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- PDB-6u18: Directed evolution of a biosensor selective for the macrolide ant... -

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Basic information

Entry
Database: PDB / ID: 6u18
TitleDirected evolution of a biosensor selective for the macrolide antibiotic clarithromycin
ComponentsErythromycin resistance repressor protein
KeywordsDNA BINDING PROTEIN/ANTIBIOTIC / macrolide / biosensor / DNA-binding / DNA BINDING PROTEIN-ANTIBIOTIC complex
Function / homology
Function and homology information


transferase activity / transcription cis-regulatory region binding / DNA-binding transcription factor activity
Similarity search - Function
: / Bacterial regulatory proteins, tetR family / DNA-binding HTH domain, TetR-type / TetR-type HTH domain profile. / Homeobox-like domain superfamily
Similarity search - Domain/homology
CLARITHROMYCIN / CITRATE ANION / Erythromycin resistance repressor protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsLi, Y. / Reed, M. / Wright, H.T. / Cropp, T.A. / Williams, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124112 United States
Citation
Journal: Acs Synth Biol / Year: 2021
Title: Development of Genetically Encoded Biosensors for Reporting the Methyltransferase-Dependent Biosynthesis of Semisynthetic Macrolide Antibiotics.
Authors: Li, Y. / Reed, M. / Wright, H.T. / Cropp, T.A. / Williams, G.J.
#1: Journal: J.Mol.Biol. / Year: 2009
Title: Structure and function of the macrolide biosensor protein, MphR(A), with and without erythromycin.
Authors: Zheng, J. / Sagar, V. / Smolinsky, A. / Bourke, C. / LaRonde-LeBlanc, N. / Cropp, T.A.
History
DepositionAug 15, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 11, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Erythromycin resistance repressor protein
B: Erythromycin resistance repressor protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,8895
Polymers43,2042
Non-polymers1,6853
Water2,432135
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5620 Å2
ΔGint-44 kcal/mol
Surface area16680 Å2
MethodPISA
Unit cell
Length a, b, c (Å)42.897, 53.161, 158.815
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Erythromycin resistance repressor protein / Macrolide 2'-phosphotransferase / Macrolide 2'-phosphotransferase MphR(A) / MphR(A) / MphR(A) ...Macrolide 2'-phosphotransferase / Macrolide 2'-phosphotransferase MphR(A) / MphR(A) / MphR(A) erythromycin resistance repressor protein / Regulator of macrolide 2'-phosphotransferase I / Repressor protein MphR(A) / TetR/AcrR family transcriptional regulator / Transcriptional regulator / TetR family


