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High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE
Published on: May 16, 2017
Single-particle visualization of assembly: I. Dimerization in a planar zone.
1Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
Journal of Microscopy
|January 19, 2005
Summary
This study analyzes the association and dissociation of bacteriophage lambda procapsids using real-time single-particle fluorescence microscopy. Findings reveal a molten agarose barrier and non-random dimer orientation influence particle behavior in the concentration plane.
Area of Science:
- Biophysics
- Microscopy techniques
- Molecular assembly analysis
Background:
- Single-particle fluorescence microscopy is crucial for studying biological assembly reactions.
- Previous work utilized molten agarose to concentrate thermally diffusing particles for analysis.
Purpose of the Study:
- To perform the first real-time, single-particle analysis of association/dissociation for thermally diffusing particles.
- To investigate the behavior of bacteriophage lambda procapsids in a concentrated solution.
Main Methods:
- Developed a method to quantify thermal motion of single particles.
- Applied single-particle fluorescence microscopy to observe procapsid dynamics.
- Utilized molten agarose to create a concentration plane near a coverglass surface.
Main Results:
- Quantified thermal motion to determine particle binding.
- Observed real-time association and dissociation events of bacteriophage lambda procapsids.
- Identified a molten agarose barrier (93-155 nm from surface) affecting particle diffusion.
Conclusions:
- The molten agarose creates a confinement barrier influencing particle interactions.
- Non-random orientation of procapsid dimers in the concentration plane was observed.
- This method enables detailed analysis of molecular assembly dynamics at the single-particle level.
