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- PDB-4rzs: Lac repressor engineered to bind sucralose, unliganded tetramer -

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Basic information

Entry
Database: PDB / ID: 4rzs
TitleLac repressor engineered to bind sucralose, unliganded tetramer
ComponentsLac repressor
KeywordsTRANSCRIPTION / sucralose / lacI / lac repressor / lactose operon repressor / protein design / allostery / Escherichia coli
Function / homology
Function and homology information


DNA-binding transcription repressor activity / cis-regulatory region sequence-specific DNA binding / transcription cis-regulatory region binding / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / identical protein binding / cytosol
Similarity search - Function
Transcriptional regulator LacI/GalR-like, sensor domain / LacI-type HTH domain signature. / Periplasmic binding protein-like domain / LacI-type HTH domain / Bacterial regulatory proteins, lacI family / LacI-type HTH domain profile. / helix_turn _helix lactose operon repressor / Lambda repressor-like, DNA-binding domain superfamily / Response regulator / Periplasmic binding protein-like I ...Transcriptional regulator LacI/GalR-like, sensor domain / LacI-type HTH domain signature. / Periplasmic binding protein-like domain / LacI-type HTH domain / Bacterial regulatory proteins, lacI family / LacI-type HTH domain profile. / helix_turn _helix lactose operon repressor / Lambda repressor-like, DNA-binding domain superfamily / Response regulator / Periplasmic binding protein-like I / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
: / Lactose operon repressor
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.71 Å
AuthorsArbing, M.A. / Cascio, D. / Kosuri, S. / Church, G.M.
CitationJournal: Nat.Methods / Year: 2016
Title: Engineering an allosteric transcription factor to respond to new ligands.
Authors: Taylor, N.D. / Garruss, A.S. / Moretti, R. / Chan, S. / Arbing, M.A. / Cascio, D. / Rogers, J.K. / Isaacs, F.J. / Kosuri, S. / Baker, D. / Fields, S. / Church, G.M. / Raman, S.
History
DepositionDec 24, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 23, 2015Provider: repository / Type: Initial release
Revision 1.1Jan 6, 2016Group: Database references
Revision 1.2Mar 2, 2016Group: Database references
Revision 1.3Nov 22, 2017Group: Refinement description / Category: software
Revision 1.4Feb 28, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_ncs_dom_lim / 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_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lac repressor
B: Lac repressor
C: Lac repressor
D: Lac repressor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,9028
Polymers163,5344
Non-polymers3684
Water41423
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11410 Å2
ΔGint-104 kcal/mol
Surface area47280 Å2
MethodPISA
Unit cell
Length a, b, c (Å)91.090, 111.600, 189.330
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11PROPROGLUGLUAA3 - 35724 - 378
21PROPROGLUGLUBB3 - 35724 - 378
12SERSERGLUGLUAA61 - 35782 - 378
22SERSERGLUGLUCC61 - 35782 - 378
13SERSERARGARGAA61 - 35582 - 376
23SERSERARGARGDD61 - 35582 - 376
14SERSERLEULEUBB61 - 35682 - 377
24SERSERLEULEUCC61 - 35682 - 377
15SERSERARGARGBB61 - 35582 - 376
25SERSERARGARGDD61 - 35582 - 376
16SERSERARGARGCC61 - 35582 - 376
26SERSERARGARGDD61 - 35582 - 376

NCS ensembles :
ID
1
2
3
4
5
6

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Components

#1: Protein
Lac repressor / Transcriptional regulator / LacI family


Mass: 40883.488 Da / Num. of mol.: 4 / Mutation: D149T, V150A, I156L, S193D
Source method: isolated from a genetically manipulated source
Details: N-terminal thrombin-cleavable 6xHis-tag / Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 MG1655 / Gene: b0345, ECDH1ME8569_0332, EcDH1_3261, lacI / Plasmid: pET14b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-AI / References: UniProt: C9QQT3, UniProt: P03023*PLUS
#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 23 / Source method: isolated from a natural source / Formula: H2O

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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: 2.94 Å3/Da / Density % sol: 58.2 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7.4
Details: 16% polyethylene glycol 3350, 200 mM ammonium nitrate, pH 7.4, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9793 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Oct 16, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 2.71→94.49 Å / Num. all: 36390 / Num. obs: 36390 / % possible obs: 68.2 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 84.064 Å2 / Rmerge(I) obs: 0.069 / Χ2: 1.001 / Net I/σ(I): 15.19
Reflection shell

