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Related Experiment Videos

Going soft and SAD with manganese.

Paula S Salgado1, Martin A Walsh, Minni R L Laurila

  • 1Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN, England.

Acta Crystallographica. Section D, Biological Crystallography
|December 21, 2004
PubMed
Summary

Manganese phasing, using lighter atoms and softer X-rays, is now practical for solving large protein structures. This study demonstrates its effectiveness for RNA-dependent RNA polymerase structure determination.

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Area of Science:

  • Structural biology
  • X-ray crystallography
  • Protein structure determination

Background:

  • Single-wavelength anomalous diffraction (SAD) phasing is crucial for determining protein structures.
  • Traditionally, heavier atoms were used for anomalous signal, limiting applications.
  • Recent advancements explore lighter atoms and softer X-rays for SAD phasing.

Purpose of the Study:

  • To investigate the feasibility of manganese phasing for large protein structure determination.
  • To showcase the application of SAD phasing with unconventional anomalous scatterers.

Main Methods:

  • Collected X-ray diffraction data for bacteriophage phi6 RNA-dependent RNA polymerase at 1.89 A wavelength.
  • Utilized SHELXD and SOLVE software to locate manganese atoms despite a low anomalous signal (1.2%).

Related Experiment Videos

  • Employed SOLVE/RESOLVE for SAD phase calculation and automated model building.

    • Main Results:

    • Successfully located three manganese atoms per asymmetric unit.
    • Obtained SAD phases and built a partial protein model, revealing the right-hand polymerase motif.
    • Demonstrated the utility of manganese phasing for a 75 kDa protein.

    Conclusions:

    • Manganese phasing is a practical method for solving large protein structures.
    • Modern synchrotron facilities and software enhance the capabilities of SAD phasing.
    • This technique expands the scope of X-ray crystallography for complex biological macromolecules.