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- PDB-8akj: Acyl-enzyme complex of cephalothin bound to deacylation mutant KP... -

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Basic information

Entry
Database: PDB / ID: 8akj
TitleAcyl-enzyme complex of cephalothin bound to deacylation mutant KPC-2 (E166Q)
ComponentsCarbapenem-hydrolyzing beta-lactamase KPC
KeywordsANTIMICROBIAL PROTEIN / acyl-enzyme complex / antibiotic resistance / beta-lactamase / antibiotic / ligand
Function / homology
Function and homology information


beta-lactam antibiotic catabolic process / beta-lactamase activity / beta-lactamase / response to antibiotic
Similarity search - Function
Beta-lactamase class A, catalytic domain / Beta-lactamase enzyme family / Beta-lactamase, class-A / Beta-lactamase/transpeptidase-like
Similarity search - Domain/homology
Chem-CEO / Carbapenem-hydrolyzing beta-lactamase KPC
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.35 Å
AuthorsTooke, C.L. / Hinchliffe, P. / Spencer, J.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/T016035/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/J014400/1 United Kingdom
Citation
Journal: J.Am.Chem.Soc. / Year: 2023
Title: Tautomer-Specific Deacylation and Omega-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 beta-Lactamase.
Authors: Tooke, C.L. / Hinchliffe, P. / Beer, M. / Zinovjev, K. / Colenso, C.K. / Schofield, C.J. / Mulholland, A.J. / Spencer, J.
#1: Journal: Acta Crystallogr.,Sect.D / Year: 2012
Title: Towards automated crystallographic structure refinement with phenix.refine.
Authors: Afonine, P.V. / Grosse-Kunstleve, R.W. / Echols, N. / Headd, J.J. / Moriarty, N.W. / Mustyakimov, M. / Terwilliger, T.C. / Urzhumtsev, A. / Zwart, P.H. / Adams, P.D.
#2: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionJul 29, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 7, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Carbapenem-hydrolyzing beta-lactamase KPC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,7098
Polymers30,8061
Non-polymers9037
Water4,468248
1
A: Carbapenem-hydrolyzing beta-lactamase KPC
hetero molecules

A: Carbapenem-hydrolyzing beta-lactamase KPC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,41716
Polymers61,6112
Non-polymers1,80614
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
Buried area4190 Å2
ΔGint-75 kcal/mol
Surface area21340 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.512, 79.579, 55.955
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Components on special symmetry positions
IDModelComponents
11A-301-

SO4

21A-454-

HOH

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Components

#1: Protein Carbapenem-hydrolyzing beta-lactamase KPC / Carbapenem-hydrolyzing beta-lactamase KPC-1


Mass: 30805.646 Da / Num. of mol.: 1 / Mutation: E166Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: bla, kpc, kpc1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): SoluBL21 / References: UniProt: Q9F663, beta-lactamase
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-CEO / 5-METHYLENE-2-[2-OXO-1-(2-THIOPHEN-2-YL-ACETYLAMINO)-ETHYL]-5,6-DIHYDRO-2H-[1,3]THIAZINE-4-CARBOXYLIC ACID / HYDROLYZED CEPHALOTHIN


Mass: 338.402 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H14N2O4S2 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 248 / 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: 2.37 Å3/Da / Density % sol: 43.75 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / Details: 2.0 M Ammonium Sulphate, 5% v/v ethanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALBA / Beamline: XALOC / Wavelength: 0.89996 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: May 1, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.89996 Å / Relative weight: 1
ReflectionResolution: 1.35→48.17 Å / Num. obs: 59361 / % possible obs: 99 % / Redundancy: 13.3 % / CC1/2: 0.999 / Rpim(I) all: 0.032 / Net I/σ(I): 14.4
Reflection shellResolution: 1.35→1.37 Å / Redundancy: 13.6 % / Mean I/σ(I) obs: 1.2 / Num. unique obs: 2871 / CC1/2: 0.726 / Rpim(I) all: 0.553 / % possible all: 97.8

