The three-dimensional structure of halorhodopsin to 5 angstrom by electroncrystallography: A new unbending procedure for two-dimensional crystals byusing a global reference structure

Electron microscopy does not, in principle, require highly ordered crystals to determine a high-resolution structure. Nevertheless. crystals of any ty pe help to constrain the molecules into a more limited range of orientation s and positions, from which it is easier to carry out structure determinati on. We describe an improved procedure for determination of crystalline diso rder, which we have applied to poorly ordered two-dimensional crystals of t he chloride pump halorhodopsin from Halobacterium salinarum. The new image analysis procedure involves the use of a reference projection calculated fr om a global three-dimensional map to carry out the initial cross-correlatio n analysis. Coupled with a greater number of images taken with field emissi on gun microscopes, this has allowed us to calculate a three-dimensional st ructure for halorhodopsin. in which the seven transmembrane helices and cer tain molecular features, such as the beta-ionone ring of retinal, are now r esolved.