Diffraction images of high-pressure cryocooled crystals prepared by three hydration methods. (a) Oil-coating method (Fig. 1a). The image shows diffuse scattering at around 5.5 Å from oil. Crystal mosaicity is 0.67°. (b) Capillary-hydration method (Fig. 1b). The innermost diffuse ring (∼5.0 Å) is generated from the polycarbonate capillary, and the second and third diffuse rings are from the solvent (HDA ice) inside the capillary. Crystal mosaicity is 0.34°. (c) Capillary-shielding method, but without removing the polyester capillary (Fig. 1d). Although the scattering from the polyester capillary is present (anisotropic peak at ∼5.0 Å), the background level is dramatically reduced compared to the oil-coating and capillary-hydration methods. Crystal mosaicity is 0.11°. (d) Capillary shielding with removal of the polyester capillary (Fig. 1e). The image shows faint diffuse scattering around 3.0 Å, which is from the small amount of external and internal solvent (HDA ice) in and around the crystal. Note that the level of diffuse scattering from the solvent is comparable to the air scattering around the beam stop. Crystal mosaicity is 0.12°. The slight increase in unit-cell dimensions and mosaicity from 0.11° (see Table 1) is due to X-ray radiation damage (Ravelli & McSweeney, 2000). (e) Crystal equilibrated to 10%(v/v) glycerol in deionized water (Fig. 1f), then prepared by the capillary-shielding method; the polyester capillary was removed before data collection. The diffuse scattering from the solvent (HDA ice) is shifted to 3.3 Å owing to the addition of glycerol and removal of NaK tartrate. Crystal mosaicity is 0.08°. (f) Diffraction image of the crystal after warming to room temperature and being refrozen to 100 K in the N2 gas cold stream at the beamline at ambient pressure (Fig. 1g). It shows strong diffraction rings from hexagonal ice formation and very poor quality diffraction from the protein crystal. The result indicates that the crystal was kept hydrated during the capillary-shielding method.