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Towards Femtosecond Electron Diffraction of Proteins

Final Report Summary - PROTEINFED (Towards Femtosecond Electron Diffraction of Proteins)

Proteins are complex molecular machines, facilitating all types of functions from enzymatic catalysis to signal transduction. Despite the availability of numerous biophysical techniques, such as NMR spectroscopy, fluorescence, X-Ray crystallography combined with theoretical modelling approaches, an experimental approach to study proteins with full spatial and temporal resolution remains lacking. Crystallography is the only technology providing full atomic resolution with the prospect of a time-resolved experiment over several orders of magnitude.

The goal of this project was to explore possibilities and limitations of ultrafast time-resolved crystallography experiments with electrons and X-Rays as structural probes.

The focus was on the novel technique femtosecond electron diffraction, a table-top experimental pump-probe technique, and its potential application for the study of protein dynamics in comparison to the study of ultrafast structural dynamics by time-resolved femtosecond X-ray crystallography using XFELs, large-scale research facilities.

At the start of the project, femtosecond electron diffraction had been recently applied to the study of a first organometallic, two-dimensional material, raising the hope of further studies of organic and biological samples. Electrons strongly interact with any kind of matter, leading to specific sample requirements, different from electron microscopy or X-ray crystallography. Membrane proteins, such as the GPCR analogue Bacteriorhodopsin, are candidates to match those requirements due to the possibility to prepare two-dimensional crystals. This family of proteins is extremely important as pharmacological target, demonstrating the tremendous potential and implications of electron diffraction experiments for the study of protein function with implications for life science and healthcare applications.

An important prerequisite for all variants of time-resolved experiments is efficient and precise sample delivery. We were able to demonstrate the successful usage of a fixed target matrix for time-resolved serial femtosecond crystallography at two XFELs in the US and Japan. This photo-crystallography chip is a nanofabricated silicone based chip with >10000 individual features for trapping protein crystals in random orientation. The design of the chip allows background free detection and low sample consumption. High hit rates with an overall rate of >50% indexable diffraction now allow collection of complete data for structure determination very quickly [1]. We adapted this serial crystallography approach in experiments at synchrotrons in the UK, Germany and recently the US.
Our approach, serial synchrotron crystallography (SSX), minimizes radiation damage and thus allows experiments at room temperature and enables the determination of protein structures under their native conditions [2]. This is broadly applicable to better understand protein function.
During XFEL experiments at SACLA we have measured ultrafast protein dynamics in a model system successfully, gaining a first glimpse of the full atomic and time-resolved structural changes important for biological function (data analysis and preparation for publication in progress).

The outgoing phase of the project was performed at the University of Toronto, jointly in the Department for Biochemistry and Chemistry, hosted by Prof. Oliver Ernst, while the return phase was spent at the Max-Planck-Institute for Structure and Dynamics of Matter/Center for Free Electron Laser Science in the department of Prof. R. J. Dwayne Miller in Hamburg. The researcher aims to build her independent research career on the achieved results at her new positions as assistant professor in physical chemistry at University of Potsdam from September 2017 on.