(Z direction) such as described in [2, 3, 6, 22, 23, 30], and, under
special circumstances of ordered phospholipids [6], conclusions on
the importance of the phospholipids to the protein structure and
function may be drawn. The 3D maps of samples in different
conformational states such as bacteriorhodopsin [24, 26] can reveal
substantial information on the detailed reaction mechanism.
4 Notes
1. Lipid stock concentrations of 1 and 10 mg/mL are useful for
many lipid-to-protein ratios used during 2D crystallization
trials. DMPC has been successfully used for 2D crystallization
of a large percentage of membrane proteins. Lipid in chloroform is preferable due to the hygroscopic nature of lipids in
powder form.
2. The stream of nitrogen gas is slowly increased to avoid splashing and drying of the lipid on the sides of the round-bottom
flask.
3. Lipid aliquots for 2D crystallization trials can frequently be
stored for 1–2 years, if not longer.
4. Approximately 4–8 crystallization conditions often provide
sufficient and critical information to optimize parameters for
the next experiment. It is important to note that simultaneous
testing of a larger number of crystallization conditions will
require a substantial amount of protein as well as many hours,
if not days, of TEM time, which is often not available in one
large time block. Thus, it is more efficient to set up fewer
crystallization conditions on a frequent basis.
5. An LPR of 0 provides critical information in the first crystallization trial on possible co-purified lipids. Even small amounts
of lipids may result in membrane formation; therefore, if membrane formation is seen at an LPR of 0 (no additional lipids
added), the protein sample requires further washes during
purification.
6. Removal of the air in the tubing is not necessary and can result
in sample loss. The air is, in fact, helpful when removing the
sample after dialysis as the tubing is less likely to bend upon
cutting.
7. The large spacing between the clips has been shown to result in
maximal recovery of the dialysate.
8. The 90
rotation between the clips will ensure that the bottom
clip remains submerged under the surface of the buffer, preventing sample loss and/or drying of the tubing.
2D Electron Crystallography of Membrane Proteins
241
special circumstances of ordered phospholipids [6], conclusions on
the importance of the phospholipids to the protein structure and
function may be drawn. The 3D maps of samples in different
conformational states such as bacteriorhodopsin [24, 26] can reveal
substantial information on the detailed reaction mechanism.
4 Notes
1. Lipid stock concentrations of 1 and 10 mg/mL are useful for
many lipid-to-protein ratios used during 2D crystallization
trials. DMPC has been successfully used for 2D crystallization
of a large percentage of membrane proteins. Lipid in chloroform is preferable due to the hygroscopic nature of lipids in
powder form.
2. The stream of nitrogen gas is slowly increased to avoid splashing and drying of the lipid on the sides of the round-bottom
flask.
3. Lipid aliquots for 2D crystallization trials can frequently be
stored for 1–2 years, if not longer.
4. Approximately 4–8 crystallization conditions often provide
sufficient and critical information to optimize parameters for
the next experiment. It is important to note that simultaneous
testing of a larger number of crystallization conditions will
require a substantial amount of protein as well as many hours,
if not days, of TEM time, which is often not available in one
large time block. Thus, it is more efficient to set up fewer
crystallization conditions on a frequent basis.
5. An LPR of 0 provides critical information in the first crystallization trial on possible co-purified lipids. Even small amounts
of lipids may result in membrane formation; therefore, if membrane formation is seen at an LPR of 0 (no additional lipids
added), the protein sample requires further washes during
purification.
6. Removal of the air in the tubing is not necessary and can result
in sample loss. The air is, in fact, helpful when removing the
sample after dialysis as the tubing is less likely to bend upon
cutting.
7. The large spacing between the clips has been shown to result in
maximal recovery of the dialysate.
8. The 90
rotation between the clips will ensure that the bottom
clip remains submerged under the surface of the buffer, preventing sample loss and/or drying of the tubing.
2D Electron Crystallography of Membrane Proteins
241