In meso Approaches to Membrane Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 272

The authors of this approach to membrane protein crystallization
(Takeda et al ., 1998) started with the preparation of BR vesicles as
described in Kouyama et al . (1994) and Denkov et al . (1998) and then
used a standard vapor diffusion method to obtain BR crystals.
Crystallization of BR from vesicles from PM can be described as follows:

(i) Resuspend the pellet of PM (preliminarily centrifuged 4000 g for

20 minutes) in a Tween 20 (0.3%) buffer (10 mM HEPES, pH 8.0,
160 mM NaCl) until a final concentration of 1 mg/ml.

(ii) C.

(iii) Centrifuge immediately for 10 minutes at 16 000 g.
(iv) Resuspend the pellet in a buffer for vesicle preparations (e.g. 10 mM
HEPES, pH 8.0, 160 mM NaCl, 0.04% NaN 3 ).
(v) Repeat the washing procedure 3-4 times from Step (ii).
(vi) Dilute PM (10 mg/ml) with a double concentrated solution of deter-
gent to a final concentration of 5 mg/ml of PM, 1-4 mg/ml of OG,
0.6-1.2 M of (NH 4 ) 2 SO 4 .
(vii) Incubate for 1-2 weeks at 32

Incubate for 20 minutes at 20 °

C.
(viii) Centrifuge for 10 minutes at 4000 g.
(ix)

°

Save supernatant and concentrate at 4 ° C.

(x) For crystallization, the protein sample is equilibrated against 80 mM

of sodium citrate (pH 5.2) buffer containing 1.9-2.2 M of (NH 4 ) 2 SO 4
in a reservoir by the standard vapor diffusion method.

(xi) Leave the crystallization batch at 4 ° C. The first crystals appear

within a week.

A birefringent hexagonal crystal has been obtained diffracting X-rays
beyond 2.5 Å resolution. This new crystal belongs to the space group
P622 with unit cell dimensions of a 114.1 Å. The

highest announced structural resolution achieved by this method is 2.3 Å.
Such an experiment is illustrated schematically in Fig. 6. A number of
experiments have been carried out in our laboratories to optimize the
method (Golubev et al .; Volkov et al ., unpublished). It has been shown

= b = 104.7 Å and c =