TECHNOTES
Reducing Background Scatter and Maximizing 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.