Related Experiment Video
Updated: Jun 8, 2025
High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE
Published on: May 16, 2017
Host Receptor Pili for Cryo-EM Single-Particle Reconstruction
Ran Meng1,2
1Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA.
Abstract:
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 Acinetobacter genomospecies 16 (A. gp16), 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 Acinetobacter genomosp. 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.
Insights
This study optimizes pili purification from Acinetobacter genomospecies 16, enhancing yields for single-stranded RNA bacteriophage (ssRNA phage) research. The improved method ensures stable, high-purity pili suitable for cryo-electron microscopy (cryo-EM) and biochemical analyses.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Single-stranded RNA bacteriophages (ssRNA phages) utilize host receptor pili for infection.
- Conventional pili purification methods often suffer from low efficiency and pili retraction, impacting yield and stability.
- Type IV pili from Acinetobacter genomospecies 16 (A. gp16) are crucial for ssRNA phage binding.
Purpose of the Study:
- To develop an optimized protocol for high-yield purification of type IV pili from A. gp16.
- To improve the stability and reproducibility of pili purification for downstream applications.
- To adapt existing F-pili purification methods for A. gp16 type IV pili.
Main Methods:
- Incorporated enhanced mechanical shearing via repeated syringe needle passage.
- Implemented strict temperature control throughout the purification process.
- Optimized Cesium Chloride (CsCl) density gradient centrifugation specific to A. gp16.
Main Results:
- Achieved significantly higher and more stable yields of purified type IV pili.
- Demonstrated the suitability of purified pili for single-particle cryo-electron microscopy (cryo-EM).
- Validated the protocol's effectiveness across various bacterial species, indicating adaptability.
Conclusions:
- The optimized protocol provides a robust method for purifying A. gp16 type IV pili.
- This method enhances pili yield and stability, facilitating advanced structural and biochemical studies.
- The protocol is adaptable for purifying pili from diverse bacterial sources.
More Related Videos
12:38Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction
Published on: August 9, 2011
09:25Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction
Published on: January 9, 2015
Related Concept Videos
01:28Cryo-electron Microscopy
01:07Electron Microscope Tomography and Single-particle Reconstruction
Electron Tomography
Electron tomography can be performed either in TEM or STEM (scanning transmission...