERIVED FROM PAM24 WHICH ENCODES 24 KDA SUBDOMAIN (SUPPLIED BY A. MAXWELL, LEICESTER), WHICH IN TURN WAS DERIVED FROM PAG111 PLASMID WHICH ENCODES FOR GYRB
Cellular location: CYTOPLASM / Gene: GYRB / Cellular location (production host): CYTOPLASM / Production host: Escherichia coli (E. coli) / Strain (production host): MM294
References: UniProt: P06982, UniProt: P0AES6*PLUS, EC: 5.99.1.3
#2: Chemical ChemComp-NOV / NOVOBIOCIN / 4-Hydroxy-3-[4-hydroxy-3-(3-methylbut-2-enyl)benzamido]-8-methylcoumarin-7-yl 3-O-carbamoyl-5,5-di-C-methyl-alpha-l-lyxofuranoside / Novobiocin


Mass: 612.624 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H36N2O11 / Comment: antibiotic*YM
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 89 / Source method: isolated from a natural source / Formula: H2O
Compound detailsTHE MUTATED RESIDUE HIS 136 HAS AN UNUSUAL ECLIPSED SIDE CHAIN CONFORMATION WHICH IS STABILISED BY ...THE MUTATED RESIDUE HIS 136 HAS AN UNUSUAL ECLIPSED SIDE CHAIN CONFORMATION WHICH IS STABILISED BY HYDROGEN BONDS.

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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: 2.2 Å3/Da / Density % sol: 45 %
Crystal growMethod: vapor diffusion, hanging drop / pH: 6.2
Details: HANGING DROP VAPOR DIFFUSION; 12MGS/ML PROTEIN/NOVOBIOCIN MIXED WITH EQUAL VOLUME OF RESERVOIR SOLUTION (12% V/V PEG200, 100MM MES PH6.2), vapor diffusion - hanging drop
Crystal grow
*PLUS
Temperature: 20 ℃ / Method: vapor diffusion, hanging drop
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
112 mg/mlprotein/novobiocin1drop
26 %(v/v)PEG2001drop
350 mMMES1drop
412 %(v/v)PEG2001reservoir
5100 mMMES1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SRS / Beamline: PX9.6 / Wavelength: 0.87
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Jun 1, 1996 / Details: VERTICALLY FOCUSSED PT-COATED MIRROR
RadiationMonochromator: SI(111) / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.87 Å / Relative weight: 1
ReflectionResolution: 2.3→19.8 Å / Num. obs: 9455 / % possible obs: 94.8 % / Observed criterion σ(I): 0 / Redundancy: 5.1 % / Biso Wilson estimate: 23.7 Å2 / Rmerge(I) obs: 0.065 / Rsym value: 0.065 / Net I/σ(I): 8.5
Reflection shellResolution: 2.3→2.38 Å / Redundancy: 5.2 % / Rmerge(I) obs: 0.131 / Mean I/σ(I) obs: 5.6 / Rsym value: 0.131 / % possible all: 97
Reflection
*PLUS
Num. measured all: 48291
Reflection shell
*PLUS
% possible obs: 97 %

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Processing

Software
NameVersionClassification
CCP4model building
TNT5Drefinement
MOSFLMdata reduction
CCP4data scaling
CCP4phasing
RefinementMethod to determine structure: DIFFERENCE FOURIER
Starting model: WILD-TYPE 24KDA FRAGMENT/NOVOBIOCIN COMPLEX

Resolution: 2.3→19.8 Å / Isotropic thermal model: TNT BCORREL / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: TNT PROGEO
Details: STRAINED GEOMETRY AND DIFFERENCE DENSITY AT PRO 23 SUGGEST IT MIGHT HAVE MORE THAN ONE CONFORMATION. ASN 178 IS IN A DISALLOWED REGION OF THE RAMACHANDRAN PLOT. IT ADOPTS A CLASSICAL C-7 ...Details: STRAINED GEOMETRY AND DIFFERENCE DENSITY AT PRO 23 SUGGEST IT MIGHT HAVE MORE THAN ONE CONFORMATION. ASN 178 IS IN A DISALLOWED REGION OF THE RAMACHANDRAN PLOT. IT ADOPTS A CLASSICAL C-7 AXIAL CONFORMATION TYPICAL OF GLYCINE RESIDUES WITH A CO(N-1)..N(N+1) H-BOND.
RfactorNum. reflection% reflectionSelection details
Rfree0.29 999 10 %RANDOM
Rwork0.195 ---
all0.206 9455 --
obs0.206 9455 95 %-
Solvent computationSolvent model: TNT / Bsol: 241 Å2 / ksol: 1 e/Å3
Refinement stepCycle: LAST / Resolution: 2.3→19.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1459 0 44 89 1592
Refine LS restraints
Refine-IDTypeDev idealNumberWeight
X-RAY DIFFRACTIONt_bond_d0.01215313
X-RAY DIFFRACTIONt_angle_deg1.36620763
X-RAY DIFFRACTIONt_dihedral_angle_d18.98860
X-RAY DIFFRACTIONt_incorr_chiral_ct0
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes0.01325
X-RAY DIFFRACTIONt_gen_planes0.01522810
X-RAY DIFFRACTIONt_it2.1152120
X-RAY DIFFRACTIONt_nbd0.051328
Software
*PLUS
Name: TNT / Version: 5D / Classification: refinement
Refinement
*PLUS
Rfactor obs: 0.195
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Type: t_plane_restr / Dev ideal: 0.015 / Weight: 10
LS refinement shell
*PLUS
Highest resolution: 2.3 Å / Lowest resolution: 2.4 Å / Rfactor Rfree: 0.132 / Rfactor obs: 0.23

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