Crystallization and preliminary crystallographic analysis of 3-carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa

Abstract

Crystals of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE; E.C. 5.5.1.5) from Neurospora crassa that diffract to high resolution have been obtained. The crystals belong to the orthorhombic space group P212121 with unit-cell dimensions a = 92.1, b = 159.7, c = 236.6A (at 103 K) and diffract at most to 2,~ resolution. The asymmetric unit of the crystals appears to contain two tetrameric CMLE molecules making up a total of 328 kDa per asymmetric unit. Both cross-linking with glutaraldehyde and cryo-cooling to 103 K have been used to facilitate data collection because the crystals are unstable in the X-ray beam; both techniques extend the crystal lifetime but cryo-cooling, unlike glutaraldehyde cross-linking, does not lower the quality of the diffraction pattern.

3-Carboxy-cis,cis-muconate lactonizing enzyme fromNeurospora crassa: MAD phasing with 80 selenomethionines

“…Based on the solvent content, there are probably two tetramers per asymmetric unit (the Matthews coef®cient V M = 2.64 A Ê 3 Da À1 corresponds to a solvent content of 53.0%; Matthews, 1968). Flotation measurements of the crystal density in a Ficoll gradient supported this model (Glumoff et al, 1996). The crystals diffracted under cryogenic conditions to 2 A Ê resolution, but¯ash-cooling was irreproducible and always resulted in crystals with a high mosaicity.…”

“…These, however, did not show signi®cant peaks above noise level (Fig. 1), as was also found in the dominant crystal type (Glumoff et al, 1996). Consequently, these could not be used to obtain the NCS operators and we therefore attempted to use the selenium sites from the SnB solution to determine the position of the operators.…”

“…Compared with the native protein, SeMet-CMLE crystallized under the same conditions of 100 mM PIPES pH 5.7, 1.56 M (NH 4 ) 2 SO 4 (Glumoff et al, 1996), but more rapidly (native protein typically crystallized in three weeks to several months, whereas the SeMet-substituted form crystallized in one to two weeks). Data were collected from¯ash-cooled crystals; we found it best to use very quick soaks in the cryoprotectant solution instead of the several minutes time previously reported (Glumoff et al, 1996). It was later found that small crystals could also in some cases be¯ash-cooled in a nitrogen stream directly from the crystallization mother liquor [i.e.…”

“…Solution studies have shown the enzyme to most likely be a homotetramer (Mazur et al, 1994). Crystals were obtained in space group P2 1 2 1 2 1 , with unit-cell parameters a = 92.1, b = 159.7, c = 236.6 A Ê (Glumoff et al, 1996). Based on the solvent content, there are probably two tetramers per asymmetric unit (the Matthews coef®cient V M = 2.64 A Ê 3 Da À1 corresponds to a solvent content of 53.0%; Matthews, 1968).…”

Stabilization of porcine pancreatic elastase crystals by glutaraldehyde cross-linking

“…Cross-linking the crystals with glutaraldehyde was successful in allowing the crystals to endure the high concentrations of dimethyl sulfoxide required for inhibitor solubilization. Glutaraldehyde cross-linking was first described by Quiocho & Richards (1964) and has since been used in various applications, namely to enhance crystal stability (Glumoff et al, 1996;Wang et al, 2003;Cohen-Hadar et al, 2006;Wine et al, 2007), to prevent lattice damage upon crycooling (Haas & Rossmann, 1970), to increase crystal diffraction quality (Vetting et al, 2009;Duan & Ye, 2009), to permit solvent exchange (Lusty, 1999) and to allow ligand soaking (Andersen et al, 2009). Furthermore, this technique has also been employed to produce catalytically active and insoluble chloroperoxidase crystals (Ayala et al, 2002), to obtain highly robust and active lipase crystals (Rajan & Abraham, 2008) and to study urea-driven denaturation using ribonuclease S crystals (Ratnaparkhi & Varadarajan, 1999).…”