Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography

Abstract

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.

A guide to sample delivery systems for serial crystallography

“… implemented an LCP‐SMX experiment on the ESRF microfocus beamline and obtained the structure of the light‐driven proton pump bacteriorhodopsin (bR) with a resolution of 2.4 Å. The authors also carried out LCP‐SFX experiments on a LCLS using the same sample . In 2017, Martin‐Garcia carried out an SMX experiment based on HVE technology at the Advanced Photon Source.…”

A Bright Future for Serial Femtosecond Crystallography with XFELs

“…One of the most successful applications of SFX has been the structural study of membrane proteins using LCP as the crystallization and crystal delivery medium, which led to structure determination of several GPCRs [13–19] including the signaling complex between GPCR and arrestin [20], the membrane enzyme diacylglycerol kinase [102], and the light-activated proton pump bacteriorhodopsin [103]. …”

Microfluidics: From crystallization to serial time-resolved crystallography

“…The fast flowrates of GDVN injectors are such that time delays longer than $100 ls would require sample illumination within the jet itself 22,141 or the use of a different sample delivery method. For example, the LCP injector has been used to facilitate the time-resolved analysis of the light sensitive membrane protein bacteriorhodopsin, 27,174 and a fixed-target approach utilizing a silicon mesh with mylar windows was used to study CO dissociation from myoglobin. 25 More recently, a combination of electrokinetic injection 164 and an on-demand droplet injector with a conveyor-belt system 169 was used with multiple laser pulses to drive the activation of varying states of photosystem II at delay times ranging from 0.5 s to 1 s. 166,167 B.…”

“…Using positive displacement extrusion, a variety of high viscosity materials including the lipidic cubic phase (LCP) matrix used in membrane protein crystallization (Figure 7(d)), 27,141,147,161,163,[171][172][173][174] a grease matrix, [175][176][177] agarose, 178 and hyaluronic acid have been used as carrier materials for serial crystallography. 179 In these methods, the aqueous slurry of crystals is mixed with the higher viscosity component.…”

“…[15][16][17][18] Furthermore, these approaches have enabled timeresolved structural determination for a much broader range of targets than had previously been accessible. [19][20][21][22][23][24][25][26][27][28][29][30] However, these large-scale serial methods suffer from the need to manipulate crystals and/ or from inefficient sample utilization. 3,[31][32][33][34][35][36] The material-intensive nature of these experiments extends the long-standing challenge of growing a well-diffracting crystal to reproducibly generating a large number of high quality, isomorphous crystals.…”