Li Dianfan, Boland Coilín, Walsh Kilian, Caffrey Martin
Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry & Immunology, Trinity College Dublin, Ireland.
J Vis Exp. 2012 Sep 1(67):e4000. doi: 10.3791/4000.
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(1-3). 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.
膜蛋白的结构-功能研究极大地受益于通过大分子X射线晶体学（MX）获得的高分辨率三维结构。MX的一个基本要素是稳定供应理想的具有衍射质量的晶体。用于膜蛋白结晶的介观或脂质立方相（LCP）方法是几种可用的膜蛋白结晶方法之一。它利用双连续中间相来生长晶体。作为一种方法，它最近取得了一些显著的成功，并引起了很多关注，现在有许多研究小组对使用它感兴趣。与该方法相关的挑战之一是主体中间相极其粘稠且有粘性，让人联想到浓稠的牙膏。因此，以可重复的方式将少量的中间相手动分配到结晶孔中需要技巧、耐心和稳健的手法。膜结构与功能生物学（MS&FB）小组开发了一种这样做的方案（1-3）。已有描述该方法的JoVE视频文章（1,4）。手动设置介观试验的方法在特殊应用方面具有明显优势，例如晶体优化和衍生化。然而，它确实是一种低通量方法。在这里，我们展示了一种用于自动进行介观结晶试验的方案。机器人具有速度快、准确性高、精度高、小型化以及能够在诸如黑暗、还原气氛或低温或高温等可视为恶劣条件下连续长时间工作的优点。一个介观机器人如果使用得当，可以通过促进结晶来极大地提高膜蛋白结构和功能研究的效率，结晶是整个结构测定流程中的缓慢步骤之一。在这篇视频文章中，我们展示了三种可商购的机器人的使用，这些机器人可以分配介观晶体形成所必需的粘性中间相。第一种机器人是由MS&FB小组开发的（5,6）。另外两种最近才可用，为了完整性在此一并介绍。本文涵盖的方案概述如图1所示。所有操作均在室温（约20°C）的环境条件下进行。
