Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Abstract

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group [1][2][3] . JoVE video articles describing the method are available 1,4 . The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline.In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group 5,6 . The other two have recently become available and are included here for completeness.An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions. Video LinkThe video component of this article can be found at https://www.jove.com/video/4000/ Protocol Preparing the Crystallization PlateSetting up to do a crystallization trial robotically begins with the preparation of the base-plate of the glass sandwich crystallization plate (Figure 2), described in detail in Reference 2. The base-plate must first be silanized and the perforated double-stick spacer that creates the wells, must be applied to the plate. The materials and supplies needed for this are itemized under Materials.1. Place the plate on a paper towel, apply a few drops of silanizing solution and distribute it evenly over the plate surf...

Crystal structure of the integral membrane diacylglycerol kinase

“…The protein solution at 12 mg/mL was homogenized with 7.8 MAG in equal parts by volume using a coupled syringe device at room temperature (RT, 20 - 22 °C). Crystallization trials were set up by transferring 50 nL of the protein/lipid cubic mesophase onto a silanized 96-well glass crystallization plate which was subsequently covered with 800 nL precipitant solution using in meso robots 31 . Wells were sealed with a glass coverslide.…”

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

“…The protein solution at 12 mg/mL was homogenised with 7.8 MAG [9, 10] in equal parts by volume using a coupled syringe mixing device at RT. Crystallisation trials were set up by transferring 50 nL of the protein/lipid cubic mesophase onto a silicanised 96-well glass crystallisation plate which was subsequently covered with 800 nL precipitant solution using an in meso robot [19]. Wells were sealed with a glass coverslide and the plates were stored in a walk-in refrigerator at 4-6 °C for crystal growth.…”

Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography

“…The host lipid would be chosen based on selected microcrystal hits from high-throughput crystallization screening (described in Refs. 22–25). See sections on “Host Lipid Selection” and “Lipid Phase Behavior” for more information.…”

“…The first stage involves high-throughput crystallization screening in nanoliter volumes using special LCP robots in order to find suitable conditions that lead to a high rate of nucleation and the formation of a large number of microcrystals. Protocols for setting up high-throughput LCP crystallization trials and crystal detection have been published elsewhere 22–25 . These protocols are currently being routinely used in many laboratories.…”