Filamentous Bacteriophage Viruses: Preparation, Magic-Angle Spinning Solid-State NMR Experiments, and Structure Determination.
Filamentous bacteriophages are elongated semi-flexible viruses that infect bacteria. They consist of a circular single-stranded DNA (ssDNA) wrapped by a capsid consisting of thousands of copies of a major coat protein subunit. Given the increasing number of discovered phages and the existence of only a handful of structures, the development of methods for phage structure determination is valuable for biophysics and structural virology. In recent years, we developed and applied techniques to elucidate the 3D atomic-resolution structures of intact bacteriophages using experimental magic-angle spinning (MAS) solid-state NMR data. The flexibility in sample preparation - precipitated homogeneous solids - and the fact that ssNMR presents no limitation on the size, weight or morphology of the system under study makes it an ideal approach to study phage systems in detail.In this contribution, we describe approaches to prepare isotopically carbon-13 and nitrogen-15 enriched intact phage samples in high yield and purity, and we present experimental MAS NMR methods to study the capsid secondary and tertiary structure, and the DNA-capsid interface. Protocols for the capsid structure determination using the Rosetta modeling software are provided. Specific examples are given from studies of the M13 and fd filamentous bacteriophage viruses.
['Morag|Omry|O|', 'Sgourakis|Nikolaos G|NG|', 'Abramov|Gili|G|', 'Goldbourt|Amir|A|']
D017104:Bacteriophage M13 D002213:Capsid D002247:Carbon Isotopes D004277:DNA, Single-Stranded D004279:DNA, Viral D017912:Inovirus D009682:Magnetic Resonance Spectroscopy D009587:Nitrogen Isotopes
Bacteriophages Magic-angle spinning Solid-state NMR Structure determination Structural virology Rosetta Assignment DNA-protein interaction