Computational protocol: Structural insights into RNA Processing by the Human RISC-Loading Complex

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Protocol publication

[…] We diluted human Dicer, RLC or RLC-subcomplex samples, either with or without cross-linking, to a concentration of about 30∼40 nM in 20 mM Tris-HCl pH 7.5, 100 mM KCl, 3 mM MgCl2, 1 mM DTT immediately before applying them to glow-discharged holey carbon grids with a thin layer of carbon over the holes. After adsorption for 1 minute, the sample was stained consecutively with three droplets of 2% (w/v) uranyl formate solution, blotted gently of the residual stain, and air-dried in a hood. We examined the specimens under an FEI Tecnai-12 Bio-Twin electron microscope equipped with a LaB6 filament and operated at 120 kV acceleration voltage, using a nominal magnification of 49,000. For tilt-pair images, the same area of a specimen was recorded first at 55° and then at 0° tilt angles. We recorded images either on SO-163 film or on a Gatan 1k×1k CCD camera using low-dose mode with an exposure dose of 20-30 e-/Å2. The defoci range used to collect the 0°-tilt image was -0.8 to -1.1 μm and that used for the 55°-tilt image was -1.5 μm. We developed the films using full-strength D19 for 12 minutes and digitized the micrographs without apparent drift and astigmatism using a Nikon Coolscan 8000 with a scanning step of 12.7 μm, resulting in a pixel size of 2.59 Å. The scanned images were first converted from their transmission values to optical densities using the PROC2D program of the EMAN image processing package . The CCD-recorded micrographs had a pixel size of 3.61 Å and were directly used in the image processing. [...] The docking of the atomic models was done either using rigid-body docking programs within the Situs2.0 software package , or by visual inspection and manual docking using Chimera . More specifically, the atomic model of human DEAD-box RNA helicase DDX3X (PDB 2i4i) was docked in the DExH/D domain using Colores within Situs2.0; the Giardia intestinalis Dicer crystal structure (PDB 2qvw) was docked in the human Dicer platform density using Fit-Model-In-Map function in Chimera guided by the biochemical evidence for the orientation, and then adjusted locally using Colacor of Situs2.0; the guide strand-containing Thermus thermophilus Argonaute crystal structure (PDB 3dlh), the Giardia intestinalis Dicer crystal structure (PDB 2qvw), and the atomic model of human DEAD-box RNA helicase DDX3X (PDB 2i4i) were docked in the GraFix prepared RLC 3D map by Fit-Model-in-Map of Chimera. All the figures with 3D models were generated using UCSF-Chimera. […]

Pipeline specifications

Software tools EMAN, Situs, UCSF Chimera
Databases miRWalk
Applications cryo-EM, Protein structure analysis
Organisms Homo sapiens