High-resolution, three-dimensional protein structure determination is a tedious process, involving protein overexpression, purification and crystallization. Many interesting biological structures, such as large complexes and membrane proteins, are all but intractable to this procedure. Moreover, information about the dynamic motions of proteins, crucial for understanding their functions, cannot be obtained from crystallography studies.

A technique to obtain snapshots of single molecules in motion would arguably revolutionize structural biology. With the construction of X-ray free-electron lasers (XFELs), such a methodological achievement seems within reach.

Taking a snapshot of a single molecule with an XFEL. Image courtesy of SLAC National Accelerator Laboratory.

The world's first XFEL, the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory in Menlo Park, California, USA, has been up and running since April 2009. This laser is a billion times brighter than previous hard X-ray sources (hard refers to the high-energy, short-wavelength end of the X-ray spectrum) and produces laser pulses with a wavelength of just 1.5 angstroms. These ultrabright, ultrafast pulses of light allow the LCLS to take a series of snapshots in rapid-fire succession with a shutter speed of about 100 femtoseconds. These unique properties have the structural biology community very excited.

But although building the LCLS was a true engineering feat, much methodological development is still needed before single-molecule structure determination will be possible. Reliable methods for injecting single molecules in the path of the XFEL beam and determining the best way to take snapshots such that a high-resolution, three-dimensional structure can be reconstructed from a diffraction pattern must be developed; these challenges will certainly be nontrivial. Analyzing the data that comes out will also be a huge hurdle and will necessitate algorithm and software development.

It may be several years or more before single-molecule structure determination becomes a reality, but progress so far is encouraging. Recently, a paper describing the LCLS's operation and performance was published (Nat. Photonics 4, 641–647; 2010). The first application papers using the LCLS, including one reporting how the electrons in neon respond to XFEL radiation (Nature 466, 56–61; 2010), are also beginning to appear in the literature. Two other XFELs are under construction: the Spring-8 XFEL in Japan is expected to be completed in 2011 and the European XFEL in Germany in 2014. We will certainly be watching for exciting further developments.