Host Receptor Pili for Cryo-EM Single-Particle Reconstruction

Single-stranded RNA bacteriophages (ssRNA phages) infect their hosts by binding to the host receptor pili. Purification of pili usually involves mechanical shearing of pili from cells followed by precipitation. However, previous methods often result in low efficiency or unstable results due to pili retraction. This protocol presents an optimized method for purifying receptor type IV pili from (), incorporating enhancements in shearing and collection steps to achieve high yields. We found that repeated passage through syringe needles increases yield, and temperature control is crucial during purification. Additionally, the CsCl density gradient was optimized specifically for this specific strain. The purified type IV pili are suitable for cryogenic electron microscopy (cryo-EM) and various biochemical experiments. Key features • Pili purification for single-particle cryo-electron microscopy (Cryo-EM) analysis • This protocol builds upon the F-pili purification method developed by Costa et al. [1] extending its application to the 16. • It is optimized for higher and more stable pili yields, as well as increased reproducibility. • The method is tested on various bacterial species and can be adapted to purify different types of pili.