Computational protocol: Structural Basis of Transcriptional Gene Silencing Mediated by Arabidopsis MOM1

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Protocol publication

[…] Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) on the beam lines ID23-1, ID29 and ID14-4. A complete data set using native protein crystal was collected at a maximum resolution of 3.2 Å. A further data set was collected at the selenium edge with a selenium-containing protein crystal. This crystal diffracted to ∼3.5 Å resolution. A Single Anomalous Dispersion phasing procedure was used to solve the phase problem using the selenium as heavy atoms. Briefly, the 8 seleno-methionines were located by the program SHELXD using the measured anomalous signals –. The sites were subsequently injected into the experimental SAD phasing procedure as defined in SHARP . Density modification was then used to improve the initial set of phases . Long tubes of electron density were readily visible in the calculated electron density map and the initial model was built using the program Coot with an alanine-only model. The protein register was defined based on the position of the methionine residues. Several rounds of refinement/rebuilding were done iteratively using the PHENIX software . All the built residues are in the favored and allowed regions of the Ramachandran plot. Residues 1700 to 1728 and 1811 to 1814 are disordered or very poorly ordered in every CMM2 molecules. The protein coordinates have been deposited in the Protein Databank with the PDB code 3VEM. […]

Pipeline specifications

Software tools SHELX, Coot, PHENIX
Application Protein structure analysis
Organisms Dipturus trachyderma, Arabidopsis thaliana