Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Abstract

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases [1][2][3][4][5] , has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field 6-21 (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting 22,23 . Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2) 24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies 4,26 . The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases. Video LinkThe video component of this article can be found at https://www.jove.com/video/4001/ Protocol 1. Laboratory Set-up Pre-harvesting 1. In preparation for harvesting, fill the dry foam Dewar with liquid nitrogen and place it beside the microscope where harvesting is to take place. 2. Submerge the storage puck, open end up, in the liquid nitrogen inside the foam Dewar and allow it to fully cool. 3. Secure a micro-mount of a size...

Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

“…Due to the relatively high concentration of MPD in the precipitant, the cubic mesophase, as expected, converted to the more fluid sponge phase from which harvesting crystals can be a challenge. However, using specialized methods developed for this purpose [20], crystals were successfully recovered, snap-cooled in liquid nitrogen and used for X-ray diffraction data collection with a synchrotron micro-beam (Table 2). Phasing was done by molecular replacement with the model of the corresponding cell-based Δ7 DgkA construct.…”

“…Wells were opened with a glass cutter and the crystals were harvested with 30-50 μm MiTeGen micromounts at 4-6 °C. Crystals were snap-cooled directly in liquid nitrogen without added cryo-protectant [20]. …”

Crystal structure of the integral membrane diacylglycerol kinase

“…Wells were opened with a tungsten-carbide glass cutter (Silverline) and the crystals were harvested using 30 - 100 μm micromounts (MiTeGen) at 4-6 °C. Crystals were snap-cooled directly in liquid nitrogen 32 .…”

“…When a second soaking was required excess bathing solution was wicked away with tissue paper 32 , fresh solution with or without additive was added through the window, and the well was sealed with ClearSeal film (Hampton). Crystals were harvested as described above.…”

Femtosecond crystallography of membrane proteins in the lipidic cubic phase

“…Some of the most important and influential developments during the past 15 years include (i) lipid syringe mixer for fast and efficient mixing of lipids with MP solution allowing for the reconstitution of MPs in LCP within minutes [13]; (ii) LCP crystallization robot that automates and miniaturizes LCP crystallization set-up [14]; (iii) glass sandwich plates that improve detection of small colourless crystals growing in LCP [14,15]; (iv) new LCP host lipids for tailoring lipid bilayer properties, such as thickness and curvature, towards specific MP properties, and to facilitate crystallization at specific conditions, such as low temperatures [16,17]; (v) high-throughput fluorescence recovery after photobleaching (LCP-FRAP) pre-crystallization assay to assess diffusion properties of MPs in LCP at different conditions and guide subsequent crystallization trials [18]; (vi) thermostability assay, LCP-Tm, to compare stability of MPs directly in LCP, enabling the selection of the most stabilizing host lipids, lipid additives, protein constructs and ligands, in order to increase the likelihood of successful crystallization [19]; and (vii) second-order nonlinear imaging of chiral crystals (SONICC), which is used to detect submicrometre-sized protein crystals [20]. Commercial availability of many of these tools and instruments, as well as published detailed protocols [21,22] and video demonstrations [23][24][25], have enabled relatively straightforward adaptation of these technologies in different structural biology laboratories, resulting in the increased number of MP structures determined by this method.…”