Computational protocol: The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4–NOT deadenylase complex

Similar protocols

Protocol publication

[…] X-ray diffraction data were collected at 100 K at beamline PXII - X10SA (Swiss Light Source). The diffraction data of the NOT1–CAF1 complex were processed using XDS () and scaled using SCALA () from the CCP4 suite (). The crystal diffracts to 2.70 Å and belongs to the space group P21. The initial phase information was obtained by molecular replacement using Phaser from the CCP4 suite and the coordinates of Hs CAF1 (PDB ID: 2D5R) as the search model. The initial densities were further improved by solvent flattening and histogram matching using RESOLVE (), as implemented in the Phenix suite (). The initial NOT1 model was built using AutoBuild from the Phenix suite and was further refined manually by iterative cycles of model building and refinement using COOT () and Refine (Phenix suite). A total of 211 water molecules were positioned in well defined positive (F0–FC) residual densities (cut-off of 3σ). Three chlorid atoms, eight magnesium atoms and three glycerol molecules were added to finalize the model (Supplementary Table S1). A similar method was used for the structure determination of free NOT1.NOT1 diffraction data were processed in the space group C2221 and scaled to 2.90 Å. The initial phase was calculated using NOT1 coordinates from the NOT1–CAF1 complex as the search model. A total of 30 water molecules were added to finalize the model. Crystallographic model refinement and data collection statistics are presented in Supplementary Table S1. Representative figures of the crystal structure were created using PyMOL (http://www.pymol.org/). […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens