Computational protocol: Cog-Wheel Octameric Structure of RS1, the Discoidin Domain Containing Retinal Protein Associated with X-Linked Retinoschisis

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Protocol publication

[…] 2 x 2 pixels from the electron micrographs were averaged for a final pixel size of 4.7Å at the specimen level. Single particle images were extracted from the micrographs using Boxer []. For two dimensional (2D) analysis, the selected particles (10,664 total) were subjected to reference-free alignment and K-means classification using the SPIDER image processing suite []. Fifty classes were specified ().To produce the RS1 3D reconstruction, class averages yielding intact rings were used to generate initial 3D models using EMAN2 []. The initial models were then refined with images containing a single well-defined particle each (2,200/10,664 total particles) with RELION while enforcing D8 symmetry []. The final resolution was determined in RELION using the Fourier Shell Correlation (FSC) function using the 0.143 FSC criterion. [...] A model of the RS1 protein was built using the Phyre2 Protein fold recognition server (http://www.sbg.bio.ic.ac.uk/phyre2/html[]). The search input was the mature peptide lacking the 23 amino acid leader sequence i.e. residues 24–224 in the intense mode. Further refinement and adjustment was performed using Coot software [] to make the appropriate disulphide bonds within the molecule. […]

Pipeline specifications

Software tools SPIDER, EMAN, RELION, Phyre, Coot
Applications cryo-EM, Protein structure analysis
Diseases Retinal Diseases, Retinoschisis