Heptameric transmembrane assembly of the CRISPR-Cas13b regulator Csx27

The type VI-B CRISPR-Cas13 system is an RNA-guided immune pathway in which the Cas13b effector is negatively regulated by the accessory protein Csx27. However, the structural and biochemical basis of Csx27 function remains poorly defined. Here, we characterize Bergeyella zoohelcum Csx27 as a membrane associated oligomeric protein. Bioinformatic prediction, detergent dependent purification, and membrane fractionation followed by western blotting support its membrane association. Native PAGE of Csx27 and BS3 cross-linking of Csx27 reconstituted in MSP2N2 nanodiscs further provide orthogonal evidence for oligomerization. Consistently, cryo-EM 2D class averages revealed ring-like particles with apparent sevenfold symmetry, and AlphaFold-assisted modeling supported a heptameric arrangement. Together, our results provide the first direct experimental evidence for the oligomeric, membrane-embedded assembly of Csx27, establishing a crucial structural foundation for elucidating its regulatory mechanism within the CRISPR-Cas13b immune response.