Biochemical reactions are extremely rapid, but the methods for imaging the enzymes that catalyse them can take hours. To get round this problem, structures can be determined at temperatures around 100 K, literally freezing an enzyme's movements and allowing the intermediates in its reaction cycle to be observed. This has been done for bacteriorhodopsin in studies reported by Edman et al. in this issue (Nature 401, 822–826; 1999) and Genick et al. (Science 286, 255–260; 1999).

Bacteriorhodopsin is a pump that uses light energy to drive protons across bacterial purple membranes. A cycle of structural changes is triggered when absorption of a photon of light causes isomerization of bacteriorhodopsin's bound chromophore, retinal (purple in the figure). Intermediates in this photocycle can accumulate in crystals at low temperatures (see Nature 392, 206–209; 1998) or in mutant proteins, as used by Genick et al., but subtler techniques are needed to trap the earliest and most elusive intermediates.

Edman et al. maintained bacteriorhodopsin crystals in the dark, bathed in liquid nitrogen (110 K). They then drove the initial step in the protein's photocycle by illuminating the crystals with green light through an optical fibre (at a wavelength of 532 nm), and exposed them to a powerful synchrotron source to aid the rapid collection of data. The authors found that, despite the isomerization of a double bond, there is very little change in the overall shape of the retinal — in the figure, electron density after illumination (blue) is compared with that before (brown).

But there are other changes. The biggest of these is the escape of a water molecule (designated W402) from the vicinity of the retinal. This molecule previously formed part of a network of water and amino-acid residues connecting retinal to the outside of the bacterial membrane. Its loss triggers changes in this network, eventually resulting in expulsion of a proton from the bacterial cell.

One snap-shot cannot reveal the whole picture of how the bacteriorhodopsin pump works. However, by building up series of freeze-frame structures for all the intermediates in the enzyme's working cycle the mechanics of this, and other, molecular machines is being revealed.