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- PDB-4wls: Crystal structure of the metal-free (repressor) form of E. Coli C... -

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Basic information

Entry
Database: PDB / ID: 4wls
TitleCrystal structure of the metal-free (repressor) form of E. Coli CUER, a copper efflux regulator, bound to COPA promoter DNA
Components
  • (COPA PROMOTER DNA NON-TEMPLATE ...) x 2
  • (COPA PROMOTER DNA TEMPLATE ...) x 2
  • HTH-type transcriptional regulator CueR
KeywordsTranscription Regulator/DNA / PROTEIN-DNA COMPLEX / MERR-FAMILY TRANSCRIPTION REGULATOR / METAL-FREE FORM / REPRESSOR / TRANSCRIPTION / Transcription Regulator-DNA complex
Function / homology
Function and homology information


DNA-binding transcription activator activity / cis-regulatory region sequence-specific DNA binding / protein-DNA complex / transcription cis-regulatory region binding / copper ion binding / DNA-binding transcription factor activity / DNA-templated transcription / positive regulation of DNA-templated transcription / identical protein binding / cytoplasm
Similarity search - Function
Cu(I)-responsive transcriptional regulator / MerR, DNA binding / Multidrug-efflux Transporter Regulator; Chain: A; Domain 2 - #10 / MerR family regulatory protein / Multidrug-efflux Transporter Regulator; Chain: A; Domain 2 / MerR-type HTH domain signature. / : / MerR HTH family regulatory protein / MerR-type HTH domain profile. / helix_turn_helix, mercury resistance ...Cu(I)-responsive transcriptional regulator / MerR, DNA binding / Multidrug-efflux Transporter Regulator; Chain: A; Domain 2 - #10 / MerR family regulatory protein / Multidrug-efflux Transporter Regulator; Chain: A; Domain 2 / MerR-type HTH domain signature. / : / MerR HTH family regulatory protein / MerR-type HTH domain profile. / helix_turn_helix, mercury resistance / MerR-type HTH domain / Putative DNA-binding domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA / DNA (> 10) / HTH-type transcriptional regulator CueR
Similarity search - Component
Biological speciesEscherichia coli DH5[alpha] (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.105 Å
AuthorsPhilips, S.J. / Canalizo-Hernandez, M. / Mondragon, A. / O'Halloran, T.V.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM038784-26 United States
CitationJournal: Science / Year: 2015
Title: Allosteric transcriptional regulation via changes in the overall topology of the core promoter.
Authors: Philips, S.J. / Canalizo-Hernandez, M. / Yildirim, I. / Schatz, G.C. / Mondragon, A. / O'Halloran, T.V.
History
DepositionOct 8, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 2, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2017Group: Author supporting evidence / Database references / Derived calculations
Category: citation / pdbx_audit_support / pdbx_struct_oper_list
Item: _citation.journal_id_CSD / _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 27, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: HTH-type transcriptional regulator CueR
B: HTH-type transcriptional regulator CueR
X: COPA PROMOTER DNA NON-TEMPLATE STRAND
Y: COPA PROMOTER DNA TEMPLATE STRAND
U: COPA PROMOTER DNA TEMPLATE STRAND (ALTERNATE CONFORMATION)
V: COPA PROMOTER DNA NON-TEMPLATE STRAND (ALTERNATE CONFORMATION)


Theoretical massNumber of molelcules
Total (without water)61,1446
Polymers61,1446
Non-polymers00
Water41423
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)52.194, 63.653, 130.205
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
DetailsChain U and V are alternate conformation of chain Y and X.

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Components

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COPA PROMOTER DNA NON-TEMPLATE ... , 2 types, 2 molecules XV

#2: DNA chain COPA PROMOTER DNA NON-TEMPLATE STRAND


Mass: 7945.113 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain COPA PROMOTER DNA NON-TEMPLATE STRAND (ALTERNATE CONFORMATION)


Mass: 7936.100 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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COPA PROMOTER DNA TEMPLATE ... , 2 types, 2 molecules YU

#3: DNA chain COPA PROMOTER DNA TEMPLATE STRAND


Mass: 8030.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain COPA PROMOTER DNA TEMPLATE STRAND (ALTERNATE CONFORMATION)