Mass: 21601.975 Da / Num. of mol.: 2 / Mutation: R122T, K132N, A151T, H184Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: mphR(A), mphR, A9R57_28010, ACN77_02555, ACN77_03810, ACN77_19745, AKG29_00740, AM465_27045, APT94_14580, B1K96_22845, B9M99_28120, B9N33_26285, B9T59_28650, BANRA_01808, BANRA_05330, BANRA_ ...Gene: mphR(A), mphR, A9R57_28010, ACN77_02555, ACN77_03810, ACN77_19745, AKG29_00740, AM465_27045, APT94_14580, B1K96_22845, B9M99_28120, B9N33_26285, B9T59_28650, BANRA_01808, BANRA_05330, BANRA_05395, BE963_00530, BEN53_26070, BET08_23460, BFD68_26430, BJJ90_27670, BK248_28255, BK292_28800, BK334_26705, BK373_25645, BK375_28450, BK383_16415, BK400_25090, BVL39_07785, BVL39_26565, C5N07_27930, C5P01_27745, C5P43_14800, C5P44_28315, C6669_28285, C6986_31345, C7235_25300, C7235_26770, CQP61_00550, CR538_26710, CRT43_27530, CRT46_27035, CWS33_26295, D0X26_00055, D0X26_28400, D3O91_28140, D9N32_01235, DB359_27590, DBY14_07355, DBY14_07475, DBY14_07595, DBY14_07715, DBY14_07835, DBY14_07955, DBY14_08075, DBY14_08195, DBY14_08400, DBY14_09000, DBY14_09120, DBY14_09240, DBY14_09360, DBY14_09480, DBY14_09600, DBY14_09720, DBY14_09840, DBY14_09960, DBY14_10080, DBY14_10200, DBY14_10320, DBY14_10440, DBY14_10560, DBY14_10680, DBY14_10800, DBY14_10920, DBY14_11040, DBY14_11160, DBY14_11280, DL545_01125, DL545_26270, DM102_26995, DM129_26600, DM129_28140, DNQ41_27340, DS732_30515, E4Z89_24290, Eco118UI_26585, ECONIH1_26780, ECTO124_05297, EGT48_01470, EIA21_26670, EJC48_00900, EJC75_00560, EJH97_25820, EPS71_24560, EQ823_24865, EQ830_27025, ERS085366_04056, ERS085404_05054, ERS150873_04751, ETN48_p0081, EVY21_24885, ExPECSC022_02556, ExPECSC036_03428, ExPECSC065_05093, EXX13_27025, EYD11_23970, FORC82_p029, HmCmsJML146_04799, HMPREF3040_01640, MS6198_B124, MS8345_A00020, NCTC13846_04922, U12A_A00163, U14A_A00115, UN86_19865, YDC107_4984
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9EVJ6
#2: Chemical ChemComp-CTY / CLARITHROMYCIN


Mass: 747.953 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C38H69NO13 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-FLC / CITRATE ANION


Mass: 189.100 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C6H5O7
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 135 / 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: 2.14 Å3/Da / Density % sol: 42.64 % / Description: elongated flat plate with beveled end
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.5 / Details: 10% PEG3350, 0.1 M ammonium citrate dibasic / PH range: 5.3 - 5.7

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Data collection

DiffractionMean temperature: 80 K / Ambient temp details: liquid nitrogen stream / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.5419 Å
DetectorType: DECTRIS EIGER R 4M / Detector: PIXEL / Date: Mar 6, 2019 / Details: Rigaku Varimax-HF Arc
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5419 Å / Relative weight: 1
ReflectionResolution: 2→29.14 Å / Num. obs: 25434 / % possible obs: 99.9 % / Redundancy: 6.1 % / Biso Wilson estimate: 23.91 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.048 / Rpim(I) all: 0.021 / Rrim(I) all: 0.052 / Net I/σ(I): 22.8
Reflection shellResolution: 2→2.05 Å / Redundancy: 5.7 % / Rmerge(I) obs: 0.292 / Mean I/σ(I) obs: 5.3 / Num. unique obs: 1883 / CC1/2: 0.959 / Rpim(I) all: 0.132 / Rrim(I) all: 0.321 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
PHENIX1.13_2998refinement
CrysalisPro40-64.40adata collection
CrysalisPro40-64.40adata reduction
Aimlessdata scaling
PHASERPhenixphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3G56

