Serial Femtosecond Crystallography of Optogenetic Function

Lead Research Organisation: Imperial College London
Department Name: Life Sciences


The ability to design synthetic light-sensitive materials that can be genetically encoded provides biologists the means and opportunity to sense and control biological function and environment. In order to learn how these materials function it has very recently become possible to make very fast 'snapshots' of the light-induced motions using X-ray crystallography, which is even possible for very short time scales that still involves the excited electrons (on femtosecond time scale). Novel 4th generation light sources can now be used to record such 'molecular movies' which is a breakthrough technology that we only very recently demonstrated. In the very near future we anticipate being able to record even better quality movies, as the European XFEL in Hamburg that allows much more data to be recorded will start user operation.
Where previously we could only use laser spectroscopy techniques we can now actually 'watch' the very first motions of proteins directly after activation. It is known that the 'outcome' of biological reactions involving either sensing or activation (via 'actuation') is determined in these very first motions that occur on time scales typically less than picoseconds. For example, in the case of a bi-directional photochromic fluorescent protein the efficiency of switching in one direction is excellent at 30%, while the efficiency of switching in the reverse direction is poor at less than 0.05%, potentially limiting optogenetics applications. We are thus in a position to ask the question what determines this dramatic difference in the outcome of the reactions, and provide structural and dynamical information that will be highly valuable for feeding back for rational optogenetics design.

Technical Summary

The objective of this proposal is to develop and execute a set of new time resolve X-ray crystallography experiments using the specific technique of Serial Femtosecond Crystallography, in order to follow the ultrafast motions of optogenetics materials with biological importance. Combined with femtosecond optical excitation and cross-correlation of arrival time (time stamping) data binning allows the reconstruction of femtosecond time resolved 'snapshot' pump-probe datasets that together form molecular movies. Everything is in place to proceed with newly identified targets that are selected from the optogenetics field on the basis of their biological significance and impact.The M13-cpGFP-CaM chimera, GEM-GECO1, is a synthetic fusion including a fluorescent protein, Calmodulin and an M13 helical domain that confers an optical readout of the cellular Ca2+ concentration which is an important physiological messenger. Published ultrafast Raman spectroscopy has shown time scales and motions associated with protein vibrations as well as the proton transfer that affects the measured steady state Stokes Shift, are excellent targets for femtosecond time resolved pump-probe TR-SFX measurements. We will develop suitable microcrystals of the construct, using seeded batch-crystallisation techniques that are also successful for photochromic fluorescent proteins or alternatively search for conditions using the liquid handling robots at Imperial. We will make the necessary optical measurements of the linear and non-linear multi-photon transformation in crystalline samples, using methods and instrumentation previously reported and used for recent successful TR-SFX of the photoactive yellow protein. These will establish the suitable optical parameters for pumping the microcrystals at the XFEL beamlines. At the Eu-XFEL, an X-ray crystallographic equivalent of an impulsive Raman spectroscopy experiment is envisioned, which retrieves frequency resolved high bandwidth vibrational coherence

Planned Impact


This proposal concerns the relatively fundamental research on the development of novel XFEL based measurements of optogenetics materials. The research addresses BBSRC the two priority areas in Synthetic biology (optogenetics) as well as in Technology developments in the biosciences (TR-SFX). Designing and modifying optogenetics materials is a key area in synthetic biology as it allows Engineering cells/organisms to include systems or parts not found in nature to impart new capacities or chemistry. For technology developments this proposal demonstrates strong multidisciplinary partnerships between bioscientists and researchers in the physical sciences, engineering and information technology disciplines.

The case for national economic importance is made two-fold. Firstly, the research is proven high-impact and at the forefront of XFEL science. This work will thus strengthen the UK competitiveness internationally. Secondly, the successful outcomes of early science in the first few years of user operation of the currently operating hard X-ray FELs (LCLS and SACLA) contributes to the UK scientific case for a national facility that is currently being developed (

The multidisciplinary work involves researchers from various backgrounds, and collaborations at XFEL stations with scientists and engineers that provide excellent training opportunities for PDRAs and participating students and collaborators. The PI has established collaboration with many groups and people working in the XFEL and ultrafast spectroscopy fields. JvT collaborates with Anders Madsen at the European XFEL MID beamline (see van Thor and Madsen, 2015, Structural dynamics), which provides further opportunities for collaborative and developmental activities involving instrumentation development and theory. Other collaborations and associations are with the UK XFEL Hub (Allen Orville), Diamond-Imperial (Isabel Moraes and So Iwata), RIKEN (So Iwata), Argonne/APS BioCARS (Keith Moffat), the BioXFEL consortium (Petra Fromme, John Spence) and others. This provides an environment for participants in this research which is excellent and at the forefront of the field.

