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Elisa Fadda-🔺

It’s the most wonderful of the year!! 🤩 🥳

Let us introduce you to (glycoshape.org and link below), a web platform that allows users to restore glycoproteins 3D structures (PDB, AF/RoseTTAFold or own) to their natural, functional state by adding the missing glycan structures. ⬇️ tootorial🧵 1/8
doi.org/10.1101/2023.12.11.571

left, white box with the GlycoShape logo and oracle logo with the spike protein in grey with glycans in blue sticking out. Right, fron page of the GlycoShape website with URL below
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Elisa Fadda-🔺

Part 2/8. The glycan database () currently counts 435 distinct glycan structures derived from 1 ms of cumulative sampling by molecular dynamics. The focus of the GDB is now on mammalian structures, yet we have examples from all species. MD trajectories are passed through the Glycan Analysis Pipeline (GAP) which outputs an ensemble of 3D structure representing the accessible conformational space.

Schematic overview of Glycan Analysis Pipeline (GAP) used to build the GlycoShape Glycan Database (GDB). Panel a) Multiple uncorrelated replica molecular dynamics (MD) simulations are performed for each glycan in the GDB, to comprehensively sample its structural dynamics. The resulting MD frames are then transformed into a graph matrix representation, as depicted in Panel b), simplified by flattening the lower half as shown in Panel c). This step enables a dimensionality reduction via principal component analysis (PCA), shown in Panel d). These data are clustered by Gaussian Mixture Model (GMM) and the results of which are displayed in terms of cluster distributions, see Panel e). Panel f) Representative 3D structures for each cluster are selected based on KDE maxima, along with comprehensive torsion angle profiles for the highest populated clusters, showing the wide breadth of the conformational space covered by GAP. Panel g) Structures derived from GAP are clearly presented on the GlycoShape GDB web platform, in addition to biological and chemical information.
Elisa Fadda-🔺

Part 3/8. The -Glyco tool in GlycoShape sources structures directly from the GDB and links them to the uploaded protein structures through a genetic algorithm. Users can upload their own PDB structures, or fetch it directly from the RCSB PDB with a PDB id, or from the AlphaFold Protein Structure Database with a UniProt id. Re-Glyco can perform all types of N- and O-glycosylation and C-mannosylation common is eukaryotes and prokaryotes.

Panel a) Definition of the φ and ψ torsion angles, with corresponding atoms labelled a to e, determining the conformation of the linkage between the protein (P shown in grey) sidechain and the reducing end of the glycan (G shown in yellow). Panel b) Heat maps showing preferential conformation of the φ, ψ torsions between Asn-b-GlcNAc and Thr-a-GalNAc. Panel c) 2D SNFG structure and (below) 3D structures of the tetra-antennary fully a3-sialylated N-glycan from the clustering analysis shown in Figure 1. Panel d) Schematic representation of the Re-Glyco workflow applied to the reconstruction of human Interleukin-5 (IL5; Uniprot P05113). In agreement with the annotation50, GlcNAc scanning identifies only the N47 sequon as potentially occupied. Accordingly N47 can be functionalized with more elaborate structures through a ‘one-shot’ glycosylation, where also T22 can be functionalized with a sialylated core1 O-glycan. Highly complex glycosylation at N47 and alternative O-glycosylation structures can be selected by sourcing directly from the GDB through the Advanced (Site-by-Site) Glycosylation tool, as shown on the alternative IL5 glycoform on the right-hand side. Molecular rendering with Mol* Viewer51 and statistical analysis and heat maps created with matplotlib (https://matplotlib.org/).
Elisa Fadda-🔺

Part 4/8. Where protein N-glycosylation is not known, -Glyco can perform a GlcNAc Scanning test which reports all the potentially occupied sequons. This tool scores a 92% success rate against experimentally confirmed sites annotated in . Moreover, the results of GlcNAc Scanning can flag flaws in the protein structure that would otherwise be impossible to discern and correct.

