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- PDB-2a63: Solution structure of a stably monomeric mutant of lambda Cro pro... -

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Basic information

Entry
Database: PDB / ID: 2a63
TitleSolution structure of a stably monomeric mutant of lambda Cro produced by substitutions in the ball-and-socket interface
ComponentsRegulatory protein cro
KeywordsVIRAL PROTEIN / helix-turn-helix / monomer / ball-and-socket
Function / homology
Function and homology information


latency-replication decision / release from viral latency / negative regulation of transcription by competitive promoter binding / negative regulation of viral transcription / 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
Regulatory protein cro
Similarity search - Component
Biological speciesEnterobacteria phage lambda (virus)
MethodSOLUTION NMR / simulated annealing
AuthorsNewlove, T. / Atkinson, K.R. / Van Dorn, L.O. / Cordes, M.H.
CitationJournal: Biochemistry / Year: 2006
Title: A Trade between Similar but Nonequivalent Intrasubunit and Intersubunit Contacts in Cro Dimer Evolution.
Authors: Newlove, T. / Atkinson, K.R. / Van Dorn, L.O. / Cordes, M.H.
History
DepositionJul 1, 2005Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 6, 2006Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 20, 2021Group: Data collection / Database references / Derived calculations
Category: database_2 / pdbx_nmr_software ...database_2 / pdbx_nmr_software / pdbx_struct_assembly / pdbx_struct_oper_list / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_software.name / _struct_ref_seq_dif.details
Revision 1.4May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Regulatory protein cro


Theoretical massNumber of molelcules
Total (without water)7,4231
Polymers7,4231
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 40all structures compatible with experimental restraints; structures chosen had the lowest energies and/or best agreement to J(NHB) data not used in explicit restraints
RepresentativeModel #1closest to the average

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Components

#1: Protein Regulatory protein cro


Mass: 7423.485 Da / Num. of mol.: 1 / Mutation: A33W, F58D, Y26Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage lambda (virus) / Genus: Lambda-like viruses / Gene: cro / Plasmid: pET21b / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 DE3 / References: UniProt: P03040

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1123D 15N-separated NOESY
122HNHA
132HNHB
144HSQC (amide hydrogen exchange)
1513D 13C-separated NOESY
1632D NOESY
2732D NOESY
3832D NOESY
1942D NOESY
NMR detailsText: Proton chemical shifts submitted with this deposition were referenced to TSP at 0.00 ppm. However, we should add a cautionary note that this referencing led to an unusually high value for the ...Text: Proton chemical shifts submitted with this deposition were referenced to TSP at 0.00 ppm. However, we should add a cautionary note that this referencing led to an unusually high value for the water signal of 4.966 at 293 K, pH 5.3. We suspect that the TSP resonance is shifted below 0 ppm in our samples, possibly as much as -0.15 ppm. In our view, this is probably due to some transient interaction of the standard with the protein.

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Sample preparation

Details
Solution-IDContentsSolvent system
12.5 mM lambda Cro A33W/F58D/Y26Q U-13C, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP90% H2O/10% D2O
25 mM lambda Cro A33W/F58D/Y26Q U-15N, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP90% H2O/10% D2O
35 mM lambda Cro A33W/F58D/Y26Q unlabelled, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP90% H2O/10% D2O
45 mM lambda Cro A33W/F58D/Y26Q U-15N, 50mM Na-phosphate, 100% D2O, 0.01% sodium azide, 1 mM TSP100% D2O
Sample conditions
Conditions-IDIonic strengthpHPressure (kPa)Temperature (K)
1no salt added 5.3ambient 293 K
2no salt added 6.1ambient 293 K
3no salt added 6.1ambient 298 K

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NMR measurement

RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M
Radiation wavelengthRelative weight: 1
NMR spectrometerType: Bruker DRX / Manufacturer: Bruker / Model: DRX / Field strength: 600 MHz

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Processing

NMR software
NameVersionDeveloperClassification
XwinNMR3.1Brukercollection
NMRPipe1.8Frank Delaglio, Stephan Grzesiek, Ad Bax, Guang Zhu, Geerten Vuister, John Pfeiferprocessing
NMRView4.1.3Bruce Johnstondata analysis
CNS1.1structure solution
CNS1.1refinement
RefinementMethod: simulated annealing / Software ordinal: 1
Details: 40 structures were calculated using 838 noe-derived restraints, 14 hydrogen bond distance restraints, 50 phi angle restraints and 17 chi1 angle restraints. All 40 calculations converged to ...Details: 40 structures were calculated using 838 noe-derived restraints, 14 hydrogen bond distance restraints, 50 phi angle restraints and 17 chi1 angle restraints. All 40 calculations converged to structures with no noe violations > 0.5 angstroms and no dihedral angle restraint violations >5 degrees. 14 of the 40 structures were discarded based on incompatibility of the rotamer of val 55 with a small J(HNHB) coupling constant, though no explicit restraint on the chi1 angle was included in the calculation. Of the 26 remaining structures, the 6 with the highest energy were discarded. In addition to having high energies, these 6 structures showed unusual conformations, particularly in turn regions, that were unreasonable and in strong disagreement with previously published structures of lambda Cro variants. The final ensemble contains 20 members. The ordered region of the protein extends from approximately residue 3 to residue 55. Pairwise RMSDs for the ordered region were 0.66 A (backbone atoms) and 1.29 A (all heavy atoms). None of the backbone angles in the ordered region of any ensemble member fell outside the most favorable and additionally allowed regions of a ramachandran plot.
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: all structures compatible with experimental restraints; structures chosen had the lowest energies and/or best agreement to J(NHB) data not used in explicit restraints
Conformers calculated total number: 40 / Conformers submitted total number: 20

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