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- PDB-3orc: CRYSTAL STRUCTURE OF AN ENGINEERED CRO MONOMER BOUND NONSPECIFICA... -

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Basic information

Entry
Database: PDB / ID: 3orc
TitleCRYSTAL STRUCTURE OF AN ENGINEERED CRO MONOMER BOUND NONSPECIFICALLY TO DNA
Components
  • DNA (5'-D(*TP*AP*TP*CP*GP*AP*TP*A)-3')
  • PROTEIN (CRO REPRESSOR)
KeywordsGENE REGULATION/DNA / CRO / PROTEIN-DNA INTERACTION / BACTERIOPHAGE LAMBDA / REPRESSOR / MONOMER-DIMER / HELIX-TURN-HELIX / COMPLEX (GENE REGULATING PROTEIN-DNA) / GENE REGULATION-DNA COMPLEX
Function / homology
Function and homology information


latency-replication decision / release from viral latency / negative regulation of viral transcription / negative regulation of transcription by competitive promoter binding / core promoter sequence-specific DNA binding / response to UV / protein homodimerization activity / DNA binding
Similarity search - Function
CRO Repressor / Regulatory protein cro superfamily / Cro / Regulatory protein cro / CRO Repressor / Lambda repressor-like, DNA-binding domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
DNA / Regulatory protein cro
Similarity search - Component
Biological speciesEnterobacteria phage lambda (virus)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsAlbright, R.A. / Mossing, M.C. / Matthews, B.W.
Citation
Journal: Protein Sci. / Year: 1998
Title: Crystal structure of an engineered Cro monomer bound nonspecifically to DNA: possible implications for nonspecific binding by the wild-type protein.
Authors: Albright, R.A. / Mossing, M.C. / Matthews, B.W.
#1: Journal: J.Mol.Biol. / Year: 1998
Title: Refined Structure of Cro Repressor Protein from Bacteriophage Lambda Suggests Both Flexibility and Plasticity
Authors: Ohlendorf, D.H. / Tronrud, D.E. / Matthews, B.W.
#2: Journal: J.Mol.Biol. / Year: 1998
Title: Crystal Structure of Lambda-Cro Bound to a Consensus Operator at 3.0 A Resolution
Authors: Albright, R.A. / Matthews, B.W.
#3: Journal: Biochemistry / Year: 1996
Title: High-Resolution Structure of an Engineered Cro Monomer Shows Changes in Conformation Relative to the Native Dimer
Authors: Albright, R.A. / Mossing, M.C. / Matthews, B.W.
#4: Journal: Science / Year: 1990
Title: Stable, Monomeric Variants of Lambda Cro Obtained by Insertion of a Designed Beta-Hairpin Sequence
Authors: Mossing, M.C. / Sauer, R.T.
History
DepositionApr 23, 1998Deposition site: BNL / Processing site: NDB
Revision 1.0Dec 2, 1998Provider: repository / Type: Initial release
Revision 1.1May 22, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Nov 29, 2017Group: Derived calculations / Category: struct_conf / struct_conf_type
Revision 1.4Aug 2, 2023Group: Database references / Refinement description / Category: database_2 / pdbx_initial_refinement_model
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
R: DNA (5'-D(*TP*AP*TP*CP*GP*AP*TP*A)-3')
S: DNA (5'-D(*TP*AP*TP*CP*GP*AP*TP*A)-3')
A: PROTEIN (CRO REPRESSOR)


Theoretical massNumber of molelcules
Total (without water)12,0933
Polymers12,0933
Non-polymers00
Water543
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)45.720, 60.660, 45.720
Angle α, β, γ (deg.)90.00, 112.70, 90.00
Int Tables number5
Cell settingmonoclinic
Space group name H-MC121
DetailsWILDTYPE CRO DOES NOT FORM STABLE MONOMERS. THE ENGINEERED MONOMER PRESENTED HERE CONTAINS A 5-RESIDUE INSERTION [DGEVK] FOLLOWING K 56. THE FIRST 2 RESIDUES OF THIS INSERTION ALLOW THE FORMATION OF A BETA-TURN. THE REMAINING 3 RESIDUES MIMIC RESIDUES OF THE WILDTYPE DIMER INTERFACE, ALLOWING STABILIZING INTERACTIONS TO BE MAINTAINED. THE OVERALL STRUCTURE OF THIS MONOMER IS QUITE SIMILAR TO A SUBUNIT OF THE WILDTYPE DIMER.

