Rational design of lipid for membrane protein crystallization

Misquitta, Y.; Cherezov, Vadim; Havas, F.; Patterson, Suzanne; Mohan, Jakkam M.; Wells, Angela J.; Hart, David J.; Caffrey, Martin

doi:10.1016/j.jsb.2004.06.008

Cited by 70 publications

(74 citation statements)

References 24 publications

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“…Thus, monoolein as the hosting lipid in a metastable phase state can be used at 4 °C 1,2,29,30 . An alternative is to use the rationally designed 7.9 MAG for low temperature crystallization 31 . We do low temperature crysallogenesis routinely with certain membrane protein targets.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…It was not until the introduction of new tools, simplified protocols and instrumentation that LCP started to show its potential of delivering high-resolution structures of MPs from different families, in particular, human GPCRs [7]. 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.…”

Section: Crystallization Of Membrane Proteins In Lipidic Cubic Phasementioning

confidence: 99%

“…Changing co-flowing gas from helium to nitrogen reduced Lc phase formation, but did not entirely prevent it. The problem was subsequently overcome by identifying two homologous lipids with shorter chains, 7.9 MAG [16] and 9.7 MAG (monopalmitolein), both of which form LCP that does not transform into Lc phase upon injection into vacuum. 7.9 MAG was specifically designed for low temperature LCP crystallization, as its equilibrium Lc-to-cubic Pn3m phase transition temperature of 68C is among the lowest for the MAG series.…”

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Femtosecond crystallography of membrane proteins in the lipidic cubic phase

Liu

Wacker

Wang

et al. 2014

Phil. Trans. R. Soc. B

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Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in highresolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins.

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“…They constitute a successful hosting lipid screen by the MS&FB group. With several targets, that include β-barrels, α-helical proteins (see below) and an integral peptide antibiotic, crystals have been grown by the in meso method using these alternative MAGs [34,[42][43][44]. In a number of cases, mono-olein either failed to produce crystals or the crystals it did produce were not of diffraction quality.…”

Section: Host Lipid Screeningmentioning

confidence: 99%

Crystallizing Membrane Proteins for Structure-Function Studies Using Lipidic Mesophases

Caffrey¹

2013

Advancing Methods for Biomolecular Crystallography

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The lipidic cubic phase method for crystallizing membrane proteins has posted some high-profile successes recently. This is especially true in the area of G-protein-coupled receptors, with six new crystallographic structures emerging in the last 3 1/2 years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years, we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico-to nano-litre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis, such that the method can now be applied to a wide variety of membrane protein types and sizes. In the present article, these assorted advances are outlined, along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to develop the method further are described.

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Rational design of lipid for membrane protein crystallization

Cited by 70 publications

References 24 publications

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

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

Femtosecond crystallography of membrane proteins in the lipidic cubic phase

Crystallizing Membrane Proteins for Structure-Function Studies Using Lipidic Mesophases

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