Computational protocol: Structure and properties of the esterase from non-LTR retrotransposons suggest a role for lipids in retrotransposition

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Protocol publication

[…] For crystallization in sitting drops (0.4 over 80 µl reservoir), sample in reaction buffer was mixed 1:1 with reservoir. For the coiled coil domain (B), a single crystal was found after 4 months over a reservoir of 0.2 M Na-thiocyanate and 20% (w/v) polyethylene glycol (PEG) 3350. The crystal was flash-frozen in liquid nitrogen, from reservoir solution supplemented with 20% glycerol. For the esterase domain (C), crystals grew over 0.1 M Na-Hepes, pH = 7.0, 0.5% (v/v) Jeffamine and 1.1 M Na-malonate. A heavy atom derivative was obtained by an overnight incubation in reservoir supplemented with 2 mM potassium dicyanoaurate. Crystals were flash-frozen from reservoir solution. Diffraction data were collected on beamline PXII of the Swiss Light Source, Villigen, Switzerland, and diffraction images were processed using XDS (). [...] The structure of the coiled coil domain was solved by molecular replacement using MOLREP () from within the CCP4 package () and a polyalanine model of an anti-parallel coiled coil as a search model, derived from PDB-ID 1A92 (). The structure of the esterase domain was solved by single isomorphous replacement with anomalous scattering (SIRAS). SHELX C/D/E was used to identify the sites for phasing and for an initial auto-building of the structure (,). Both structures were then (re-)built automatically also to remove any potential model bias in the case of the coiled coil domain, using ARP/wARP () and BUCCANEER (). The model was finished manually in COOT (), alternating with rounds of refinement using PHENIX (). Final refinement rounds were done in PHENIX, refining TLS parameters in addition to individual B-factors and including hydrogens. Stereochemical properties were analyzed with MOLPROBITY (). Modeling of the rotated L281 and of the enclosed palmitate in the context of the closed esterase monomer (ZfES_BA, connecting residues M135-L199 from chain B with residues R200-I302 from chain A) was also achieved in COOT followed by energy minimization in PHENIX. Cavity volumes were extracted using the Voss Volume Voxelator () using inner and outer probe radii of 1.2 and 5.0 Å, respectively. Figures were generated in PyMOL ( using the APBS plug-in () to visualize electrostatic surface potentials. […]

Pipeline specifications

Software tools XDS, Molrep, CCP4, SHELX, ARP/wARP, Buccaneer, Coot, MolProbity, 3V, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Danio rerio