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Components

#1: DNA chain DNA (5'-D(*TP*AP*TP*CP*GP*AP*TP*A)-3')


Mass: 2425.629 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: 50% OCCUPANCY IN THE OUTERMOST POSITIONS
#2: Protein PROTEIN (CRO REPRESSOR)


Mass: 7242.207 Da / Num. of mol.: 1 / Mutation: INSERTION (K56-DGEVK)
Source method: isolated from a genetically manipulated source
Details: WILDTYPE CRO DOES NOT FORM STABLE MONOMERS. THE ENGINEERED-MONOMER PRESENTED HERE CONTAINS A 5-RESIDUE INSERTION [DGEVK] FOLLOWING K 56. THE FIRST 2 RESIDUES OF THIS INSERTION ALLOW THE ...Details: WILDTYPE CRO DOES NOT FORM STABLE MONOMERS. THE ENGINEERED-MONOMER PRESENTED HERE CONTAINS A 5-RESIDUE INSERTION [DGEVK] FOLLOWING K 56. THE FIRST 2 RESIDUES OF THIS INSERTION ALLOW THE FORMATION OF A BETA-TURN. THE REMAINING 3 RESIDUES MIMIC RESIDUES OF THE WILDTYPE DIMER INTERFACE, ALLOWING STABILIZING INTERACTIONS TO BE MAINTAINED. THE OVERALL STRUCTURE OF THIS MONOMER IS QUITE SIMILAR TO A SUBUNIT OF THE WILDTYPE DIMER.
Source: (gene. exp.) Enterobacteria phage lambda (virus) / Genus: Lambda-like viruses / Plasmid: PUCRO.MDG / Gene (production host): CRO MUTANT K56-[DGEVK] / Production host: Escherichia coli (E. coli) / Strain (production host): X90 / References: UniProt: P03040
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / 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: 3 Å3/Da / Density % sol: 48.45 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: COCRYSTALS WERE OBTAINED BY MIXING A 1.5 MOLAR EXCESS OF THE 7BP DNA FRAGMENT WITH CRO K56-[DGEVK], COMBINING WITH AN EQUAL VOLUME OF PRECIPITANT BUFFER (140MM AMMONIUM ACETATE, 31% PEG ...Details: COCRYSTALS WERE OBTAINED BY MIXING A 1.5 MOLAR EXCESS OF THE 7BP DNA FRAGMENT WITH CRO K56-[DGEVK], COMBINING WITH AN EQUAL VOLUME OF PRECIPITANT BUFFER (140MM AMMONIUM ACETATE, 31% PEG 3350, 100MM ACETATE BUFFER PH 4.6), THEN EQUILIBRATING AGAINST THE PRECIPITANT BUFFER VIA THE HANGING-DROP METHOD AT ROOM TEMPERATURE., vapor diffusion - hanging drop, temperature 293K
Components of the solutions
IDNameCrystal-IDSol-ID
1AMMONIUM ACETATE11
2PEG 335011
3ACETATE BUFFER11
4AMMONIUM ACETATE12
5PEG 335012
6ACETATE BUFFER12
Crystal grow
*PLUS
Details: drop solution was mixed with an equal volume of precipitant
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
116 mg/mlprotein1drop
220 mM1dropK2HPO4
30.1 mMEDTA1drop
413 mg/mlDNA duplex1drop
580-200 mMammonium acetate1reservoirprecipitant
629-33 %PEG33501reservoirprecipitant
7100 mMacetate1reservoirprecipitant

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Data collection

DiffractionMean temperature: 295 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418
DetectorType: SDMS / Detector: AREA DETECTOR / Date: Feb 1, 1993 / Details: COLLIMATOR
RadiationMonochromator: GRAPHITE CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 3→20 Å / Num. obs: 2452 / % possible obs: 99.7 % / Redundancy: 5.3 % / Biso Wilson estimate: 51.1 Å2 / Rsym value: 0.053
Reflection
*PLUS
Num. measured all: 13041 / Rmerge(I) obs: 0.053

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Processing

Software
NameVersionClassification
EDPDB(ZHANG)model building
TNT5EBrefinement
SDMSdata reduction
SDMSdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1ORC

1orc


Resolution: 3→20 Å
Isotropic thermal model: TNT BCORREL (MODIFIED TO INCLUDE DNA)
σ(F): 0 / σ(I): 13041
Stereochemistry target values: TNT PROTGEO (MODIFIED TO INCLUDE DNA)
Num. reflection% reflection
all13041 -
obs2452 99.7 %
Solvent computationSolvent model: BABINET / Bsol: 160 Å2 / ksol: 0.6 e/Å3
Refinement stepCycle: LAST / Resolution: 3→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms501 322 0 3 826
Refine LS restraints
Refine-IDTypeDev idealNumberWeight
X-RAY DIFFRACTIONt_bond_d0.0156890.38
X-RAY DIFFRACTIONt_angle_deg2.6569590.66
X-RAY DIFFRACTIONt_dihedral_angle_d19.553990.7
X-RAY DIFFRACTIONt_incorr_chiral_ct0
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes0.006131.5
X-RAY DIFFRACTIONt_gen_planes0.013822
X-RAY DIFFRACTIONt_it2.5487074
X-RAY DIFFRACTIONt_nbd0.0185110
Software
*PLUS
Name: TNT / Version: 5EB / Classification: refinement
Refinement
*PLUS
Rfactor obs: 0.224
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev idealWeight
X-RAY DIFFRACTIONt_dihedral_angle_d
X-RAY DIFFRACTIONt_dihedral_angle_deg19.550.7
X-RAY DIFFRACTIONt_plane_restr0.0132

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