Thursday, May 02, 2013

Protein crystallography using x-ray free-electron lasers



Just over 100 years ago, Max von Laue reported the first x-ray diffraction patterns from crystals. Soon after, Lawrence Bragg interpreted diffraction spots. Their work provided the foundation for x-ray crystallography, which today allows us to build three-dimensional images of molecular structures. Several scientific fields were born as a result, including structural biology, where the molecular machinery of life is inspected with increasing complexity. To solve protein structures, scientists around the world have historically used the intense and tunable x-ray beams produced by synchrotron radiation facilities. Now there is a new source: the x-ray free-electron laser (FEL), which is set to overcome the challenges in studying protein crystallography, vastly increasing the range of proteins whose structures can be solved.

X-ray FELs provide incredibly intense pulses of x-rays with up to 1013photons in a duration of tens of femtoseconds (fs). The first hard x-ray FEL, the Linac Coherent Light Source at the SLAC National Accelerator Laboratory in California, went into operation in 2009. This facility routinely produces x-ray pulses of around 3mJ energy in a duration of 70fs or less. Focused beams can achieve x-ray intensities of greater than 1020W/cm2. This is many orders of magnitude greater than that required to form a plasma from any material, and indeed this beam vaporizes anything in its path.

Remarkably, these destructive pulses overcome the problem of radiation damage in protein crystallography and x-ray microscopy. With conventional sources, such as synchrotron particle accelerators (or storage rings), ionizing x-rays break bonds and cause changes to the structure as the exposure takes place. This limits the total dose that can be used to record images or diffraction patterns, since we cannot compensate for a weakly diffracting crystal by exposing the protein for longer. Therefore, crystallographers need to grow large, well-diffracting crystals of proteins, which can require months or years of sustained effort.

Using this technique they can characterize diffraction from protein crystals that are too small for conventional measurement. They quantified diffraction patterns from nanocrystals of the photosystem I complex (proteins involved in photosynthesis) that are smaller than 200nm in diameter—only six unit cells across. Because each pulse vaporizes the sample, they needed to replenish it in time for the next pulse, arriving at a rate of 120Hz. They did this by continuously flowing a suspension of nanocrystals in a microjet that intersected the beam path (see Figure 1). They collected 120 diffraction frames per second. The crystals are randomly oriented and not synchronized to the x-ray pulses, so the odds of hitting one depend on the concentration of crystals in the fluid suspension (the x-ray flash freezes all motion). They collected millions of frames, from which they extracted tens to hundreds of thousands of individual snapshot diffraction patterns. 

The method of serial femtosecond crystallography is well suited to studies of irreversible reactions that are time-resolved. We can 'pump' samples with an optical pulse synchronized to the x-ray to an accuracy of greater than 100fs, varying the delay between the pump and the x-ray measurement pulse to build up an ultrafast movie. The sub-micron-sized crystals make it possible to diffuse a reagent in a shorter time, increasing the accuracy. Future developments could include improved sample delivery to reduce the consumption of protein, and implementing anomalous diffraction schemes for de novo phasing, where no base model is required to solve the protein structure. The small number of unit cells in the crystals also offers intriguing possibilities for phasing, by measuring intensities between Bragg spots. This approaches x-ray FEL's ultimate aim of measuring the smallest crystals of all: single molecules.



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