The avian reovirus protein σA plays a dual role. It functions primarily as a structural protein that forms part of the transcriptionally active core. In addition, it has also been implicated in the resistance of the virus to interferon by binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase (Martinez-Costas et al., 2000 ; Gonzalez-Lopez et al., 2003 ). The structure of the mammalian reovirus core has been solved by X-­ray crystallography (Reinisch et al., 2000 ). In this structure, the structural equivalent of avian reovirus σA, σ2, is present in 150 copies per core in three different locations (σ2-i, σ2-ii and σ2-iii) and acts as a core-stabilizing clamp. The protein interacts extensively with λ1 (the mammalian reovirus equivalent of λA) at two different binding sites (the binding sites for σ2-ii and σ2-iii are similar). There are only sparse contacts between σ2 monomers and between σ2 and the other core protein λ2 (the equivalent of avian reovirus λC). In agreement with the mammalian reovirus ‘core’ structure, cryo-electron microscopy of avian reovirions confirmed that they contain 150 σA ‘nodules’ contacting mainly λA, with minor contacts formed with the λC turrets and other σA molecules (Zhang et al., 2005 ). They also contact the μB proteins of the outer capsid (equivalent to μ1 in mammalian reovirus). However, with respect to double-stranded RNA binding, σA appears to play an analogous role to the mammalian reovirus protein σ3, as this protein (the structural equivalent of avian reovirus σB) exhibits double-stranded RNA-binding activity (Yue & Shatkin, 1997 ), albeit not as strongly as avian reovirus σA (Martinez-Costas et al., 2000 ).

For large-scale expression, the Escherichia coli strain XL1-Blue was freshly transformed with the pMALCS2 vector and four 750 ml cultures were grown aerobically at 310 K to an optical density of 0.6 measured at 600 nm in LB medium. Expression was induced by adding 1 mM isopropyl β-d-thiogalactopyranoside and allowed to proceed for 3 h. Harvested cells were resuspended in 40 ml lysis buffer (20 mM Tris–HCl pH 7.3, 200 mM sodium chloride, 1 mM dithiothreitol) containing a protease-inhibitor cocktail specific for bacterial cell extracts (Sigma–Aldrich, Madrid, Spain) and the suspension was frozen at 253 K. The cellular pellet was lysed by a double pass through an emulsifier (Avestin Emulsifier C5, Avestin Europe GmbH, Mannheim, Germany). After removal of the insoluble material by centrifugation, sodium chloride and poly(ethyleneimine) were added to final concentrations of 1 M and 0.1%(w/v), respectively, and the suspension was incubated at 277 K for 1 h to precipitate nucleic acids. Following a second centrifugation step, bovine ribonuclease A (>70 Kunitz units per milligram; Sigma–Aldrich, Madrid, Spain) was added to the supernatant to a final concentration of around 30 µg ml⁻¹ and incubated for 30 min at room temperature. 3 ml suspended amylose resin (New England Biolabs, Ipswich MA, USA) was then added and gently mixed and the suspension was left for 10 min at room temperature before being poured into an empty column. The resin was washed three times with three column volumes of MBP buffer (20 mM Tris–HCl pH 7.5, 200 mM sodium chloride, 1 mM EDTA) and elution was performed by washing twice with 10 ml 10 mM maltose in MPB buffer. Calcium chloride and protease factor Xa (New England Biolabs, Ipswich MA, USA) were added to final concentrations of 1 µg ml⁻¹ and 1 mM, respectively. Incubation was carried out at room temperature for 20 h. The digested protein was dialysed overnight against TE buffer (10 mM Tris–HCl pH 8.5, 1 mM EDTA) and applied onto a 1 ml Uno-Q column (Biorad, Barcelona, Spain). Pure σA eluted in the flowthrough, while maltose-binding protein and other impurities were retained on the column. The protein was concentrated with Centricon concentrators (Millipore, Madrid, Spain), using three washes with TE buffer to eliminate small-molecule impurities.

In vapour-diffusion experiments, sizable crystals were obtained after 6–12 months at pH 5.5–6.5 and 278 K using 100 mM sodium citrate, sodium phosphate or 2-(N-morpholino)ethanesulfonic acid–NaOH as a buffer and 0.1–0.6 M ammonium sulfate as precipitant (Fig. 1 ). For data collection, crystals were briefly incubated in mother liquor containing 25%(v/v) glycerol and then flash-frozen in liquid nitrogen.

A high prevalence of multiple lattices was observed in the diffraction patterns obtained from these crystals. However, fairly complete data sets with reasonable statistics were collected from several crystals using synchrotron radiation, in the best case yielding a data set to 2.3 Å resolution (Table 1 ). For each crystal, 180 1° oscillation-angle images were collected, with exposure times of 1–2 s for each image, using the insertion-device beamline ID23-1 at the European Synchroton Radiation Facility. Data sets from different crystals were not merged, as the crystals were found to be insufficiently isomorphous to each other. The crystals were assigned to space group P1 and analysis of the Matthews coefficient suggested the presence of between six and 18 protein molecules in the asymmetric unit (with a solvent fraction of between 81 and 43%, respectively). We have recently obtained a convincing molecular-replacement solution containing 12 copies of a model based on the mammalian reovirus core protein σ2, which would give 62% solvent content.