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The article discusses the capture of high-resolution intermediates of photoactive yellow protein (PYP), a bacterial blue light photoreceptor, with time-resolved serial femtosecond crystallography (TR-SFX), which uses ultrashort pulses from x-ray free electron lasers (XFELs). Topics include the challenges of TR-SFX, TR-SFX's advantages over synchrotron-based, time-resolved Laue crystallography, and difference electron density (DED) maps created by using microcrystals of PYP as a model system.

De novo membrane protein structure determination is often limited by the availability of large crystals and the difficulties in obtaining accurate diffraction data for experimental phasing. Here we present a method that combines in situ serial crystallography with de novo phasing for fast, efficient membrane protein structure determination. The method enables systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without the need for direct crystal harvesting. The requisite data quality for experimental phasing is achieved by accumulating diffraction signals from isomorphous crystals identified post-data collection. The method works in all experimental phasing scenarios and is particularly attractive with fragile, weakly diffracting microcrystals. The automated serial data collection approach can be readily adopted at most microfocus macromolecular crystallography beamlines. Huang et al. report a fully automated method for collecting and merging data from thousands of in meso-grown microcrystals. This in situ approach provides a fast and efficient way to determine the structures of membrane proteins from fragile microcrystals. [ABSTRACT FROM AUTHOR]

A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia. • X-ray free-electron laser unveils alternative protein structure of lipoprotein • Advanced molecular dynamics explore conformational space among solved structures • Virtual ligand screening shows possible lead fragments for potential drug therapies Using X-ray free-electron laser (XFEL) technology, Zook et al. describe an alternative protein conformation of the virulence determinant Flpp3 and use molecular dynamics (MD) to investigate conformational landscapes between these conformations. MD identifies several intermediate conformations that allow these conformational transitions to occur at physiologically relevant temperatures and provide insight to potential future drug therapies for the potentially deadly disease tularemia. [ABSTRACT FROM AUTHOR]