Learn Technotes Reducing Background Scatter and Maximizing Diffraction Signal to Noise

TECHNOTES
Reducing Background Scatter and Maximizing Diffraction Signal to Noise

Methods to minimize the diffuse background and maximize your diffraction signal to noise

The diffuse background intensity in a diffraction pattern comes from many sources, both inside and outside the crystal, including:

  • Internal and external solvent.
  • Thermal motions of the protein and hydration water (thermal diffuse scatter).
  • Static crystal disorder (e.g., associated with flexible loop regions or poor packing).
  • The mount holding the crystal.
  • Air along the beam path between source and detector.

How can you minimize the diffuse background and maximize your diffraction signal to noise?

  • Use MicroMounts™ and/or our other sample mounting products in standard (not Dual) thickness to obtain the lowest possible scatter from your mount.
  • Minimize the amount of liquid around your crystal. Use a mount with an aperture that’s a bit smaller than the crystal, and carefully remove excess liquid using a paper wick or by gently tapping on the steel rod of the mount.
  • Use an X-ray beam that is smaller than your sample, to minimize the amount of scatter from surrounding liquid and the mount.
  • Orient your crystal on the mount to minimize data collection at angles that place the plane of the mount along (instead of perpendicular to) the X-ray beam.
  • Flash cool your crystal and collect data at T=100 K.
  • Dehydrate the crystal to reduce diffuse scatter from internal water, using the MicroRT™ system.
  • Use a larger crystal to maximize the ratio of sample volume to liquid, mount and air volume within the X-ray beam.
  • Use a helium filled bag or chamber to reduce scatter in the gas along the beam path.

 

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