Abstract
Revealing the structure of complex biological macromolecules, such as proteins, is an essential step for understanding the chemical mechanisms that determine the diversity of their functions. Synchrotron based X-ray crystallography and cryo-electron microscopy have made major contributions in determining thousands of protein structures even from micro-sized crystals. They suffer from some limitations that have not been overcome, such as radiation damage, the natural inability to crystallize a number of proteins, and experimental conditions for structure determination that are incompatible with the physiological environment. Today, the ultra-short and ultra-bright pulses of X-ray free-electron lasers have made attainable the dream to determine protein structures before radiation damage starts to destroy the samples. However, the signal-to-noise ratio remains a great challenge to obtain usable diffraction patterns from a single protein molecule. With the perspective to overcome these challenges, we describe here a new methodology that has the potential to overcome the signal-to-noise-ratio and protein crystallization limits. Using a multidisciplinary approach, we propose to create ordered, two dimensional protein arrays with defined orientation attached on a self-assembled-monolayer. We develop a literature-based flexible toolbox capable of assembling different kinds of proteins on a functionalized surface and consider using a graphene cover layer that will allow performing experiments with proteins in physiological conditions.
Highlights
With the perspective to overcome these challenges, we describe here a new methodology that has the potential to overcome the signal-to-noise-ratio and protein crystallization limits
Using a multidisciplinary approach, we propose to create ordered, two dimensional protein arrays with defined orientation attached on a self-assembled-monolayer
We develop a literature-based flexible toolbox capable of assembling different kinds of proteins on a functionalized surface and consider using a graphene cover layer that will allow performing experiments with proteins in physiological conditions
Summary
For 2D cryo-TEM studies, most often the protein crystals are grown embedded within a lipid bilayer, whereas the first XFEL experiments used dices of SiN windows for harvesting 2D crystals, which are covered with a thin C film These first experiments indicated that in order to overcome the present resolution limits of 7 Aand truly exploit the unique XFEL properties, an improvement of both sample preparation and data analysis is of the essence. In this perspective, we put forward a new sample delivery method based on fabricating sample supports for hosting the target protein in a near-native environment. We present an experimental approach based on free electron laser diffraction before destruction, with the final aim of our work being the determination of the protein structure in physiological conditions
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