Diffraction-ID: 1

Resolution (Å)Highest resolution (Å)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. unique obs% possible all
2.71-2.780.4613.877131193.1
2.78-2.850.5043.6620933549.3
2.85-2.940.4924.18393464017.2
2.94-3.030.4644.59588292626
3.03-3.130.4914.528784134538.5
3.13-3.240.4544.8612743190756.8
3.24-3.360.4934.4319051287488.2
3.36-3.490.3615.4119136314399.6
3.49-3.650.2497.61208883018100
3.65-3.830.16510.3119640287299.8
3.83-4.040.12113.9218387274799.8
4.04-4.280.08917.71169012613100
4.28-4.580.06520.914318245699.6
4.58-4.940.06223.7515062229599.9
4.94-5.410.06224.5138942123100
5.41-6.050.0625.2612421193199.9
6.05-6.990.0526.589892172299.6
6.99-8.560.04332.79350146899.9
8.56-12.110.04133.956958115899.7
12.110.0432.71378567998.4

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XSCALEdata scaling
PHASERphasing
REFMAC5.8.0049refinement
PDB_EXTRACT3.15data extraction
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.71→94.49 Å / Cor.coef. Fo:Fc: 0.939 / Cor.coef. Fo:Fc free: 0.91 / WRfactor Rfree: 0.2371 / WRfactor Rwork: 0.1952 / FOM work R set: 0.8108 / SU B: 33.348 / SU ML: 0.29 / SU R Cruickshank DPI: 0.3965 / SU Rfree: 0.4112 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.411 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : RESIDUAL ONLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2392 3638 10 %RANDOM
Rwork0.1989 32749 --
obs0.2029 32745 68.11 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 104.82 Å2 / Biso mean: 79.965 Å2 / Biso min: 12.14 Å2
Baniso -1Baniso -2Baniso -3
1--0.26 Å20 Å2-0 Å2
2--1.2 Å2-0 Å2
3----0.94 Å2
Refinement stepCycle: LAST / Resolution: 2.71→94.49 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9420 0 24 23 9467
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0199637
X-RAY DIFFRACTIONr_bond_other_d0.0060.029286
X-RAY DIFFRACTIONr_angle_refined_deg1.5661.96713144
X-RAY DIFFRACTIONr_angle_other_deg1.109321221
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.0151303
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.3824.765361
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.819151517
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.2221557
X-RAY DIFFRACTIONr_chiral_restr0.0750.21624
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.02111160
X-RAY DIFFRACTIONr_gen_planes_other0.0050.022061
X-RAY DIFFRACTIONr_mcbond_it2.8354.2055215
X-RAY DIFFRACTIONr_mcbond_other2.8354.2055214
X-RAY DIFFRACTIONr_mcangle_it4.6656.3046514
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A189590.08
12B189590.08
21A169750.07
22C169750.07
31A174530.07
32D174530.07
41B167380.05
42C167380.05
51B172020.06
52D172020.06
61C171120.05
62D171120.05
LS refinement shellResolution: 2.707→2.777 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.739 105 -
Rfree-10 -
all-115 -
obs--2.96 %
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)
13.5607-0.584-0.64682.21340.07991.6642-0.1121-0.19740.43270.41870.01660.3672-0.3064-0.03080.09550.2402-0.0455-0.05930.05570.02570.2524-2.3886-23.993918.634
23.56651.0734-0.97691.9864-0.65621.88430.0307-0.3289-0.03080.3609-0.05750.31710.0633-0.13240.02680.294-0.0205-0.01460.06120.00120.1107-3.9129-46.383826.5419
31.47360.3623-0.33941.6452-1.65443.9990.1566-0.1103-0.54460.3114-0.1042-0.05930.23880.8149-0.05240.24490.09030.02920.2888-0.14130.67330.9815-45.776553.6158
41.6979-0.8808-1.68860.50810.56194.737-0.07080.1878-0.10720.1079-0.0830.1117-0.5058-0.18770.15370.2307-0.10590.10540.1488-0.05690.268319.9504-22.924947.3596
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A3 - 501
2X-RAY DIFFRACTION2B3 - 501
3X-RAY DIFFRACTION3C61 - 501
4X-RAY DIFFRACTION4D61 - 501

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