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Figure 5
Semi-automatically building an oligosaccharide using LO/Carb. (a) focuses on the glycosylation site of a structure refined using data extending to 1.6 Å resolution (PDB entry 4gos; Jeon et al., 2014BB60), after removing the carbohydrate. The current model is shown (sticks) along with the 2mFoDFc density map (blue) and difference density map (green/red). The positive difference density corresponds to the missing carbohydrate structure, which we know comprises five sugars: two N-acetyl-D-glucosamines (NAG), one β-D-mannose (BMA) and two α-D-mannoses (MAN). The carbohydrate is N-linked to the protein residue Asn112. The carbohydrate structure is built by first placing a NAG conformer into the density next to Asn112 (b) using the `Get Monomer' tool in Coot (found in the `File' menu). After deleting the H atoms, Jiggle Fit (hotkey `J') is used to quickly position and orient the sugar correctly, before manual adjustment and real-space refinement (hotkey `r') to fit the model into the density (c). The O atom which visibly clashes with an N atom in Asn112 can then be removed from the glycosylation site (d), and the new sugar merged into the existing model (using the `Merge Molecules' tool found in the `Calculate' menu). LO/Carb functionality is accessible via the `Glyco' menu (which is activated by selecting `Carbohydrate' from the `Modules' section in the `Extensions' menu), through which it is possible to automatically add and fit the additional NAG (e) and finally the remaining BMA and two MAN sugars (f). Note that in this case REFMAC5 will automatically create the N-link record when the model is next refined, owing to it being a standard link present in the monomer library.

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ISSN: 2059-7983
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