Technology Development for Biological Imaging with XFELs

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Project Summary / Abstract Determining the structure and conformational dynamics of large protein complexes as well as other biological nanoparticles at room temperature with near atomic resolution has the potential to greatly impact structural biology and our knowledge of biomolecular function and interactions. A major bottleneck in structural biology is that while many critical cellular functions are performed by membrane proteins, they have proven intractable to structure determination by traditional x-ray crystallography, in which x-ray radiation damage is mitigated by spreading the radiation dose over many molecules in a crystal. Consequently, most membrane protein structures remain unknown to date. While cryo-electron microscopy (cryo-EM) has been successful in obtaining high-resolution structural information from large biomolecules and nanoparticles, it requires freezing of the sample to mitigate electron-induced radiation damage and cryogenic measurement makes it impossible to visualize fast conformational changes. X-ray free electron lasers (XFELs), which produce ultra-short and ultra-bright x-ray pulses, allow us to break this nexus between resolution and radiation damage by utilizing the “diffraction-before-destruction” principle and promise imaging at unprecedented spatio-temporal resolution. Over the last decade since the opening of the world's first XFEL, the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory, protein structure determination at room temperature to near-atomic resolution by serial- femtosecond nanocrystallography (SFX) has been demonstrated. However, several challenges and limitations remain that need to be addressed to fully utilize the capabilities of these new light sources and the upcoming next generation XFELs for structural biology. The overall objective of this proposal is to enable new science by addressing several of the current technological and methodological challenges in x-ray diffractive imaging of biological samples with XFELs, in particular in the areas of sample preparation for membrane proteins that, generally, suffer from low abundance and/or are hard to crystallize, sample introduction technologies enabling high data acquisition rates, and novel approaches to time-resolved structure determination of membrane proteins. This work will also drastically reduce sample consumption and will increase the diversity of membrane protein and other biological nano- objects that can be studied with XFELs. The proposed work also aims to develop new capabilities for time- resolved structural studies at XFELs to include cross-membrane potential triggered membrane protein dynamics, enabling investigation of a broader range of biomolecular and cellular reactions and the associated structural change over a large range of times scales from microseconds to milliseconds. If successful, this work would greatly aid our experimental capabilities to study and understand function of protein complexes and biological nanoparticles in a wide range of fields including human health and biosecurity.