With diffraction quality crystals a rarity and some compounds and proteins almost impossible to crystallize, or in a crystal form without ligand in binding cavity, structural biology is often thought of as a field for the most patient. Why? Notwithstanding whether a particular target protein would form crystals of quality sufficient for structural elucidation, some proteins such as the transmembrane G-protein coupled receptors are too flexible in backbone structure to yield crystals of diffraction quality for important processes to be understood at the atomic level, for example, the formation and breaking of chemical bonds.
Thus, structural biology is a technology or instrument enabled field requiring fresh ideas on how to reduce the conformational flexibility of proteins, or introduction of new instruments capable of mapping changes in electron density of proteins to atomic coordinates, as it perform its function in its native environment, for example, in the model system environment of a dilute solution. To this end, a new experiment technique, X-ray free electron laser spectroscopy, offers the capability to obtain structural imagery information by illuminating a target protein with multiple ultra-short duration laser flashes in dilute solution environment; thereby, enabling the elucidation of a protein structural dynamics in solution – a feat not available to the gold standard approach of X-ray crystallography.
Note the use of the concept of “structural imagery information”. Specifically, X-ray free electron laser spectroscopy (XFEL) does not provide direct imaging information such as those available from an optical microscope. Instead, it collects information about how the laser flashes scatters from the target protein and reconstruct a structural image of the protein in action. Essentially, it is similar to X-ray diffraction in concept, but differs in the important area of not requiring a crystal for spectroscopic examination as well as having the ability to determine dynamic structural changes of proteins in dilute solution.
Besides the ability to examine both solid and liquid samples, and thus, determine the dynamic changes of structures of proteins and biomolecules in the more native environment of solutions vis-à-vis the previous approach of capturing a protein binding to a ligand in the solid state, XFEL requires significantly less molecules for structural elucidation. Specifically, by using significantly more laser flashes per unit time compared to X-ray diffraction, XFEL is able to probe ensemble structural changes of protein molecules at a lower concentration because the enhanced incident beam enables more scattering and diffraction information to be obtained by detectors from a smaller total number of molecules, which enable the technique to probe protein solutions at low concentration.
Similarly, the requirement for less molecules also translates to the ability to obtain good structural information from crystals of micron size relative to those of larger sizes necessary for X-ray diffraction studies. Considering the difficulty of obtaining large crystals of good quality, the ability to use more readily available small crystals may open up entire vista for structural biology: specifically, the chance to determine conformational changes of proteins at each stage of ligand binding as well as understanding how different isoforms of protein work, the latter of special importance to fields such as calcium channel research where alternative splicing generates a plethora of protein isoforms. Previously, chief hindrance to a structural view of protein function lies in the inability to elucidate structural insights from small crystals or membrane proteins not willing to yield to various crystallization attempts.
But XFEL is not coming to the lab any time soon given its need for a linear accelerator to generate the speeds needed for electron beams to be used for X-ray generation. Currently, there are only a few XFEL instruments in the world and they are concentrated in large accelerator complexes such as the Linac Coherent Light Source, which first introduce the technique to the scientific community in 2009. Soon, an European XFEL would be the brightest and fastest X-ray source in the world capable of firing 27000 flashes of light per second.
Hence, compared to the gold standard technique of X-ray diffraction, X-ray free electron laser (XFEL) spectroscopy captures structural details of proteins and molecules in action in dilute matrixes through gathering vast ensemble of scattering information from the multitude of laser flashes directed to the sample. Add to its ability to obtain reconstructed structural images of proteins or other molecules with a small sample, the technique affords researchers the hitherto unavailable capability of analyzing coordinated movements of differing domains of proteins in different stages of catalysis or ligand binding. Nevertheless, availability of XFEL only at national level facilities with linear accelerator meant that the method would likely find use as a confirmatory technique in lieu of X-ray diffraction, which is more readily available at many universities. Hence, lack of access by researchers may limit the impact of this useful analytical technique capable of obtaining structural information from molecules in dilute solutions or small crystals.
Categories: instrument, structural biology, biochemistry,
Tags: structural biology, diffraction quality, X-ray diffraction, crystallization, dilute solution, X-ray free electron laser, conformational flexibility, electron beam, proteins,