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

Date Published: October 01, 2019

Publisher: International Union of Crystallography

Author(s): Rebecka Andersson, Cecilia Safari, Petra Båth, Robert Bosman, Anastasya Shilova, Peter Dahl, Swagatha Ghosh, Andreas Dunge, Rasmus Kjeldsen-Jensen, Jie Nan, Robert L. Shoeman, Marco Kloos, R. Bruce Doak, Uwe Mueller, Richard Neutze, Gisela Brändén.

http://doi.org/10.1107/S2059798319012695

Abstract

A novel method is presented to screen for suitable crystallization conditions and produce large amounts of microcrystals of membrane proteins in lipidic cubic phase for serial crystallography experiments.

Partial Text

Reconstitution of the protein into LCP is achieved using standard protocols, in which the protein is mixed with an appropriate host lipid such as monoolein in a suitable ratio, often between 40% and 50% of aqueous phase for most lipids, in two Hamilton gas-tight 100 µl syringes until the suspension is homogeneous and transparent (Liu et al., 2014 ▸). A nine-well glass plate is prepared with 0.1–1 ml precipitant solution in each of the wells. A short removable needle such as a Mosquito LCP narrow-bore needle is connected to the syringe with the LCP suspension and a string of between 5 and 50 µl of LCP suspension is dispensed into each well [Fig. 3 ▸(a)]. The glass plate is then sealed with a ClearVue plastic sheet cut to an appropriate size to fit inside the rim of the plate. This makes it possible to cut open an individual well without disrupting the rest of the plate. During the process of screening for optimal crystallization conditions, it is recommended to start by varying the protein concentration of the LCP and the precipitant concentration in the wells. For this, it is suitable to dispense a small amount (∼5 µl) of LCP per well. Different ratios of LCP to precipitant solution affect the crystallization outcome and should be investigated for each protein. Thus, as a next step, the effect of varying the volume of the crystallization solution in each well, ranging between 0.1 and 1 ml, and the amount of LCP per well, ranging between 5 and 50 µl, should be investigated. This is typically performed in steps of 0.1 ml and 5–10 µl, respectively. If further fine-tuning is needed, the thickness of the LCP string can also be varied by changing the gauge size of the needle that is connected to the LCP-containing syringe.

We present a novel method to screen for optimal conditions and produce large amounts of membrane-protein microcrystals in LCP in nine-well glass plates for serial crystallo­graphy experiments at XFELs and synchrotrons. Compared with the standard procedure of crystallization in glass syringes, this allows easy visualization of the progress of crystallization without interrupting the process. With better focus and higher magnification, crystallization trials can be monitored and evaluated more easily with regard to crystal quality, shape and density (compare Fig. 2 ▸ with Fig. 1 ▸). The well-based system can be scaled up to produce large volumes of LCP microcrystals that can be collected and packed into a Hamilton glass syringe, as shown in Fig. 3 ▸.

The development of serial crystallography at XFELs and synchrotrons has opened up completely new possibilities within structural biology, as we can study protein structures at room temperature and investigate structural dynamics using time-resolved experiments. A severe limitation for these experiments is the need for large amounts of well ordered microcrystals. Membrane proteins are in many cases particularly difficult to crystallize. For a number of membrane proteins, high-resolution crystal structures have successfully been solved from crystals formed using LCP-reconstituted protein (Cherezov et al., 2007 ▸; Zhang et al., 2015 ▸; Johansson et al., 2019 ▸). As an added advantage for serial experiments, the LCP matrix provides a suitable carrier medium for slow-running high-viscosity injectors. There is, however, a need to simplify the process of producing large amounts of LCP microcrystals for serial crystallography experiments.

 

Source:

http://doi.org/10.1107/S2059798319012695

 

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