A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization

Wöhri, Annemarie B.; Johansson, Lars Gunnar; Wadsten-Hindrichsen, Pia; Wahlgren, Weixiao Yuan; Fischer, Gerhard W.; Horsefield, Rob; Katona, Gergely; Nyblom, Maria; Öberg, Fredrik; Young, Gillian; Cogdell, Richard J.; Fraser, Niall J.; Engström, Sven; Neutze, Richard

doi:10.1016/j.str.2008.06.003

Cited by 61 publications

(60 citation statements)

References 33 publications

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“…Thus, the sponge phase fails to retain its original shape and its edges are characteristically smooth. Precipitants that include Jeffamine, PEG 400, 2-methyl-2,4-pentandiol, pentaerythritol propoxylate, butanediol and hexanediol, can result in a transition from the cubic to the sponge phase 4,26 . …”

Section: Representative Resultsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Boland

Aragão

et al. 2012

JoVE

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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...

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Section: Representative Resultsmentioning

confidence: 99%

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confidence: 99%

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

Boland

Aragão

et al. 2012

JoVE

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“…In meso method Crystallization in lipidic phases has only recently been developed but has already become an essential tool in the arsenal of membrane protein crystallization, especially for GPCRs [70,71] . The cubic phase is a bicontinuous lipidic meso phase formed spontaneously by mixing monoacylglycerols (MAGs) and water at a given ratio [72] ( Figure 4B).…”

Section: Bicelle Methodsmentioning

confidence: 99%

Ice breaking in GPCR structural biology

Zhao

2012

Acta Pharmacol Sin

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G-protein-coupled receptors (GPCRs) are one of the most challenging targets in structural biology. To successfully solve a high-resolution GPCR structure, several experimental obstacles must be overcome, including expression, extraction, purification, and crystallization. As a result, there are only a handful of unique structures reported from this protein superfamily, which consists of over 800 members. In the past few years, however, there has been an increase in the amount of solved GPCR structures, and a few highimpact structures have been determined: the peptide receptor CXCR4, the agonist bound receptors, and the GPCR-G protein complex. The dramatic progress in GPCR structural studies is not due to the development of any single technique, but a combination of new techniques, new tools and new concepts. Here, we summarize the progress made for GPCR expression, purification, and crystallization, and we highlight the technical advances that will facilitate the future determination of GPCR structures.

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“…An advantage of the L 3 approach is that the liquid properties of the sponge phase at room temperature can be used directly in hanging-or sitting-drop vapor-diffusion crystallization by commercially available robots. Recently, a sponge phase sparse matrix crystallization screen consisting of different conditions became available [30]. However, unlike the LCP method, this one has not led to a breakthrough in structural biology of membrane protein.…”

Section: Crystallization From Sponge Phases (L 3 -Phase)mentioning

confidence: 99%