tte last few years have witnessed great advances in structural biology, as shown by the evolution in the number of entries in the protein structure database (http:// from approximately 1,000 entries between 1972 and 1990 to 35,000 today. Most of these protein structures have been solved by X-ray crystallography, some others by NMR. X-ray crystallography exploits the diffracting power of large (approximately millimeter-sized) 3D protein crystals irradiated by an X-ray beam; NMR takes advantage of the response of protein atomic nuclei to changing magnetic fields in solution, ttese two techniques have the undeniable advantage of giving atomic resolution data on the specimen under study. However, a large majority of biological macromolecules work in complexes too large and too flexible to be easily approached byX-ray crystallography or NMR. In contrast, 3D electron microscopy (EM) can deal with a wide range of specimen sizes (from single proteins to whole organelles; Fig. 10.1) and does not require sample crystallization. Although at present

small ribonucleoprotein electron microscope herpesvirus mitochondria ribosome

10000 A

Fig.10.1. Different scales: some examples of biological objects recently analyzed by 3D electron microscopy (EM) illustrate the versatility of the technique, able to deal with a large variety of geometries and sizes. From left to right, rendering of 3D maps for: spliceosomal U1 small nuclear ribonucleoprotein (Stark et al., 2001) (reproduced with permission from Macmillan Publishers Ltd: Nature, copyright 2001); Escherichia coli ribosome (Valleetal., 2003b) (reproduced with permission from Elsevier, copyright 2003); herpesvirus capsid (Zhou et al., 2000) (reproduced with permission from AAAS, copyright 2000) and mitochondria (Frey and Mannella, 2000) (reproduced with permission from Elsevier,copyright 2000).

Springer Series in Biophysics J.L.R. Arrondo and A. Alonso Advanced Techniques in Biophysics © Springer-Verlag Berlin Heidelberg 2006

3D-EM is limited to worse resolution than crystallography or NMR, its versatility has turned it into an excellent technique to analyze the structure of large biological machines. Even the moderate attainable resolution in routine 3D-EM maps permits us to localize structural and functional domains, and to combine this information with previously solved atomic resolution data.

Was this article helpful?

0 0

Post a comment