The small cocoons were by fat the smallest protein crystals ever images with X-ray crystallography, and the results point the way to employing even smaller crystals, or even un-crystallized proteins and other biomolecules for structure analysis.
Employing SLAC’s Linac Coherent Light Source, a DOE Office of Science User Facility, the scientists hit closely 500,000 cocoons with X-ray pulses, creating diffraction patterns in a detector that were compiled to form an image of the cocoon’s structure with a resolution of 0.2 nanometres.
The potential to observe a structure of protein provides clues about how the molecule behaves, information that can be applied to an extensive range of fields like agriculture, industrial and medicine processes. This research looked at Cydia pomonella granulovirus, which infects the caterpillars of the coding moth and is also employed as a biological pesticide.
“Over the past 50 years, researchers have determined the structures of more than 100,000 proteins,’ says Henry Chapman, a researcher at Deuthsches Electronen-Synchroton in Germany and head of the research group. “By far the most vital tool for this is the X-ray crystallography.”
Usually, researchers performing X-ray crystallography prepare crystals comprising numerous copies of the protein they intend to study. LCLS has enabled them to study much smaller crystals than before, with the potential to capture images with ultrafast X-ray pulses before they are damaged by the intense radiation. This eradicates a major roadblock to studying proteins that are difficult to make into large crystals.
In this situation, the virus has a cocoon prepared of naturally formed crystallized proteins. The crystals each comprised about 9,000 copies of the protein, about one-thousand times smaller than crystals used before. Prior to such result, the typical crystal size employed for structural determination at LCLS has been about five microns, with the smallest on the order of one micron, says Sebastien Boutet, SLAC researcher and co-author on the paper.
‘We have pushed the potentialities of LCLS down in the sub-micron region by employing extreme emphasizing that prepares a more intense beam on the small sample,” Boutet says. LCLS began the Single Particle Imaging Initiative to work toward atomic-scale imaging for numerous sorts of biological samples. To address technical limitations, such efforts have comprised detailed beam and detector characterization. Such effort will benefit from future developments like building novel detectors and upgrading mirrors to enhance beam quality.
This study lays out a path for employing free-electron lasers to get structures of proteins and other biomolecules without having to crystallize them at all. In the future, the scientific group expects to look at even smaller structures with the same level of detail. “Simulations based on our estimates suggest that our method can probably be employed to determine the structure of even smaller crystals consisting of just hundreds or thousands step further molecules,” says Chapman.
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