Strategies for carbohydrate model building, refinement and validation

Abstract

Sugars are the most stereochemically intricate family of biomolecules and present substantial challenges to anyone trying to understand their nomenclature, reactions or branched structures. Current crystallographic programs provide an abstraction layer allowing inexpert structural biologists to build complete protein or nucleic acid model components automatically either from scratch or with little manual intervention. This is, however, still not generally true for sugars. The need for carbohydrate-specific building and validation tools has been highlighted a number of times in the past, concomitantly with the introduction of a new generation of experimental methods that have been ramping up the production of protein–sugar complexes and glycoproteins for the past decade. While some incipient advances have been made to address these demands, correctly modelling and refining carbohydrates remains a challenge. This article will address many of the typical difficulties that a structural biologist may face when dealing with carbohydrates, with an emphasis on problem solving in the resolution range where X-ray crystallography and cryo-electron microscopy are expected to overlap in the next decade.

Current developments in Coot for macromolecular model building of Electron Cryo‐microscopy and Crystallographic Data

“…Asparagine (Asn)‐linked glycosylation is the most common type of N‐glycosylation of eukaryotic proteins, and it is also found in viruses, including HIV and Ebola . Agirre reports that the fraction of Asn‐linked glycosylation in the PDB as of 2013 is 5.5% (and increasing) .…”

Carbohydrate 3D structure validation

“…The last decade saw the introduction of new experimental techniques that have almost doubled the structural throughput of glycoproteins. About 10% of the structures deposited annually contain carbohydrates but, while those covalently-linked accounted for ~2.5% of the total in the early 2000Õs, this number has increased to ~5% since 2010 [1]. It is apparent that the structural biology community has been caught off-guard; a number of communications have raised issues on the way carbohydrates are represented in structural databases [2][3][4], with numerous problems affecting nomenclature, structure and conformation which, in combination, may affect more than 30% of the glyco-related structural data deposited in the Protein Data Bank (PDB).…”

“…D-mannopyranose is encoded as two three-letter codes, MAN (a-anomer) and BMA (b-anomer). Some dictionary generation programs may produce an improbable high-energy conformer as starting coordinates, or create torsion restraints that lock it into that, or other high-energy conformation [1]. Model building and refinement programs do not take conformational preferences into account, which has a deleterious knock-on effect on other aspects of the model, from interactions to linkage torsions.…”

“…Finally, while the conformation, geometry, structure and interactions of amino acids have been analysed and reviewed frequently and regularly, the glycorelated structural literature has been largely restricted to the biochemical field, with very little impact in structural biology. This, hopefully, has started to change [1,4,7,8].…”

X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

“…The overall glycan geometry was validated using programs including PDB CArbohydrate REsidue check (pdb-care; http:// www.glycosciences.de/tools/pdb-care/), CArbohydrate Ramachandran Plot (carp; http://www.glycosciences.de/tools/carp/) and Privateer (Agirre et al, 2015;Agirre, 2017). Glycans were built into the initial models for the 3.9 and 3.5 Å resolution IOMA-10-1074-BG505 crystal structures using 2F o À F c maps calculated with model phases and using composite-annealed OMIT maps calculated with phases from which the model was omitted (Adams et al, 2010).…”