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. 2018 Aug 1;74(Pt 8):463-472.
doi: 10.1107/S2053230X18004016. Epub 2018 Jul 26.

Making glycoproteins a little bit sweeter with PDB-REDO

Bart van Beusekom  1 , Thomas Lütteke  2 , Robbie P Joosten  1
Affiliations

Making glycoproteins a little bit sweeter with PDB-REDO

Bart van Beusekom et al. Acta Crystallogr F Struct Biol Commun. .

Abstract

Glycosylation is one of the most common forms of protein post-translational modification, but is also the most complex. Dealing with glycoproteins in structure model building, refinement, validation and PDB deposition is more error-prone than dealing with nonglycosylated proteins owing to limitations of the experimental data and available software tools. Also, experimentalists are typically less experienced in dealing with carbohydrate residues than with amino-acid residues. The results of the reannotation and re-refinement by PDB-REDO of 8114 glycoprotein structure models from the Protein Data Bank are analyzed. The positive aspects of 3620 reannotations and subsequent refinement, as well as the remaining challenges to obtaining consistently high-quality carbohydrate models, are discussed.

Keywords: PDB-REDO; carbohydrates; glycoproteins; pdb-care; validation.

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Figures

Figure 1
Figure 1
An example of incorrect modelling of N-glycosylation in the structure of the laccase McoG (PDB entry 5lm8; Ferraroni et al., 2017 ▸). The first NAG residue is attached to the Oδ atom (OD1 in PDB nomenclature) of Asn103, rather than to the Nδ atom. The clear electron density (2mF o − DF c at 1.2σ; clipped around the residues for clarity) and the fact that the neighbouring N-glycosylation on Asn60 is correct suggest that this is a simple oversight. Errors of this type are automatically corrected in PDB-REDO by swapping the names of the Oδ and Nδ atoms and updating the linkage description accordingly. This figure was produced using CCP4mg (McNicholas et al., 2011 ▸).
Figure 2
Figure 2
The sialoglycopeptide from glycophorin A (chain D of PDB entry 2cwg; Wright & Jaeger, 1993 ▸) required multiple reannotation events: a LINK record was added to connect the threonine side chain to the central carbohydrate residue, the central residue NDG was renamed A2G (α-d-GalpNAc) and a LINK record between GAL and A2G was rewritten to correctly describe the leaving atom O1. The dotted line represents a glycosidic linkage that was already present in the PDB entry but is not displayed by CCP4mg.
Figure 3
Figure 3
Distribution of θ angles in the PDB and in PDB-REDO. The data are displayed as box-and-whiskers plots with the whiskers extending to 1.5 times the interquartile range. In the PDB the θ angle that describes the carbohydrate-ring conformation increases gradually, while in PDB-REDO this effect is magnified.
Figure 4
Figure 4
An N-linked glycan attached to Asn35 of human Toll-like receptor 4 (PDB entry 2z62; Kim et al., 2007 ▸). Electron density is contoured at 1σ for the 2mF o − DF c map (grey) and 3σ for the mF o − DF c difference density map (green, positive; red, negative). (a) Model as found in the PDB with the (1–6)-linked fucose incorrectly modelled as FUL. The density near the O6 atom of asparagine-linked NAG is partially filled by four water molecules in a symmetry-related copy of the model (purposely not shown), which reduces the amount of positive difference density. (b) PDB-REDO model and map. The fucose residue is renamed FUC. Subsequent refinement improves the fit to the electron density, but distorts the ring conformation and flattens the hand of the C1 atom. (c) Manually rebuilt model after refinement by PDB-REDO. The (1,6)-linked fucose is flipped to correctly fit the density, as is the acetylamino group of the second NAG residue. Together with adding a (1–3)-linked fucose, these corrections remove all strong difference density. (d) The CARP plot for (1–6)-linked fucose shows the distribution of FUC-(1–6)-NAG glycosidic linkage torsion angles in the PDB. The relevant bonds in the glycosidic linkage are marked in (a). The models are marked as follows: PDB, P; PDB-REDO, R; manually rebuilt, M. Only the manually rebuilt model has common glycosidic torsion angles.
Figure 5
Figure 5
Distribution of chiral volume deviations (absolute values) of anomeric centre atoms C1 in the PDB and in PDB-REDO. The optimal value for chiral volume is either −2.22 (for an α-linkage) or +2.22 (for a β-linkage); corrections of chirality are therefore expected to change the chiral volume by about 4.5. A clear improvement can be observed for the vast majority of cases (all points below the diagonal). Three distinct clusters are observed in the plot: cases with the correct hand in both the PDB and PDB-REDO (bottom left), cases with the wrong hand in the PDB but the correct hand in PDB-REDO (bottom right) and cases where the hand was incorrect in the PDB and the anomeric centre ended up flat in PDB-REDO (middle right).
Figure 6
Figure 6
Comparison of knowledge-based potentials for glycosidic linkages commonly found in N-glycans in the PDB and in PDB-REDO. Potentials are calculated by CARP (Lütteke et al., 2005 ▸) and are given in arbitrary units; lower values are better.
Figure 7
Figure 7
Distribution of hydrogen-bond parameters for different hydrogen-bonding types. The hydrogen-bond length distribution (top) for cases involving carbohydrates is much broader than for protein-only hydrogen bonds. The hydrogen-bond angle distributions (bottom) show that typical carbohydrate–carbohydrate hydrogen bonds have much sharper angles, indicating that they are relatively weak and not suited for generating hydrogen-bond restraints.
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