Muscle is widely-used software for making multiple alignments of biological sequences.

Muscle achieves highest scores on Balibase, Bralibase and Balifam benchmark tests and scales to thousands of sequences or structures on a commodity desktop computer.

Muscle supports generating an ensemble of alternative alignments with the same high accuracy obtained with default parameters. By comparing downstream predictions from different alignments, such as trees, a biologist can evaluation the robustness of conclusions against alignment variation caused by ambiguities and errors.

Multiple structure alignment

Structure alignment (“Muscle-3D”) is supported as well as conventional amino acid sequence alignment. Input for structure alignment is a “mega” file generated by the pdb2mega command of reseek (https://github.com/rcedgar/reseek).

# for up to ~100 structures
reseek -pdb2mega STRUCTS -output structs.mega
muscle -align structs.mega -output structs.afa

# for up to ~10,000 structures
reseek -convert STRUCTS -bca structs.bca
reseek -pdb2mega structs.bca -output structs.mega
reseek -distmx structs.bca -output structs.distmx
muscle -super7 structs.mega -distmxin structs.distmx -reseek -output structs.afa

Downloads and installation

Binary files are self-contained, no dependencies. To install, download the binary and make sure the execute bit is set.

https://github.com/rcedgar/muscle/releases

Documentation

Muscle v5 home page
Manual

Building MUSCLE from source

https://github.com/rcedgar/muscle/wiki/Building-MUSCLE

References

Edgar RC., Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nature Communications 13.1 (2022): 6968.
https://www.nature.com/articles/s41467-022-34630-w.pdf

Edgar RC. and Tolstoy I., Muscle-3D: scalable multiple protein structure alignment (2024) BioRxiv.