RESUMEN
Scanning transmission electron microscopy (STEM) of unstained, freeze-dried biological macromolecules in the dark-field mode provides an image based on the number of electrons elastically scattered by the constituent atoms of the macromolecule. The image of each isolated particle provides information about the projected structure of the latter, and its integrated intensity is directly related to the mass of the selected particle. Particle images can be sorted by shape, providing independent histograms of mass to study assembly/disassembly intermediates. STEM is optimized for low-dose imaging and is suitable for accurate measurement of particle masses over the range from about 30 kDa to 1,000 MDa. This article describes the details of the method developed at the Brookhaven National Laboratory STEM facility and illustrates its application to the mass mapping of large globin complexes.
Asunto(s)
Globinas/química , Globinas/ultraestructura , Microscopía Electrónica de Transmisión de Rastreo/métodos , Animales , Estabilidad de Medicamentos , Liofilización , Microscopía Electrónica de Transmisión de Rastreo/estadística & datos numéricos , Peso Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Oligoquetos/química , Desnaturalización Proteica , Programas InformáticosRESUMEN
The infectious component of hepatitis B (HB) virus (HBV), the Dane particle, has a diameter of approximately 44 nm and consists of a double-layered capsid particle enclosing a circular, incomplete double-stranded DNA genome. The outer capsid layer is formed from the HB surface antigen (HBsAg) and lipid, whereas the inner layer is formed from the HB core Ag assembled into an icosahedral structure. During chronic infection HBsAg is expressed in large excess as noninfectious quasispherical particles and tubules with approximately 22-nm diameter. Here, we report cryo-EM reconstructions of spherical HBsAg particles at approximately 12-A resolution. We show that the particles possess different diameters and have separated them into two predominant populations, both of which have octahedral symmetry. Despite their differing diameters, the two forms of the particle have the same mass and are built through conformational switching of the same building block, a dimer of HBsAg. We propose that this conformational switching, combined with interactions with the underlying core, leads to the formation of HBV Dane particles of different sizes, dictated by the symmetry of the icosahedral core.