Computational protocol: Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9

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Protocol publication

[…] MORF179–190 crystallized by sitting drop vapor diffusion (1 μl protein plus 1 μl reservoir) at 4°C with a reservoir containing 0.1 M Tris, pH 8.0 and 2.4 M ammonium sulfate (space group P1) or 0,1 M Bicine, pH 9.0 and 2.4 M ammonium sulfate (space group P21). Crystals were cryo-protected by a mixture of 90% (v/v) lithium sulfate and 10% of the respective reservoir solution. For derivatization experiments, crystals were incubated for 2 min in the respective cryo-protectant solution supplemented with 0.5 M NaBr and flash frozen in liquid nitrogen.MORF986–186 crystallized by sitting drop vapor diffusion (0.1 μl protein plus 0.1 μl reservoir plus 30 nl additive) at 4°C with a reservoir containing 0.08 M sodium cacodylate, pH 6.5, 14.4% (w/v) PEG 8000, 20% (v/v) glycerol, 0.16 M calcium acetate and 0.3 M NaCl as an additive. Crystals were cryo-protected in reservoir solution and flash frozen in liquid nitrogen. Diffraction data were collected at 100 K at beamline 14.1 of the BESSY II storage ring, Berlin, Germany (). All diffraction data were processed with XDS ().The structure of MORF179–190 was solved by single-wavelength anomalous dispersion (SAD) with 26 Br-sites in space group P1 employing SHARP/AUTOSHARP (). Initial phases were improved by density modification with SOLOMON (). The initial experimental electron density was of good quality and allowed the building of all four MORF179–190 molecules in the asymmetric unit with PHENIX.AUTOBUILD (,). The second MORF179–190 crystals grown at pH 9.0 were processed in space group P21 and identified as pseudo-merohedrally twinned by PHENIX.XTRIAGE (). The structure of the latter crystals was solved by molecular replacement with PHASER employing the coordinates of MORF179–190. The structure of MORF986–186 was solved by molecular replacement with PHASER employing the coordinates of a MORF179–190 monomer lacking the N-terminal β-strand. Structural models were completed through alternating rounds of automated refinement using PHENIX.REFINE () and manual model building using COOT (). The pseudo-merohedrally twinned data were refined with PHENIX.REFINE in space group P21 with a twin fraction of 0.14 and the twin operator h, −k, l. […]

Pipeline specifications

Software tools XDS, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Arabidopsis thaliana