Mass: 8039.222 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / Non-polymers , 2 types, 25 molecules AB

#1: Protein HTH-type transcriptional regulator CueR / Copper efflux regulator / Copper export regulator


Mass: 14596.817 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli DH5[alpha] (bacteria) / Strain: DH5alpha / Fragment: RESIDUES 1-128 / Gene: cueR, ybbI, b0487, JW0476 / Plasmid: PET24A / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P0A9G4
#6: Water ChemComp-HOH / water / Water


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

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Details

Sequence detailsThe crystallized DNA contains a pseudo-palyndromic region and has the following sequence: 5'- ...The crystallized DNA contains a pseudo-palyndromic region and has the following sequence: 5'- TGACCTTCCCCTTGCTGGAAGGTTTA -3', 3'- ACTGGAAGGGGAACGACCTTCCAAAT -5'. The two conformation A and B result from the alternate DNA conformations observed in the crystals. The pseudo-palyndrome sequence is ACCTTCC:GGAAGGT. Two orientations of the DNA were observed in the crystal. Chain X and Y in conformer A represent the the original sequences of the pseudo-palyndrome. Chain U and V in conformer B represent the alternate DNA conformation. We showed this to be the fact by using Bromine-subsituted bases in the top strand only. The results of which showed Br density peaks in both strands, leading to the alternate conformation of several bases. The refinements were performed, with Phenix, with both alternates present. the pseudo-palindrome in the sequence is: 5'-nnACCTTCCnnnnnnnGGAAGGTnnn-3', 3'-nnTGGAAGGnnnnnnnCCTTCCAnnn-5'. The Bromine peaks (seen on both strands, even though only the top strand, chain X, had Bromines) guided us on how to build the alternate chain which is: U: 5'-AAACCTTCCAGCAAGGGGAAGGTCA-3', V: 3'-TTTGGAAGGTCGTTCCCCTTCCAGT-5'.

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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: 2.41 Å3/Da / Density % sol: 49.02 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.2M HEPES, PH 7.5, 0.1M CACL2 AND 16% PEG4000, VAPOR DIFFUSION, HANGING DROP TEMPERATURE 295K
PH range: 7.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 0.97853 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Jun 11, 2012
RadiationMonochromator: SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97853 Å / Relative weight: 1
ReflectionResolution: 2.1→52.19 Å / Num. obs: 25034 / % possible obs: 97.2 % / Observed criterion σ(I): 6 / Redundancy: 4.1 % / Rmerge(I) obs: 0.084 / Net I/σ(I): 8.7
Reflection shellResolution: 2.1→2.22 Å / Redundancy: 4.1 % / Rmerge(I) obs: 0.402 / Mean I/σ(I) obs: 2.6 / % possible all: 97.3

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Processing

Software
NameVersionClassification
XDSdata reduction
SCALAdata scaling
PHENIXphasing
PHENIX(PHENIX.REFINE: 1.9_1692)refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: XXXX

Resolution: 2.105→45.513 Å / SU ML: 0.3 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 26.89
RfactorNum. reflection% reflection
Rfree0.2638 1275 5.1 %
Rwork0.2115 --
obs0.2142 24991 96.59 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.105→45.513 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1794 2120 0 23 3937
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.013558
X-RAY DIFFRACTIONf_angle_d1.0875133
X-RAY DIFFRACTIONf_dihedral_angle_d26.6731487
X-RAY DIFFRACTIONf_chiral_restr0.049576
X-RAY DIFFRACTIONf_plane_restr0.005408
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1053-2.18960.31881440.26822594X-RAY DIFFRACTION97
2.1896-2.28920.29531280.26432662X-RAY DIFFRACTION98
2.2892-2.40990.3241510.25072620X-RAY DIFFRACTION98
2.4099-2.56090.33371460.25772611X-RAY DIFFRACTION97
2.5609-2.75860.30471530.25352622X-RAY DIFFRACTION97
2.7586-3.03620.29551440.2482629X-RAY DIFFRACTION97
3.0362-3.47540.25751230.22062647X-RAY DIFFRACTION96
3.4754-4.37810.24231470.17772640X-RAY DIFFRACTION95
4.3781-45.52390.21281390.17542691X-RAY DIFFRACTION93

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