29 Nov 2022
29 Nov 2022
Status: this preprint is currently under review for the journal MR.

Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR

Sarah Kuschert1, Martin Stroet2, Yanni Ka-Yan Chin1, Anne Clair Conibear3, Xinying Jia1, Thomas Lee2, Christian Reinhard Otto Bartling4, Kristian Strømgaard4, Peter Güntert5,6,7, Karl Johan Rosengren8, Alan Edward Mark2, and Mehdi Mobli1 Sarah Kuschert et al.
  • 1Centre for Advanced Imaging, The University of Queensland, Brisbane, QLD 4072, Australia
  • 2School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia
  • 3Institute of Applied Synthetic Chemistry, Technische Universität Wien, Getreidemarkt 9/163, Wien 1060, Vienna, Austria
  • 4Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
  • 5Laboratory of Physical Chemistry, ETH Zürich, 8093 Zürich, Switzerland
  • 6Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany
  • 7Department of Chemistry, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan
  • 8School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia

Abstract. Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server ( with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of sidechains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein-protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally.

Sarah Kuschert et al.

Status: final response (author comments only)

Comment types: AC – author | RC – referee | CC – community | EC – editor | CEC – chief editor | : Report abuse
  • RC1: 'Comment on mr-2022-22', Anonymous Referee #1, 14 Dec 2022
    • AC1: 'Reply on RC1', Mehdi Mobli, 16 Jan 2023
  • RC2: 'Comment on mr-2022-22', Anonymous Referee #2, 09 Jan 2023
    • AC2: 'Reply on RC2', Mehdi Mobli, 20 Jan 2023
  • RC3: 'Comment on mr-2022-22', Bruno Kieffer, 20 Jan 2023

Sarah Kuschert et al.

Sarah Kuschert et al.


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Short summary
The 20 genetically encoded amino acids provide the basis for most proteins and peptides that make up the machinery of life. This limited repertoire is vastly expanded by the introduction of non-canonical amino acids (ncAAs). To study the structure of a protein containing an ncAA requires new computational representation that are compatible with existing modelling software. We have developed an online tool for this, to aid future structural studies of this class of complex biopolymers.