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Scientists at the US Department of Energy's Argonne National Laboratory have followed up the earlier idea that crystallisation is no longer a prerequisite for successfully determining a crystal structure. They have devised an alternative approach to aligning large groups of molecules that makes their target samples equally amenable to investigation using a synchrotron X-ray source. Crystallography usually relies on being able to make crystals of a compound of interest. In protein studies that frequently proves impossible and as such protein crystallographers have barely scratched the surface of the proteome, the range of human proteins that might be important in a wide range of diseases for which drugs are sought. Conventionally, crystallization allows investigators to create the necessary periodic structure that will strongly diffract X-rays and spit out the standard diffraction pattern with which readers are familiar. Of course, disorder means no pattern. Argonne physicist Robin Santra explains how he and his colleagues have brought order to proteins that fail to crystallise using strong laser fields. "A non-resonant, linearly polarized laser field will align a molecule by interaction with the molecule's anisotropic polarizability; the most polarizable axis within the molecule will align parallel to the laser polarization axis," the researchers explain in the journal Applied Physics Letters this month, "Since the laser polarization direction is under simple control with a waveplate, so is the direction of the molecule's most polarizable axis with respect to the laboratory frame." The researchers point out that the technology is closely tied to optical trap investigations used to hold single molecules for examination using other techniques. In optical trapping, however, the alignment aspect of the trapping process is usually overlooked. Linda Young adds that, with this approach, "Using X-rays, we can ivestigate their properties in a totally new way." The new laser technique allows for millions of molecules suspended in a gaseous state to be aligned so that, when bombarded with X-rays, they all diffract in the same way as if they were locked in a crystal but without the inconvenience of crystallization. The alignment approach allows Bragg-like diffraction spots to be obtained from a material in the gas phase rather than the concentric rings that would otherwise be observed from scattering X-rays in isotropic gas. In the preliminary work leading up to this study, the researchers had considered analysing single molecules with short bursts of X-ray photons using an optical trap technique and achieving resolutions of a few Ångstroms. However, it became apparent that the alignment of tens of millions of molecules would provide much more detailed information. As such, the team has carried out a proof of principle on the nonhazardous, symmetric-top shaped molecule bromotrifluoromethane (CF3Br), with a view to moving to proteins at a later stage. "Understanding the structure of the approximately 1 million human proteins that cannot be crystallized is perhaps the most important challenge facing structural biology," Young says, "A method for structure determination at atomic resolution without the need to crystallize would be revolutionary." The team demonstrated proof of principle using a hard X-ray probe with the small molecule, CF3Br, aligned in the gas phase using high-intensity optical lasers at non-resonant wavelengths focused to 40 micrometres and with pulses as short as 95 picoseconds from an 800 nanometre Ti:sapphire laser. The X-rays used were linearly polarized of 120 picosecond pulses focused to 10 micrometres and tuned to a key resonance of the bromine atoms. Having obtained viable data the team then showed theoretically that the technique could be used to obtain an X-ray structure. The researchers point out that there are technical challenges to overcome prior to demonstrating laser-aligned X-ray diffraction with proteins, but that diffraction from an aligned ensemble of smaller molecules is not far off. "We are currently considering slightly larger molecules, say ten times larger, and have just acquired a new high repetition rate laser system that will enable such studies," Young told SpectroscopyNOW, "However, for small molecules, such as CF3Br, a higher laser intensity is required and we must await a beamtime upgrade to do structure/diffraction studies." Related links:
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