MBIB researchers were part of a team that used SLAC’s X-ray free-electron laser (XFEL) – the Linac Coherent Light Source (LCLS), a DOE Office of Science User Facility – to get atomic views of the toxin BinAB, used as a larvicide to control mosquito-borne diseases such as malaria, West Nile virus and viral encephalitis. The structure of this bacterial toxin was solved using de novo phasing: the protein crystals were tagged with heavy metal markers, tens of thousands of diffraction patterns were collected using the XFEL, and the information was combined to obtain a three-dimensional map of the electron density of the protein.
The Berkeley team, headed by Senior Scientist Nicholas Sauter (pictured, right), acquired and processed data for the study, published in Nature last week. The collaboration was led by David Eisenberg of UCLA and included researchers from University of Grenoble Alpes, UC Riverside, LCLS at SLAC National Accelerator Laboratory, California Baptist University, and Stanford University. This research uncovers the mechanism of action of toxin BinAB, and gives insight into how these crystals, secreted from bacteria along with spores, kill mosquitos when ingested. As mentioned by SLAC scientist Sebastien Boutet in their press release, this work demonstrates “the first time de novo phasing has been used on a crystal of great interest at an X-ray free-electron laser.” LCLS commenced operations in 2009, but new computational methods had to be written by software teams at Lawrence Berkeley National Laboratory and Stanford University to enable this type of analysis.
“This was a collaborative effort,” Sauter said, “We wrote the program that measures diffraction spots on the tens-of-thousands of images obtained using XFEL, using custom techniques designed to improve accuracy. Then we had the initial ideas for merging all the information together, ultimately using a technique called post-refinement that dates 
back to 1979, but which had to be completely reimagined for XFEL data.” Mona Uervirojnangkoorn, a researcher in the lab of Axel Brunger at Stanford, wrote the code for the program with input from the Berkeley team. Finally, Aaron Brewster (pictured, left), a project scientist in the Sauter group, used Uervirojnangkoorn’s code to analyze the data collected at LCLS.
“The demands on accuracy were unprecedented. The heavy metal markers contributed a very small effect, and we had to combine all the information together before we could see something recognizable as a protein structure,” said Sauter. This protein structure contains clues to its mechanism of action that can allow scientists to manipulate the toxin to combat other species of mosquitos, such as the Aedes mosquitos that transmit Zika and dengue fever. For more on this story, read the SLAC press release.
