Free-electron lasers reveal architecture of proteins in unprecedented detail

Researchers from the Max Planck Institute for Medical Research (MPI; Heidelberg, Germany) and the Max Planck Advanced Study Group (Hamburg, Germany) have analyzed protein crystals using short pulses of x-ray light from the world's first hard x-ray free-electron laser, the U.S. Department of Energy's Linac Coherent Light Source (LCLS) at Stanford University (Stanford, CA).

The study demonstrates the immense potential of free-electron lasers for obtaining the structures of macromolecules from tiny crystals when illuminated with ultrashort free-electron laser x-ray pulses, even though the crystals are destroyed in the process. In the current study, their structural analysis reveals details with a spatial resolution of 0.2 millionths of a millimeter. What's more, their data compared well with those collected from large, well characterized crystals using conventional x-ray sources. 

Schematic representation of the experimental setup at the Linac Coherent Light Source, where millions of tiny crystals are injected into the free-electron laser beam in a thin, liquid jet
Schematic representation of the experimental setup at the Linac Coherent Light Source, where millions of tiny crystals are injected into the free-electron laser beam in a thin, liquid jet. Diffraction patterns are generated when a crystal intersects a free-electron x-ray flash and are captured on a detector (left). (Image courtesy of the Max Planck Institute for Medical Research)

Free-electron lasers can obtain structural information from tiny crystals that refuse to reveal their secrets by conventional structural methods due to the damage induced by the radiation used for the structure analysis. Although the tiny crystals are completely destroyed by the intensity of the free-electron laser, the ultrashort pulses can pass through the sample before the onset of detectable damage and thus provide the necessary scattering signal of the still-intact molecules.

In this diffraction-before-destruction approach, crystals are replenished for serial data collection by injecting them into the free-electron laser beam using a liquid jet, developed by scientists from Arizona State University (Tempe, AZ), exposing one crystal after the other instead of rotating a single large crystal as in conventional crystallography. This concept of serial femtosecond crystallography has been demonstrated before by the same team of researchers at the Linac Coherent Light Source, using the CAMP instrument, developed by the Max Planck Advanced Study Group. The relatively long wavelength x-rays then limited the attainable level of structural detail.

Recently, a new instrument at the Linac Coherent Light Source, the Coherent X-ray Imaging endstation, has allowed the use of short wavelength x-rays and thus made it possible to infer atomic detail in the molecular architecture. To benchmark the method, the team investigated the small protein lysozyme, the first enzyme ever to have its structure revealed.

Structure of the protein lysozyme: The spatial arrangement of the 129 amino acids is schematically depicted in the form of spirals (helices) and arrows (pleated sheets)
Structure of the protein lysozyme: The spatial arrangement of the 129 amino acids is schematically depicted in the form of spirals (helices) and arrows (pleated sheets). (Image courtesy of the Max Planck Institute for Medical Research)

The team collated 10,000 snapshot exposures from crystals that measured only a thousandth of a millimeter, and showed that the data compared well with those collected using conventional approaches and hundred-fold larger lysozyme crystals—with no significant signs of radiation damage. Since small crystals are typically easier to produce than large ones, the work is relevant for all studies of molecules that are difficult to crystallize—including 60 percent of all proteins, many of which are prime targets for medical therapies.

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