You know those high-speed cameras on the television show Mythbusters, the ones the hosts use to slow a bullet to a crawl or catch an explosive shock wave in mid-flight? Imagine using a camera like that to capture molecular dynamics. You could catch the motion of enzymes as they flex to bind ligands, watch photoreactive proteins change shape in response to light, or map a virus's topology.
Turns out, imaging at that level isn't science fiction: The first such “high-speed” instrument was installed near Stanford University in 2009 and has been pumping out a steady stream of images since 2011.
But it's not exactly a camera; the Linac Coherent Light Source (LCLS) is actually a laser, the world's first “hard” X-ray free electron laser (XFEL). There is a second XFEL, the SPring-8 Angstrom Compact free electron laser (SACLA) in Japan, and a third is under construction in Germany. Though specifications vary, these lasers all fire blindingly bright, short-wavelength X-ray light into samples in pulses just femtoseconds (10-15 seconds) long. Like the flashes of a stroboscopic camera, researchers have used these pulses to develop an entirely new paradigm in protein structural biology: serial femtosecond nanocrystallography, or SFX for short.Let's talk about “SFX”
X-ray crystallography isn't exactly new— of the 96,980 structures currently stored in the Protein Data Bank (PDB), nearly 86,000 were solved by X-ray crystallography. Clearly, this is an imaging method that works—so, why is there need for a new one?
The reality is, crystallography suffers from a number of drawbacks. First, it requires a crystal, and a big one at that, typically at least 0.1 mm long. But not every protein crystallizes well, especially large proteins or those residing in membranes, which limits the technique's reach. Second, X-ray radiation damages crystals as it images them, causing the images to blur. To circumvent that problem, the crystals typically are chilled in liquid nitrogen, but resolution still inevitably suffers. Finally, crystals are, by definition, static snapshots of a single protein configuration. Researchers interested in protein dynamics typically use NMR for their structural studies, and indeed, most of the non-X-ray structures in the PDB were acquired by that method.
SFX addresses all of these issues, says John Spence, Regents Professor at Arizona State University (ASU) and Director of Science at the NSF BioXFEL Science and Technology Center, who with ASU colleague Petra Fromme and Henry Chapman from the Center for Free-Electron Laser Science in Hamburg, led the team that first implemented the technique in 2011 to produce an 8.5 Å image of photosystem I. According to Spence, the first advantage of SFX is that it can “outrun” radiation damage.
LCLS pulses come 120 times per second, but each pulse is just 50 femtoseconds long. To put that number into context, says Spence, the time it takes for an atom to vibrate once at room temperature is 100 femtoseconds, and the time it takes an electron to orbit once around an atom is about 1 femtosecond.
The energy delivered in those pulses is staggering. According to Janos Hajdu, Professor of Cell and Molecular Biology at Uppsala University, his team has achieved power densities in excess of 1020 W/cm2 in a tightly focused XFEL beam. “If you focused all sunshine hitting the Earth to a millimeter-square, you get the same power density that one would get if you focused a pulse from the LCLS to 1 μm2 during the duration of the pulse,” Hajdu says. The peak brilliance of the beam, he adds, is 9 to 10 orders of magnitude greater than synchrotron radiation, the current crystallography standard.
Because those pulses are so short and so bright—each contains some 1012 photons—the method is able to produce a sharp image before the crystal itself is damaged. A fraction of a second later, the crystal explodes, but not before the detector records the diffraction pattern. “We call [that] ‘diffract-and-destroy’, or ‘diffract-then-destroy’,” Spence says.