Projection structure of halorhodopsin from Halobacterium halobium at 6 A resolution obtained by electron...

Two-dimensional crystals of halorhodopsin (HR), in space group p42(1)2 (a = 102 A) have been obtained using the overexpressing Halobacterium halobium strain D2. An HR membrane fraction with the same buoyant density as purple membrane (HR-PM) was obtained by homogenization and sucrose gradient purification and used for electron cryomicroscopic analysis. Electron micrographs and electron diffraction patterns of HR-PM were recorded at liquid nitrogen temperatures. The micrographs showed significant diffraction out to 9 A resolution optically and to 6 A after computer processing. By combining data from electron micrographs and electron diffraction patterns, a projection map of HR was calculated. The crystal form of the isolated HR consists of one membrane in which alternating halorhodopsin tetramers are oriented in opposite directions across the membrane. It is not known whether this occurs by misinsertion of some of the molecules in vivo, or by adventitious fusion at some point during isolation. The projected structure of the HR molecule to a resolution of 6A is almost identical to that found for bacteriorhodopsin (BR). This physical structural similarity thus complements the known sequence relatedness to BR.