Postcrystallization treatment. Rationale- We have often obtained crystals of membrane proteins but found the resolution limit to be on the order of 10-30 A, so that we only see a few diffraction spots around the beamstop. While these crystals are useless for structure determination, in principle they represent a tremendous step towrd our goal in terms of arrangement of the molecules and reduction of entropy. 10-30 A is small compared to the dimensions of the molecules (100-200 A), so this level of order means we have hundreds of millions of molecules lined up in a three-dimensional array, but with a little bit of rotational and translational "jitter" in their positions. Now suppose instead of going back to repeat the crystallization under different conditions in hope of getting better order, we can take the already-formed crystals and change conditions slightly so the the pre-positioned molecules click into position with good order. This strategy also allows for the fact that the best conditions for growing a crystal may not be the conditions giving the best order once the crystal is grown. As for dehydration, in the case of the orthorhombic bc1 crystals, we dehydrate by mounting the crystal in a loop, holding the loop in the air at room temperature for 1-3 minutes, then freezing in liquid nitrogen. This does two things- reduce the water activity enough to prevent ice formation, and collapse the lattice to the point where it is more rigid. To make this more reproducible, so it doesn't depend on things like relative humidity, air drafts, and size of the crystal, we fish out the crystal on a loop in a Hampton-Research "cryocap" pin base. Then screw the cryocap into a cryovial with 200 ul of "humectant" solution in the bottom to define humidity, and incubate overnight at the temperature the crystals were grown before freezing. In a typical experiment there would be 5-10 crystals in vials with different humectant, ranging from 25% to 70% w:w glycerol. At 70% the diffraction is lousy and there is a powder-pattern ring at 4.37? A from precipitated PEG. At 25% there are ice rings and little or no diffraction. somewhere in between there is good diffraction. The unit cell parameters are extremely variable, with the a edge going from 160 down to 120 A, best diffraction at 128 A. And there is a new contact at 128, which was well separated in the larger cells. So we think the lattice collapses with dehydration until it hits a hard stop when this contact is made, then further collapse causes chaotic deformation and worse diffraction. However the complex II crystals are damaged by any dehydration, and the lattice parameters are pretty constant. In that case I think the lattice is already rigid. Maybe a slight increase in cell volume would help. So we treat with mother-liquor plus glycerol and freeze immediately. I think a 2-dimensional optimization is necessary: The water activity has to be between the limits set by ice formation and PEG precipitation. Then the cell volume should be just right to give tight lattice contacts without disruptive disordering. Dehydration varies both simultaneously. By adjusting the glycerol concentration before the dehydration step, the curves can be shifted over so that a given water activity corresponds to a larger or smaller cell volume.