1 Crystallisation and Derivatisation
Chemicals:   Hen egg-white lysozyme (M ~ 14600 g/mol, Fluka cat. no. 62970) CH3COONa (M = 82.03 g/mol, Sigma cat. no. S2889) CH3COOH (M = 60.0 g/mol, Sigma cat. no. 537020) NaCl (M = 58.44 g/mol, Sigma cat. no. S7653) Ethylene glycol (M = 62.07 g/mol, Merck, cat. no. 109621) KAuCl4 (M = 377.88 g/mol, Aldrich, cat. no. 33,454-5) Milli-Q water
Tetragonal crystals of HEWL were grown as described by Weiss et al. (2000) by mixing 4 ul of protein solution (30 mg/ml in water) and 4 ul of reservoir solution containing 50 mM Na acetate pH 4.5 and 5% (w/v) NaCl and equilibrating the drop against the reservoir. The crystals belong to space group P4s2i2 (space group number 96) and exhibit the usual unit-cell parameters of a = 78.8 A and c = 37.2 A (Figure 3). They appeared within few days after setting up the experiment. Prior to flash cooling to 100 K, they were transferred into a solution containing 25% (v/v) ethylene glycol, 10% (w/v) NaCl and 100 mM Na acetate pH 4.5. They typically diffracted X-rays to a resolution better than 1.6 A.
	c Si
300^m	300^m
Figure 3: Tetragonal HEWL crystals.
A 10 mM solution of KAuCU in reservoir solution was freshly prepared and one crystal was soaked in this solution for 1 minute (Sun et al., 2002). This crystal was then also cryo-protected in a solution containing 25% (v/v) ethylene glycol, 10% (w/v) NaCl and 100 mM Na acetate pH 4.5. The diffraction properties of such derivatized crystals are significantly worse than the ones for the native crystals but still very much acceptable (Figure 4).