Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases

Technology

Crystallography

Summary

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.

Description

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. JoVE video articles describing the method developed by the Membrane Structural & Functional Biology (MS&FB) Group are available (Caffrey & Porter, 2010; Li, et al., 2012).

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 (Cherezov, et al., 2004; Peddi 2007). The other two have recently become available and are included here for completeness.

Publication

Caffrey M, Porter C. Crystallizing membrane proteins for structure determination using lipidic mesophases. J. Vis. Exp. 45:e1712 (2010). PubMed ID: 21113125 | PMC Link

Li D, Boland C, Walsh K, Caffrey M. "Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases." J. Vis. Exp. 67:e4000 (2012). PubMed ID: 22971907 | Search SBKB Publications portal | PMC Link

Cherezov V, Peddi A, Muthusubramaniam L, Zheng YF, Caffrey M. A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Crystallogr. D Biol. Crystallogr. 60:1795–1807 (2004). PubMed ID:15388926

Peddi A. High-throughput automated system for crystallizing membrane proteins in lipidic mesophases. IEEE Trans. Autom. Sci. Eng. 4:129–140 (2007).

Contact

Martin Caffrey, Email: martin.caffrey@tcd.ie

Center for Membrane Proteins in Infectious Diseases

Link




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Last edited:Mon 07 Oct 2013 - 3 years, 6 months ago