Procedure 7 2D Crystals of PSI Synechococcus sp OD 24

Trimer PSI complexes were isolated from the thermophilic cyanobacterium Synechococcus sp. OD 24 as described in Reference 12.

1. Change the detergent Triton X-100 by polyethyleneglycol (PEG) 6000/MgCl2 precipitation.

2. Repeat this step 3 times.

3. Resuspend the precipitated PSI in 10 mM HEPES, pH 7.0, containing 0.5% OTG to a protein concentration of 2 mg/mL.

4. Add DMPC solution to achieve a LPR of 1.

5. Dialyze the mixture against a detergent-free buffer containing 25 mM ammonium ferric citrate.

Dialysis cell temperature: 26°C for 24 hours; increase to 37°C over 12 hours; 37°C for 24 hours; and decrease to 26°C over 10 hours. Total dialysis time is 70 hours.

Digital image processing of negatively stained and frozen-hydrated specimens prepared using Procedure 7 revealed ortho-rhombic crystals with unit cell dimensions a = 13.8 nm, b = 14.5 nm, and p12i symmetry. The same procedure has also been used with trimeric PSI isolated from mesophilic cyanobacterium Synechococcus PCC 7002 by isoelectric focusing (46) to provide 2D crystals (Tsiotis, unpublished results).

Purple sulfur and nonsulfur bacteria possess membrane-bound LH complexes, which serve to transfer energy to the RC, where charge separation occurs. LH complex (170 pg), isolated from a carotenoid-less mutant of the purple nonsulfur bacterium Rhodospirillum rubrum G9 as described previously (13), was dissolved in 100 pL of 50 mM NH4HCO3, 1% octyl-

P-glucoside (OG), pH 7.8. After dialysis against buffer containing 0.8% OG, 5 mM MgCl2, and 50 mM NaCl in the dark at 4°C for 5 days, 2D crystals were obtained, which had a hexagonal pattern with a lattice constant of 12.3 nm. Modification of this procedure, by the use of sonicated vesicles of dioleoyl-9-10 phosphatidylcholine lipid solubilized in OG in a ratio 1:1 to proteins, allowed the formation of crystals which diffracted at 8.5 A (23). Digital image processing of frozen-hydrated specimens revealed crystals with a p22121 symmetry and unit cell with dimensions of a = 12.8 nm, b = 19.4 nm.

As an alternative to the dialysis apparatus shown in Figure 2, an inexpensive mi-crodialysis arrangement with Eppendorf® tubes or dialysis buttons may be used (Figure 3). This method enables the dialysis of small volumes (<50 pL) of protein—lipid— detergent. Another interesting microdialy-sis device using a bent glass capillary tube, shown in Figure 4 has been described by Kuhlbrandt (26). A glass tube of 2.5 mm inner diameter and about 6.5 mm outer diameter is bent by 90° near to one end. The end is melted into a smooth surface, which forms a tight seal with the dialysis

Figure 2. Continuous dialysis system for the growing of 2D crystals.

membrane. The dialysis membrane is fixed with a ring of silicon tubing, and 20 to 50 mL dialysate is fed into the capillary from the open end using a syringe (Figure 4). The dialysate is shaken down against the dialysis membrane to remove air bubbles, and the device is placed in a glass beaker with dialysis buffer. One interesting advantage of this device is that a sample for electron microscopy can be taken at anytime with a finer glass capillary without disturbing the progress of the experiment.

Figure 3. An Eppendorf cap for the growing of 2D crystals by microdialysis.
Figure 4. Glass capillary tube for the growing of 2D crystals by microdialysis.
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