Filamentous Bacteriophage Viruses: Preparation, Magic-Angle Spinning Solid-State NMR Experiments, and Structure...

Area of Science:

• Structural biology
• Biophysics
• Virology

Background:

• Filamentous bacteriophages are vital models for studying viral structure and assembly.
• Determining phage structures is crucial for understanding their biological functions.
• Existing structural data is limited, necessitating advanced methodologies.

Purpose of the Study:

• To present a robust method for determining the 3D atomic-resolution structures of intact filamentous bacteriophages.
• To detail sample preparation and magic-angle spinning (MAS) solid-state NMR techniques for phage structural analysis.
• To provide protocols for capsid structure determination using Rosetta modeling software.

Main Methods:

• Preparation of isotopically enriched (¹³C, ¹⁵N) intact phage samples.
• Application of magic-angle spinning (MAS) solid-state NMR spectroscopy.
• Utilizing Rosetta software for capsid structure determination.
• Analysis of capsid secondary and tertiary structure, and DNA-capsid interface.

Main Results:

• Successful preparation of high-yield, high-purity phage samples.
• Elucidation of capsid secondary and tertiary structures using MAS ssNMR.
• Detailed analysis of the DNA-capsid interface.
• Demonstration of the method's applicability to M13 and fd phages.

Conclusions:

• Magic-angle spinning (MAS) solid-state NMR is a powerful technique for atomic-resolution structure determination of intact filamentous bacteriophages.
• The described methods overcome limitations of size and morphology for structural studies.
• This approach advances biophysical and structural virology research on phage systems.