Panel a) Structure of the CD16b (PDB 1E4J, resolution 2.50 Å) with all the N-glycosylation sequons labelled. The bold labels indicate that the sequons are occupied in neutrophil-bound CD16b56. The green check marks indicate the sequons predicted to be occupied by the Re-Glyco GlcNAc-Scanning tool, while the red x-marks indicate the sequons that Re-Glyco deems unoccupied. Panel b) Structural alignment of the CD16b (PDB 1E4J) in cyan with the CD16b (PDB 6EAQ, resolution 2.22 Å) in magenta. Both sequons occupied in PDB 6EAQ, namely N45 and N161, are correctly predicted as occupied by Re-Glyco. Panel c) Structure of the CD16b (PDB 1E4J) modified by swapping OD1 and ND2 coordinates and alternative rotameric orientation of the N74 sidechain N-glycosylated by Re-Glyco with a different selection of N-glycan structures from the GlycoShape GDB at each site. Panel d) Close-up view of the OD1 and ND2 orientation of N45 in the CD16b from PDB 1E4J (cyan) and from PDB 6EAQ (magenta). Panel d) Close-up of the orientation of the N74 sidechain in the CD16b from PDB 1E4J. The distance between the CG of N45 and the CA of F33 is 4.7 Å. Rendering of the 3D structures in panels a,b, d and e and rotamer search performed with pymol (https://pymol.org/2/). Rendering of 3D structure in panel c with VMD (https://www.ks.uiuc.edu/Research/vmd/) and N-glycans 2D structures with DrawGlycan-SNFG (http://www.virtualglycome.org/DrawGlycan/).
Elisa Fadda-🔺

Part 5/8. Extensive testing with structures from the EBI-EMBL database confirms earlier work indicating that predicted structures are suitable for reintroducing native glycosylation,. Here we also propose strategies to introduce modifications to the underlying proteins, where required to allow glycosylation.

Panel a) Histogram analysis of the distribution of pLLDT scores of residues clashing during the GlcNAc Scanning of 3,415 proteins from the AlphaFold Protein Structure Database, with a total of 12,789 glycosylation sites annotated in Uniprot. The distribution of pLDDT values for all residues in the protein tested is shown with a dotted line. The distribution of the pLDDT values for the residues clashing with the GlcNAc during GlcNAc Scanning, where clashes were resolved by Re-Glyco is shown with powder blue histograms and a blue line. The distribution of the pLDDT values for the residues where clashing was not resolved by Re-Glyco and for the residues in the immediate vicinity (± 2) is shown with rose histograms and a red line. Panel b) 3D structure of the EXTL3 monomer from PDB 8OG1 (green cartoons) represented within the homodimer from PDB 7AU2 (white surface). The resolution for each structure is shown in the labels. Asn residues within sequons are shown with van der Waals (vdW) spheres, where N atoms are in blue and O atoms are in red. The N-glycosylation sequons known to be occupied are shown in bold. A green checkmark indicates that the site is predicted to be occupied by Re-Glyco, while a red cross mark indicates that the site is predicted to be unoccupied by Re-Glyco due to major steric clashing. Panel c) (pls see paper.. doesn't fit here)
Elisa Fadda-🔺

Part 6/8. Ultimately, we hope that through its user-friendly and intuitive interface will allow glycobiology to finally enter all aspects of scientific discovery through structure, by restoring glycoproteins to their functional state, and explicitly highlight the presence of glycans and their functions in the biology of health and disease.

Any feedback, please do not hesitate to contact us. :ablobcatrave:

Elisa Fadda-🔺

Part 7/8. We would like to thank first and foremost Rich Pitts and Mike Riley at ORACLE for Research for their feedback, encouragement and support, 👏 👏 and ORACLE for Research for generous allocation of computational resources. :ablobcatrave:

GlycoShape is currently hosted by ORACLE for Research. It has been a long time coming! Thank youuuuu!

Elisa Fadda-🔺

Part 8/8. We are hugely grateful to all the colleagues and friends in the FAB beta testing team :ablobcatrave: :ablobcatheart: that spent some quality time on GlycoShape and gave us key feedback and encouragement, highlighted nasty bugs, and gave us great new ideas and projects for the next release. 🤩

We owe you all big style! Thank youuuuu!

Gabriele Pollara

@Elisa what a fabulous opening line! Great thread and piece of work too!

Elisa Fadda-🔺

@gpollara thank you do much Gabriele! 😊😊

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