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6Z21

6z21


Resolution: 1.35→48.168 Å / SU ML: 0.15 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 19.13 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1776 3026 5.1 %
Rwork0.1504 56261 -
obs0.1518 59287 98.75 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 121.61 Å2 / Biso mean: 21.3087 Å2 / Biso min: 8.39 Å2
Refinement stepCycle: final / Resolution: 1.35→48.168 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1999 0 125 248 2372
Biso mean--32.06 35.58 -
Num. residues----268
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
1.35-1.37110.33551210.3622247997
1.3711-1.39360.29241520.3241248297
1.3936-1.41760.29871360.2839249598
1.4176-1.44340.23361500.2456248998
1.4434-1.47120.27641370.2333250198
1.4712-1.50120.23241430.2114249498
1.5012-1.53380.23621400.1936250799
1.5338-1.56950.21741370.1869251398
1.5695-1.60880.17261570.1693250798
1.6088-1.65230.18851390.1696253498
1.6523-1.70090.20221330.1595252499
1.7009-1.75580.17351320.1514255699
1.7558-1.81850.16871300.1454255899
1.8185-1.89140.16891090.1395258299
1.8914-1.97740.19031370.1352257099
1.9774-2.08170.15141480.128257799
2.0817-2.21210.15631280.12122586100
2.2121-2.38290.14711510.11962584100
2.3829-2.62270.14991250.11822608100
2.6227-3.00220.1611380.12232641100
3.0022-3.78220.15061450.12672667100
3.7822-48.1680.17131380.14932807100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
14.19790.4136-0.47366.6331-6.41616.3996-0.08180.524-0.1288-0.46830.002-0.13230.18980.00930.07830.1467-0.02210.00760.2629-0.05050.095211.573710.3139-4.394
23.22031.2798-0.13731.7199-0.10681.9406-0.08250.35460.1962-0.17660.10610.0756-0.0649-0.1369-0.04150.07810.014-0.00610.19510.0110.09599.001418.62272.201
31.56410.99892.02621.6512-0.11244.9594-0.1815-0.19860.22730.15870.00110.1724-0.5443-0.04270.15460.09850.01790.0120.2183-0.05140.127514.895626.612228.9921
46.5652.34670.98658.62751.23982.47710.093-0.0984-0.37180.02690.0236-0.00720.2355-0.0747-0.11870.093-0.0243-0.00510.20960.01710.080120.747514.311134.6445
55.7257-2.1762.79580.9238-1.20161.5699-0.0382-0.29230.03760.07720.02090.0354-0.0747-0.06690.02360.0902-0.01630.00020.1622-0.02310.087212.012122.203625.7594
61.47440.2799-0.120.8877-0.05080.62830.01460.00950.06630.0128-0.0068-0.0395-0.04720.0297-0.00850.0693-0.0092-0.00140.1520.00140.06514.167919.939814.0126
75.20555.1588-2.20017.1245-2.56831.2434-0.10230.1742-0.0774-0.1420.0764-0.11620.0538-0.02720.01750.0701-0.0061-0.00720.1766-0.01280.043414.318712.41177.208
80.4603-0.62010.11883.2863-0.97321.9924-0.02310.1610.0105-0.2763-0.1752-0.42340.06270.27230.1960.1013-0.00150.02750.2859-0.00240.14420.441513.7211-0.5286
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 25 through 41 )A25 - 41
2X-RAY DIFFRACTION2chain 'A' and (resid 42 through 68 )A42 - 68
3X-RAY DIFFRACTION3chain 'A' and (resid 69 through 98 )A69 - 98
4X-RAY DIFFRACTION4chain 'A' and (resid 99 through 118 )A99 - 118
5X-RAY DIFFRACTION5chain 'A' and (resid 119 through 155 )A119 - 155
6X-RAY DIFFRACTION6chain 'A' and (resid 156 through 251 )A156 - 251
7X-RAY DIFFRACTION7chain 'A' and (resid 252 through 275 )A252 - 275
8X-RAY DIFFRACTION8chain 'A' and (resid 276 through 294 )A276 - 294

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