The Leeuwenhoek lecture 2006. Microscopy goes cold: frozen viruses reveal their structural secrets

Abstract

The electron microscope provides a powerful tool for investigating the structure of biological complexes such as viruses. A modern instrument is fully capable of atomic resolution on suitable non-biological specimens, but biological materials are difficult to preserve, owing to their fragility, and to image, owing to their radiation, sensitivity. The act of imaging the specimen severely damages it. Originally, samples were prepared by staining with a heavy metal salt, which provides a stable specimen but limits the amount of details that can be retrieved. Now particulate specimens, such as viruses, are prepared by rapid freezing of unstained material and observed in a frozen state with low doses of electrons. The resulting images require extensive computer processing to extract fully detailed three-dimensional information about the specimen. The whole process is referred to as single-particle electron cryomicroscopy. Using this approach, the structure of the human hepatitis B virus core was solved at the level of the protein fold. By comparing maps of RNA-and DNAcontaining cores, it was possible to propose a model for the maturation and control of the envelopment of the virus during assembly. These examples show that cryomicroscopy offers great potential for understanding the structure and function of complex biological assemblies.

The Coming of Advanced Materials: A Personal View of the Contributions by Cambridge Scientists

“…[17] Klug, who won the chemistry Nobel Prize in 1982, enabled the structures of viruses to be determined by ingenious use of the electron microscope. The first ever complete virus structure-that of bushy stunt-was determined by his colleague, R. A. Crowther, [18] who has recently reviewed the development of cryoelectronmicroscopy over the intervening years-see also refs. [13,19], where Ahmed Zewail and I set this work in a broader context.…”

Ultrafast Light and Electrons: Imaging the Invisible

“…Minimizing radiation damage by embedding the biological macromolecules and machines in vitreous ice affords a non-invasive, high-resolution imaging technique for visualizing the 3D organization of eukaryotic cells, with their dynamic organelles, cytoskeletal structure, and molecular machines in an unperturbed context, with an unprecedented resolution. I refer the reader to the papers by Henderson [35], Sali et al [73], Crowther [16], and Glaeser [30], and the books by Glaeser et al [31] and by Frank [27]. The Nobel paper by Roger Kornberg [48] on RNA polymerase II (pol II) transcription machinery is a must for reading.…”

Self‐Assembling Viral Mimetics: One Long Journey with Short Steps

“…reconstitute independently of nucleic acid components. [12][13][14] Thus, in principle, any virus can be assembled to encase a foreign but similarly sized material without compromising the native morphology of the capsule, and hence devising an artificial vector comes down to the construction of an encapsulating self-assembling system. Although structural parameters behind the viral architecture are fairly well described to provide first design principles for capsule mimetics, an artificial version of a viral assembly has yet to emerge.…”

“…The latter define the morphological dimensions of assembled virions. Yet, despite their apparent diversity, all viral forms are discrete self‐assemblies underlain by the same architectural parameters, which are, most importantly, symmetry driven and are programmed to be able to reconstitute independently of nucleic acid components 12–14. Thus, in principle, any virus can be assembled to encase a foreign but similarly sized material without compromising the native morphology of the capsule, and hence devising an artificial vector comes down to the construction of an encapsulating self‐assembling system.…”