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Related Experiment Videos

Crystallization of ccdB

M H Dao-Thi1, L Wyns, F Poortmans

  • 1Laboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituut Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium.

Acta Crystallographica. Section D, Biological Crystallography
|October 3, 1998
PubMed
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Researchers optimized crystallization of the CcdB protein, a DNA gyrase poison, yielding a novel crystal form. This new form enables structure determination via the multiple isomorphous replacement (MIR) method, crucial for understanding protein function.

Area of Science:

  • Structural Biology
  • Protein Crystallography
  • Molecular Biology

Background:

  • CcdB protein is a well-known inhibitor of DNA gyrase (topoisomerase II).
  • Previous CcdB crystal forms were unsuitable for detailed structural analysis using methods like MIR.
  • Understanding CcdB's structure is key to its mechanism of action.

Purpose of the Study:

  • To investigate and optimize crystallization conditions for CcdB protein.
  • To obtain a novel crystal form of CcdB suitable for structure determination.
  • To characterize the crystallographic properties of a CcdB double mutant.

Main Methods:

  • Systematic screening of crystallization parameters (precipitant type, concentration, pH, protein concentration).
  • X-ray diffraction analysis of novel crystal forms.

Related Experiment Videos

  • Determination of space group and unit-cell parameters.
  • Analysis of diffraction resolution and data quality.

    • Main Results:

    • A novel CcdB crystal form in space group C2 was obtained, suitable for MIR.
    • Unit-cell parameters for the new form: a = 74.94, b = 36.24, c = 35.77 Å, β = 115.27°.
    • High-resolution diffraction data (up to 1.5 Å) were achieved with flash-frozen crystals.
    • A CcdB double mutant (S74C/G77Q) crystallized in space group I222, diffracting to 2.5 Å.

    Conclusions:

    • The identified novel crystal form significantly advances the potential for CcdB structure determination.
    • The crystallographic data obtained provide a foundation for future structural studies of CcdB.
    • The characterization of the double mutant offers insights into protein engineering for crystallography.