Crystallization in gels

In the middle of the 80's, Marie-Claire Robert and Françoise Lefaucheux at the Laboratoire de ... originally implemented for small molecules [1]. Indeed, these ...
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Crystallization in gels: principles and practicle tips Claude Sauter, Bernard Lorber, Elodie Touzé, Abel Moreno, Adel Kadri, Anne Théobald-Dietrich & Richard Giegé UPR 9002 – CNRS – IBMC, 15 rue R. Descartes, F-67084 Strasbourg, France http://www-ibmc.u-strasbg.fr/arn/Giege/ Background

During the last decade, experiments have been carried out on biological objects ranging from small proteins like insulin to large particules like viruses, using different types of gels (mainly polysiloxane and agarose hydrogels) and various crystallization methods. As with small molecules, following positive effects have been observed:

In the middle of the 80's, Marie-Claire Robert and Françoise Lefaucheux at the Laboratoire de Mineralogie et de Cristallographie de Paris (LMCP) were the first to apply to the crystallization of hen lysozyme techniques originally implemented for small molecules [1]. Indeed, these physicists had previously shownon one hand that crystals of inorganics grown in a gel do not sediment because they are immobilized in the network and on the other that their overall quality may be enhanced. In a gel, convection is reduced and mass transfer is restricted to diffusion. Crystals grown in such a medium exhibit well shaped facets, larger volumes and possess better diffraction properties when compared to crystals grown in pure solution. The transfer of this technology from chemistry to biochemistry has opened the way to the preparation of better macromolecular crystals for crystallographic analyses in the frame of structural biology projects.

Batch experiment with hen lysozyme: (left) Crystals grow in a polysiloxane gel (0.8% w/v); (right) The interferogram reveals spherical depletion zones around the crystals in the absence of convection (Pictures: M.-C. Robert)

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Absence of convection Crystal growth in diffusive regime Absence of sedimentation (crystals in suspension in their mother liquor ) 3D crystal growth Mechanical protection of crystals during soaking, mounting, transport... Enhanced diffraction properties

Crystallization in gels is a simple, cost-effective and efficient way to optimize crystal growth conditions [2]. Below we give some practical tips for the preparation of macromolecular crystals in agarose gels.

Agarose gel in action

Gels for smoother soaking

Agarose is a very convenient gel to work with, especially when using a gel with a low gelling temperature (~28ºC) which is more compatible with the presence of heat-sensitive macromolecules. A 2% (w/v) agarose stock solution is stored at 4ºC. Before setting-up crystallization trials, this solution is melted at 85-90ºC (a) and kept at 35-45ºC (b). Then it is added to the crystallizing agent or to the macromolecule solution to a final concentration of 0.15-0.5% (w/v). This mixture can either be used in vapor diffusion, dialysis, batch, free interface diffusion (FID) or counter-diffusion.

In this example, a substrate analogue was soaked into DRS-1 crystals (Moreno et al., in preparation). Crystals grown by vapor-diffusion in solution started to crack after 2h (A). In contrast, crystals prepared in the presence of 0.2% agarose gel (w/v) did not show any apparent damage even after 15d of soaking (B). Their diffraction pattern (C) reveal sharp spots extending beyond 2 Å resolution (ESRF – beamline ID14-1), leading to the observation of the substrate bound to the enzyme active site (D). The same idea can be applied to heavy atom or cryoprotectant soaking. A

B

C

D

Low gelling point agarose a) 85-90ºC

b) 35-45ºC

solution

0.2% agarose gel

Gels for safer cryocooling

Examples of crystallizations in gel using various techniques Batch: A) Concanavalin and B) turkey lysozyme in capillaries under hyperbaric conditions, C) hen lysozyme, D) and D') glutaminyl-tRNA synthetase from D. radiodurans in microbatch under oil without and with gel, respectively; Vapor diffusion: E) Orthorhombic and F) monoclinic crystals of aspartyl-tRNA synthetase from T. thermophilus (DRS); Free Interface Diffusion: G) Tobacco bushy stunt virus (TBSV); Counter-diffusion: H) Orthorhombic crystals of DRS-1; Dialysis: I) Thaumatin grown in microgravity in the Advanced Protein Crystallization Facility.

A

B

The presence of the gel matrix in the mother liquor and inside the solvent channels, as illustrated on the left [3], can facilitate cryocooling. Monoclinic crystals of DRS-1 grown in gel (middle) were easily frozen and led to complete data collected at 100K [4], whereas crystals grown in solution severly suffered from flash freezing (dramatic loss of resolution, increase of mosaicity, formation of ice rings). A similar behavior was observed with crystals of A. fulgidus Holliday Junction Cutting Enzyme (right) prepared in gel by counter-diffusion (Biertümpfel et al., in preparation). Agarose fibers in a lysozyme crystal! (Gavira & Garcia-Ruiz, 2002)

C

Gels for nicer crystals and better diffraction D

F

D'

H

E

The diffraction properties of orthorhombic DRS crystals grown by vapor diffusion in solution and in gel, and by counter-diffusion (Gel Acupuncture Method or GAME) were compared [5]. Data were collected in parallel with samples of equivalent size at a maximal resolution of 2.5 Å. Overall, the diffraction patterns from crystals grown in diffusive conditions (Gel and Game) exhibit a higher signal-to-noise ratio. A similar effect was already reported in the case of thaumatin [6].

I

G

References [1] M.-C. Robert & F. Lefaucheux (1988). Crystal growth in gels: Principle and applications. J. Crystal Growth, 90, 358367. [2] M.-C. Robert, O. Vidal, J.-M. Gracia-Ruiz & F. Otalora (1999). Crystallization in gels and related methods in Crystallization of nucleic acids and proteins (2d edition), A. Ducruix & R. Giegé (eds), OUP, Oxford, p.149-175. [3] J.-A. Gavira & J.-M. García-Ruiz (2002). Agarose as crystallisation media for proteins II: Trapping of gel fibres into the crystals. Acta Cryst. D58, 1653-1656. [4] D.-W. Zhu, B. Lorber, C. Sauter, J.D. Ng, P. Bénas, C. Le Grimellec & R. Giegé (2001). Growth kinetics, diffraction properties and effect of agarose on the stability of a novel crystal form of Thermus thermophilus aspartyl-tRNA synthetase-1. Acta Cryst. D57, 552-558. [5] A. Moreno, A. Théobald-Dietrich, B. Lorber, C. Sauter & R. Giegé (2005). Effects of macromolecular impurities and of crystallization method on the quality of eubacterial aspartyl-tRNA synthetase crystals. Acta Cryst. D61, in press. [6] B. Lorber, C. Sauter, M.-C. Robert, B. Capelle & R. Giegé (1999). Crystallization within agarose gel in microgravity improves the quality of thaumatin crystals. Acta Cryst. D55, 1491-1494.

In conclusion... The crystallization in gel - is easy to perform - can be adapted to all current crystallization methods - provides better growth conditions - facilitates crystal handling (transport, soaking, fishing, cryocooling) - leads to crystals of superior quality ...Just do it!