Obtaining movies of molecular movement of proteins through serial femtosecond crystallography

Seeing is believing; thus, it would be much better if we could observe real-time molecular level movement of proteins during catalysis, where, for example, binding of a substrate to an active site leads to molecular cleavage and enzymatic action. But, this feat is not realized until recent advent of serial femtosecond crystallography (SFX).


In an article in Scientific American, May 2017, pp. 58-61, “Split second reactions”, a technique for capturing the molecular motions of proteins and other biomolecules by pulsing them with ultrashort duration X-ray pulses, which when reflected and scattered from the nanometre sized crystals, would generate sufficient data for the reconstruction of the protein structure at a specific moment in time. By stitching the different structural pictures of the proteins at different time points, a molecular movie depicting the motions and movements of the protein during catalysis could be obtained.


In realizing the technique, researchers solved four major problems: (i) how to pulse ultrashort duration X-ray pulses in the femtosecond timescale, (ii) how to align a stream of protein nanocrystals into a single file for X-ray diffraction, (iii) developing the optics for capturing the multitude of reflections and scattering of X-ray pulses from the protein nanocrystals, and (iv) developing data analysis algorithms and software for converting the large trove of scattering and reflection data into molecular pictures useful for understanding the molecular details of biocatalysis or photosynthesis or drug action.


Given the need to generate high energy X-rays that could destroy the protein crystals, the method is currently only limited to large accelerator facilities such as the Linac Coherent Light Source in California, USA. More importantly, the need to analyse large amount of data puts the technique out of reach of the common laboratory as experts and specialists are needed for solving the multitude of crystal structures obtained.


On the other hand, the ability of SFX to obtain structural details of protein crystals of nanometre sized range meant that the technique opens up a large set of small protein crystals for analysis, which are previously deemed to be too small to yield useful data. While high energy X-rays tend to destroy the protein crystals, the use of ultrashort duration femtosecond X-ray pulses help solves the problem as the method only result in small incremental damage to the crystals. However, accumulation of multiple damage from a string of X-ray pulses meant that the technique is a destructive technique, and the protein crystals used are all eventually destroyed by the strong X-ray pulses.


Hence, understanding the molecular details of protein motion in catalysis or substrate binding has been the long sought after dream of protein crystallography, and a technique, serial femtosecond crystallography is available to help capture the multitude of reflections and scatterings of a stream of protein nanocrystals placed in the injector path of the accelerator based method. While allowing high quality movies of molecular motion to be stitched together using what is essentially a protein crystallography technique, the method is not available to most laboratories given the need for specialized equipment. However, development of shared facilities at accelerator complexes could help make this useful but data rich technique available to more investigators and researchers keen to explore the molecular details of enzymatic action. Finally, by enabling crystal structures to be obtained from nanocrystals, the method help opens up the field of protein crystallography, which is hampered by the inability to obtain good quality structures from small crystals, thereby, preventing exploration of a suite of questions that hinges on the resolution of a protein structure.


Category: instrument, structural biology, physics, chemistry,

Tags: serial femtosecond crystallography, data analysis, nanocrystals, linear accelerator, X-ray diffraction, destructive technique, protein crystallography,


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