3g56


Resolution: 2→29.14 Å / SU ML: 0.2052 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 22.6185 / Stereochemistry target values: GeoStd + Monomer Library
RfactorNum. reflection% reflection
Rfree0.2207 1308 5.15 %
Rwork0.2019 24066 -
obs0.2029 25374 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 33.72 Å2
Refinement stepCycle: LAST / Resolution: 2→29.14 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2806 0 117 135 3058
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0042991
X-RAY DIFFRACTIONf_angle_d0.61434101
X-RAY DIFFRACTIONf_chiral_restr0.0374487
X-RAY DIFFRACTIONf_plane_restr0.0032515
X-RAY DIFFRACTIONf_dihedral_angle_d12.90861743
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.080.29111390.23472633X-RAY DIFFRACTION99.96
2.08-2.170.23521240.21542647X-RAY DIFFRACTION99.96
2.17-2.290.24431600.2062618X-RAY DIFFRACTION100
2.29-2.430.26011580.21072623X-RAY DIFFRACTION100
2.43-2.620.21821500.20492643X-RAY DIFFRACTION100
2.62-2.880.24981490.21542658X-RAY DIFFRACTION100
2.88-3.30.25051310.22242692X-RAY DIFFRACTION99.65
3.3-4.160.1851410.18732696X-RAY DIFFRACTION99.72
4.16-29.140.18941560.18242856X-RAY DIFFRACTION99.74
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)
10.45628642433-0.0918977118840.1628969874840.178348389099-0.161588824160.161329947227-0.00741809745683-0.05484476292990.0121091840407-0.0127562897868-0.0265039203767-0.05572975121010.06815034437350.0430008171825-2.19151803655E-60.277707673744-0.00925810786718-0.02561967724360.1957369435940.006625053795860.2051271028923.544478626-0.37993476945729.9189657136
20.134113994919-0.1226224917380.1606225895280.215411213848-0.1651190207910.1955558782690.0244960223821-0.1567637945530.0450756335434-0.0419468796681-0.00133743766053-0.08307622823280.0922141661488-0.04421703059861.40037000163E-50.232003212829-0.0466672287826-0.0226846783110.2933393745530.01679005860320.23000698783212.55393805385.1336915552427.0232964446
30.0675864595984-0.04573617924880.05279654159130.0368799805507-0.00959588924910.150688336312-0.07338825976310.0161460855770.035195854746-0.01139122944220.017914328272-0.02167946664660.00794997102076-0.0437568762898-0.0009645821998730.154817551302-0.0422868024848-0.04026755187990.213088652748-0.0143459537710.18972565693610.721115018519.901048860221.3778594341
41.10792636573-0.492269938911-0.5863387202880.8599669501380.5046520365090.5835908349680.2414467832310.0582423233991-0.111651675092-0.0765493157767-0.121508641565-0.08122211037960.259730063315-0.1123405407670.5524100291130.327925560898-0.00740727031665-0.1349687295610.131453274801-0.01370341176020.2321936083383.01349074539-5.91442482461-1.44473768578
50.0461885431514-0.00395083567994-0.01524483152260.0104506177378-0.01158017198520.02157062168660.0656741129079-0.1899952595050.00970818144549-0.2021961612-0.05135662122690.02215043593450.0764576331006-0.2777961970164.04066310476E-50.174988263289-0.00891005667362-0.01356985264740.233539368339-0.0257621134150.269543401064-10.259836081415.775004941313.0302780352
60.0310485902476-0.03463982419790.005971896985040.0438937684381-0.01176383576860.006014626323990.107063971070.01834050813620.06449411813910.123709841581-0.005312111082860.0299475959620.1635404757910.0189060418232.11829051747E-50.308496207054-0.0854164261359-0.04219472465390.2753926092470.03900516692270.2818605738786.34004055251-1.6123745270912.3297490233
70.461320434516-0.2346811987970.3661595980940.440506183747-0.04918368019260.3509877656080.02087874143-0.232565108430.0285725344513-0.0893296697675-0.0343703760828-0.004016648232630.00920752434212-0.100422366340.001978178321410.152859939351-0.0444092757729-0.02428200616650.212368275897-0.01345473545150.2035777916560.27812938377214.808432808315.5554732465
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 7 through 91)
2X-RAY DIFFRACTION2chain 'A' and (resid 92 through 141)
3X-RAY DIFFRACTION3chain 'A' and (resid 142 through 190)
4X-RAY DIFFRACTION4chain 'B' and (resid 8 through 71)
5X-RAY DIFFRACTION5chain 'B' and (resid 72 through 92)
6X-RAY DIFFRACTION6chain 'B' and (resid 93 through 112)
7X-RAY DIFFRACTION7chain 'B' and (resid 113 through 188)

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