Computational protocol: Structure of a rabbit muscle fructose-1,6-bisphosphate aldolase A dimer variant

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Protocol publication

[…] The structure was solved by molecular replacement with the program AMoRe (Navaza, 1994) using a monomer from the structure of wild-type human muscle aldolase A (PDB code 1ald; Gamblin et al., 1991) as the initial search model. The orientation of the monomer obtained by molecular replacement was improved by rigid-body refinement using the Crystallography & NMR System (CNS) program suite (Brünger et al., 1998). σA-weighted electron-density maps with coefficients 2F o − F c calculated from the model after rigid-body refinement in the program CNS showed good electron density for all areas of the monomer with the exception of the N-terminal (1–­4) and C-terminal regions (345–363), which failed to appear even after subsequent rounds of refinement. To minimize model bias in the electron-density maps, the side chain of residue 128 was modeled as an alanine. The density was clearly that of a valine in the 2F o − F c map and was modeled as such in subsequent rounds of refinement. Iterative rounds of model building using Coot (Emsley & Cowtan, 2004) were performed alternately with rounds of positional, group temperature-factor and simulated-annealing refinement in the program CNS. A test set consisting of 10% of the data was used for R free calculations. In the final rounds of refinement water molecules were included using a 3σ cutoff. A total of 655 water molecules were added to the model as well as six sulfates which were bound at the surface of the protein. In the final round of refinement, a composite-omit electron-density map was used to fit the models of DHAP and sulfate to the active site. The final structure had a working R factor of 0.189 and a free R factor of 0.209. Analysis of the Ramachandran plot defined by PROCHECK (Laskowski et al., 1993) showed good statistics for the model, with 91.8% of residues in the most favorable regions and 8.2% of residues in additionally allowed regions for D128V aldolase. [...] The change in intersubunit orientation of the dimer in comparison to the tetramer was measured using the program DynDom (Hayward et al., 1997). The buried surface area between monomers was calculated for the structures of aldolase (PDB code 1ado) and D128V aldolase using CNS with a probe radius of 1.4 Å (Lee & Richards, 1971). […]

Pipeline specifications

Software tools PROCHECK, DynDom, CNS
Application Protein structure analysis
Organisms Dipturus trachyderma, Oryctolagus cuniculus, Homo sapiens
Diseases Anemia, Hemolytic