Knowledge and Outreach

The knowledge generated by the proposed experiments will be communicated through the standard routes: research papers and conference presentation, but in addition also press releases and outreach. The home page ( shows a link to a news item that describes our recent TR-SFX publication in Science.
The PI has a strong track record in publishing in top tier journals, and is a frequent presenter at international conferences (
In addition, the PI has organised international conferences, in London and Telluride (the TSRC conference on Protein Dynamics).


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Description Ultrafast X-ray crystallography of the photoactive yellow protein with femtosecond delays using an X-ray free electron laser has successfully probed the dynamics of an early Franck-Condon species. The femtosecond pump-probe application of protein crystallography represents a new experimental regime that provides an X-ray structural probe for coherent processes that were previously accessible primarily using ultrafast spectroscopy. We address how the optical regime of the visible pump, that is necessary to successfully resolve ultrafast structural differences, affects the motions that are measured using the technique. The sub-picosecond photochemical dynamics in proteins involves evolution of a mixture of electronic ground and excited state populations. Additional to photoisomerisation that is considered to proceed through activated barrier crossing, within the dephasing time structural motion include vibrational coherence arising from excited states, the ground state and a ground state intermediate under experimental conditions used for ultrafast crystallography. Intense optical pulses are required to convert population levels in crystals that allow detection by X-ray crystallography, but the compromise currently needed for the optical bandwidth and power has consequences with regard to the contributions to the motions that are experimentally measured with femtosecond delays
We have been developing methods of analysis for the emerging capabilities of ultrafast time resolved crystallography, which have been reported in several research articles (van Thor, 2019, Hutchison and van Thor, 2019, Hutchison and van Thor, 2017, Sanches-Gonzales et al., 2017, Hutchison et al., 2017). We have proposed a new analysis methods to extract population information from ultrafast crystallography observation (van Thor, 2019, Hutchison and van Thor, 2019). The method involves a transform from the X-ray coordinate frame to the optical frame where knowledge of intrinsic birefringence and non-linear cross section allows a direct population determination. Additional methodology and theory that has been reported includes a theoretical calculation of coherence parameters measured in real space through coordinate refinement and adds the ability for optical control using modification of the laser field parameters.
Exploitation Route Femtosecond resolution pump-probe experiments are now routinely carried out at X-ray Free Electron Lasers, enabled by the development of cross-correlation "time-tools" which correct the picosecond-level jitter between the optical and X-ray pulses. These tools provide very accurate, <10 fs, measurement of the relative arrival time, but do not provide a measure of the absolute coincidence time in the interaction. Cross-correlation experiments using transient reflectivity in a crystal are commonly used for this purpose, and to date no quantitative analysis of the accuracy or stability of absolute coincidence time determination has been performed. We have performed a quantitative analysis of coincidence timing at the SACLA facility through a cross-correlation of 100 ± 10 fs, 400 nm optical pulses with 7 fs, 10.5 keV X-ray pulses via transient reflectivity in a cerium-doped yttrium aluminum garnet crystal. We have modelled and fit the transient reflectivity, which required a convolution with a 226 ± 12 fs uncertainty that was believed to be dominated by X-ray and laser intensity fluctuations, or assuming an extinction depth of 13.3 µm greater than the literature value of 66.7 µm. Despite this, we are able to determine the absolute coincidence time to an accuracy of 30 fs. We discuss the physical contributions to the uncertainty of coincidence time determination, which may include an uncharacterised offset delay in the development of transient reflectivity, including cascading Auger decays, secondary ionisation and cooling processes. Additionally, we present measurements of the intrinsic short-term and long-term drifts between the X-rays and the optical laser timing from time-tool analysis, which is dominated by a thermal expansion of the 25 m optical path between tool and the interaction region, seen to be ~60 fs over a period of 5 h.
Sectors Energy