A weak spot in the parasite that causes African sleeping sickness, a disease that kills an estimated 30,000 people each year, has been mapped using an x-ray laser, pinpointing a promising new target for treating the fly-borne illness. An international group used the SLAC National Accelerator Laboratory’s Linac Coherent Light Source (LCLS) to study African trypanosomiasis, a disease transmitted by tsetse flies infected with the single-celled parasite Trypanosoma brucei. About 60 million people across Africa are at risk for contracting the disease. Researchers have revealed the detailed structure of an important protein involved in the transmission of African sleeping sickness. They used the LCLS x-ray laser to create diffraction patterns (shown in background) that were then reconstructed into the molecular structure (shown at center). The research is an important step toward developing a new drug to target the disease, which is carried by tsetse flies and is responsible for tens of thousands of deaths each year. Courtesy of Greg Stewart/SLAC National Accelerator Laboratory. The increasingly drug-resistant parasite’s artillery includes an enzyme that breaks down the proteins of its victims. Scientists hope to stop the disease by mimicking a natural inhibitor that keeps this enzyme in check until the parasite invades the victim’s bloodstream. However, the enzyme is so similar to one in humans that blocking it could also be harmful to the patient. To design a drug that attacks only the parasite, researchers needed more detailed information about its structure. Typically, researchers determine these protein structures by shining an x-ray beam onto a protein crystal. But it’s difficult to grow crystals large enough to withstand this radiation for long enough to capture the necessary information. A new method, known as serial femtosecond crystallography, overcomes the radiation-damage problem by using short x-ray laser pulses that radiate a steady stream of small crystals. These pulses are so fast that a snapshot of information can be recorded before each crystal is destroyed. A map of intensities merged using the CrystFEL software suite from almost 200,000 diffraction patterns obtained from in vivo grown crystals of Trypanosoma brucei cathepsin B. This map is used to synthesize the 3-D molecular structure of the enzyme. Courtesy of Karol Nass/CFEL. The crystals were grown inside live insect cells, and the enzyme frozen in its natural inhibited state. The crystals were streamed into the laser’s path, producing patterns in a detector that were used to reconstruct the enzyme. The resulting structure shows how the enzyme is inhibited prior to activation and provides clues that may be useful for developing drugs to treat sleeping sickness. “This is the first new biological structure solved with a free-electron laser,” said Henry Chapman of the Center for Free-Electron Laser Science in Hamburg, Germany, one of the leaders of the research team. Previous research by the team appeared in Nature Methods (doi: 10.1038/nmeth.1859). “In my opinion, we provided the most complete blueprint available so far for the development of a synthetic inhibitor to block this enzyme,” said Lars Redecke, a structural biologist at the Joint Laboratory for Structural Biology of Infection and Inflammation of Hamburg and Lübeck universities in Germany. “This is really a landmark in structural biology, and a significant step toward developing a new drug.” Colored electron micrograph of the bloodstream form of the Trypanosoma brucei parasite (light blue) that causes African trypanosomiasis (also called sleeping sickness) in humans in the presence of erythrocytes (red) and lymphocytes (yellow). After infection of the mammalian host from the bite of a tsetse fly, the parasite lives in the bloodstream before it invades the central nervous system and the brain to cause fatal effects. Courtesy of Michael Duszenko/University of Tübingen. Redecke’s team is working to crystallize proteins relevant to other parasites and viruses — including strains of hepatitis and flu — that also could be studied at LCLS. He said he expects this field of research to grow. “Our study will encourage others to use free-electron lasers to obtain new structural information of biologically relevant molecules,” Redecke said. Other institutions involved in the study were Hamburg, Lübeck and Tübingen universities; the German Electron Synchrotron (DESY) and Max Planck Institute for Medical Research in Germany; Gothenburg and Uppsala universities in Sweden; Arizona State University; and Lawrence Livermore National Laboratory. The study was performed using the Coherent x-ray imaging (CXI) instrument at LCLS. The results were published in Science Express. For more information, visit: www.slac.stanford.edu