Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

Abstract

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

Keywords: lipidic cubic phase; membrane proteins; protein crystallization; serial crystallography.

Figure 1

LCP microcrystallization in glass syringes. (a) Crystals of ba ₃-type cytochrome c oxidase visualized at 7.5× magnification in a stereo microscope. (b) At a higher magnification of 15, crystals can only be visualized in some parts of the LCP owing to the shape of the syringe and the orientation of the LCP string. Poor focus makes it difficult to determine the crystal quality, shape and size.

Figure 2

LCP microcrystallization in glass wells. (a) A string of LCP suspension (here 40 µl) is dispensed into each well of a nine-well glass plate. Crystals of ba ₃-type cytochrome c oxidase are visualized in a stereo microscope after three days of incubation at 20°C (b) under polarized light at a magnification of 40 and at magnifications of (c) 50 and (d) 135. Crystal formation can be monitored over time and the crystal quality, shape and density are clearly visible.

Figure 3

Well-based crystallization procedure. (a) A short needle is connected to the syringe to dispense the LCP suspension into the wells of a nine-well glass plate for crystallization. (b) The microcrystals are collected by pooling the LCP strings from different wells with a plunger. The LCP blob is transferred (c) from the precipitant solution to (d) a 500 µl Hamilton syringe from the back. (e) A 500 µl plunger is inserted into the syringe and slowly pushed forward to remove any residual precipitant solution. (f) A packed syringe of ba ₃-type cytochrome c oxidase crystals.

Figure 4

Cytochrome c oxidase electron-density maps. Electron-density maps calculated from the BioMAX CcO data extending to 3.6 Å resolution. (a) 2F _o − F _c electron-density map contoured at 1.0σ around haem a ₃. (b) OMIT map calculated around haem a ₃ using phenix.polder (Liebschner et al., 2017 ▸) contoured at 3.0σ around haem a ₃.

Figure 5

LCP microcrystals of other target proteins produced in wells. (a) SRII microcrystals 10–40 µm in size produced without purple membrane that diffract to 2.7 Å resolution at a synchrotron. (b) Microcrystals of bacterial reaction center of around 50 µm in size that diffract to 2.4 Å resolution at an XFEL.