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A graphic and command line protocol for quick and accurate comparisons of protein and nucleic acid structures with US-align

Nature Protocols (2025)Cite this article

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Abstract

With the success of structural biology and the advancements in deep-learning-based structure predictions, rapid and accurate structural comparisons among macromolecular structures have become increasingly important in structural bioinformatics. US-align is a highly efficient, versatile, open-source program for sequential and nonsequential structure comparisons of proteins, RNAs and DNAs in pairwise and multiple alignment forms and applicable to both monomeric and multimeric complex structures. The core algorithm of US-align is built on a highly optimized, iterative superimposition and dynamic programming alignment process, guided with a unified and sequence length-independent scoring function, TM-score. The unique design of US-align not only ensures its high accuracy and speed compared with other state-of-the-art methods designed for specific alignment tasks but also makes it the only protocol that can be applied to multiple alignment tasks and allow a structural comparison across different molecular types, the latter of which is critical for template-based heteromolecular structure prediction and function annotations. Here we describe how to install and effectively utilize US-align as a command line tool, as an online web server, and as a plugin to commonly used molecular graphic systems such as PyMOL. US-align installation takes a few minutes to setup, while the actual alignment implementation can be completed typically within 1 s.

Key points

  • US-align is a highly efficient, versatile and open-source program for the sequential and nonsequential structure comparisons of proteins, RNAs and DNAs in pairwise and multiple alignment forms. It is applicable to both monomeric and multimeric complex structures.

  • Its unique design ensures high accuracy and speed compared with other state-of-the-art methods designed for specific alignment tasks and enables it to be applied to multiple alignment tasks, allowing structural comparison across different molecular types.

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Fig. 1: Diagrams of the US-align algorithm and its different alignment modes.
Fig. 2: Installation of the PyMOL plugin for US-align.
Fig. 3: An illustrative example of pairwise monomer alignment by the US-align command line program.
Fig. 4: Illustrative examples of MSTA and fNS alignments by the US-align command line program.
Fig. 5: An illustrative example of CP alignment by the US-align command line program.
Fig. 6: Illustration of TM-score calculations with different options of US-align.
Fig. 7: An example of pairwise alignment by the US-align PyMOL plugin.
Fig. 8: Example output of US-align web server.

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Data availability

PDB files for PDB IDs 101m, 1mba, 4jhm, 4iaj, 1eh1, 1evv, 6jxm, 3am1, 1ajk and 2ayh are available through https://rcsb.org. The non-redundant PDB library (that is, the I-TASSER template library) is updated on a weekly basis and available at https://zhanggroup.org/library/PDB.tar.bz2. Files to demonstrate the ‘-TMscore’ option of US-align are available at https://zhanggroup.org/TM-score/help.zip.

Code availability

The US-align web server and source code are available at https://zhanggroup.org/US-align/. The code in this Protocol has been peer reviewed.

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Acknowledgements

The authors thank X. Wei and Z. Perry for technical assistances to compile US-align for Mac OS. This work used the Advanced Cyberinfrastructure Coordination Ecosystem: Services and Support (ACCESS) program, which is supported by National Science Foundation (2138259, 2138286, 2138307, 2137603 and 2138296). This work is supported in part by the National Institute of Allergy and Infectious Diseases (AI134678 to L.F. and Y.Z.), Ministry of Education (T1 251RES2309 to Y.Z.), and the National University of Singapore startup grants (WBS #A-8001129-00-00, #A-0010130-15-00, #A-8000974-00-00 to Y.Z.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Author information

Authors and Affiliations

  1. CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Chengxin Zhang

  2. Gilbert S Omenn Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA

    Chengxin Zhang & Lydia Freddolino

  3. Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA

    Chengxin Zhang & Lydia Freddolino

  4. Department of Computer Science, School of Computing, National University of Singapore, Singapore, Singapore

    Yang Zhang

  5. Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore

    Yang Zhang

  6. Department of Biochemistry, School of Medicine, National University of Singapore, Singapore, Singapore

    Yang Zhang

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  1. Chengxin Zhang
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  2. Lydia Freddolino
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Contributions

Y.Z. conceived the project. C.Z. developed the program and prepared the server. C.Z., L.F. and Y.Z. drafted the manuscript and approved the final version. All collaborators of this study who fulfilled the criteria for authorship inclusion required by Nature Portfolio journals have been included as authors. Roles and responsibilities were agreed among collaborators ahead of the research. Local and regional research relevant to this study is referenced.

Corresponding authors

Correspondence to Lydia Freddolino or Yang Zhang.

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Nature Protocols thanks John Dzimianski and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Zhang, C. et al. Nat. Methods 19, 1109–1115 (2022): https://doi.org/10.1038/s41592-022-01585-1

Zhang, Y. et al. Nucleic Acids Res. 33, 2302–2309 (2005): https://doi.org/10.1093/nar/gki524

Zhang, Y. et al. Proteins 57, 702–710 (2004): https://doi.org/10.1002/prot.20264

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Zhang, C., Freddolino, L. & Zhang, Y. A graphic and command line protocol for quick and accurate comparisons of protein and nucleic acid structures with US-align. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01189-x

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