Automatically Fixing Errors in Glycoprotein Structures with Rosetta

Abstract

Summary Recent advances in single-particle cryo-electron microscopy (cryoEM) have resulted in determination of an increasing number of protein structures with resolved glycans. However, existing protocols for the refinement of glycoproteins at low resolution have failed to keep up with these advances. As a result, numerous deposited structures contain glycan stereochemical errors. Here, we describe a Rosetta-based approach for both cryoEM and X-ray crystallography refinement of glycoproteins which is capable of correcting conformational and configurational errors in carbohydrates. Building upon a previous Rosetta framework, we introduced additional features and score terms enabling automatic detection, setup and refinement of glycan-containing structures. We benchmarked this approach using twelve crystal structures and showed that glycan geometries can be automatically improved while maintaining good fit to the crystallographic data. Finally, we used this method to refine carbohydrates of the human coronavirus NL63 spike glycoprotein and of an HIV envelope glycoprotein, demonstrating its usefulness for cryoEM refinement.

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

“…N-linked glycans were hand-built into the density where visible and the models were refined and relaxed using Rosetta . Glycan refinement relied on a dedicated Rosetta protocol, which uses physically realistic geometries based on prior knowledge of saccharide chemical properties (Frenz et al, 2019), and was aided by using both sharpened and unsharpened maps. Models were analyzed using MolProbity (Chen et al, 2010), EMringer (Barad et al, 2015), Phenix (Liebschner et al, 2019) and privateer (Agirre et al, 2015) to validate the stereochemistry of both the protein and glycan components (Table S1).…”

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

“…N-linked glycans were hand-built into the density where visible and the models were refined and relaxed using Rosetta . Glycan refinement relied on a dedicated Rosetta protocol, which uses physically realistic geometries based on prior knowledge of saccharide chemical properties (Frenz et al, 2019), and was aided by using both sharpened and unsharpened maps. Models were analyzed using MolProbity and privateer (Agirre et al, 2015).…”

An antibody against the F glycoprotein inhibits Nipah and Hendra virus infections

“…The model was subsequently rebuilt manually using Coot 81 and refined using Rosetta [82][83][84] . Glycan refinement relied on a dedicated Rosetta protocol, which uses physically realistic geometries based on prior knowledge of saccharide chemical properties 85 and was aided by using both sharpened and unsharpened maps. Models were analyzed using MolProbity 68 , EMRinger 86 and Privateer 87 .…”