The Leeuwenhoek lecture 2006. Microscopy goes cold: frozen viruses reveal their structural secrets.
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 DNA-containing 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.
Crowther
R A
RA
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. mrc-lmb@cam.ac.uk
D005615:Freezing; D008853:Microscopy / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D014764:Viral Proteins / Q000648:ultrastructure; D014780:Viruses / Q000648:ultrastructure*
D014764:Viral Proteins
J Virol. 1999 Dec;73(12):10122-8
10559327
J Virol. 1983 Aug;47(2):337-43
6620456
Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2194-8
7892246
J Virol. 2003 Jan;77(1):416-22
12477846
Biochem Pharmacol. 2003 Dec 15;66(12):2273-9
14637185
Intervirology. 2001;44(2-3):98-114
11509871
Adv Virus Res. 2005;64:125-64
16139594
Cell. 1982 Jun;29(2):403-15
6180831
Proc R Soc Lond B Biol Sci. 1987 Mar 23;230(1259):107-46
2884666
EMBO J. 1998 Dec 1;17(23):6839-45
9843489
Nature. 1997 Mar 6;386(6620):91-4
9052787
Nature. 1984 Mar 1-7;308(5954):32-6
6322001
Science. 2003 Feb 7;299(5608):893-6
12574631
Nature. 1956 Mar 10;177(4506):473-5
13309339
Nature. 1997 Mar 6;386(6620):88-91
9052786
Q Rev Biophys. 1988 May;21(2):129-228
3043536
Virology. 2004 May 20;323(1):108-19
15165823
Cell. 1994 Jun 17;77(6):943-50
8004680
Mol Cell. 1999 Jun;3(6):771-80
10394365
Philos Trans R Soc Lond B Biol Sci. 1971 May 27;261(837):221-30
4399207
Nature. 1968 Jan 13;217(5124):130-4
23610788
J Biol Chem. 2006 Jun 23;281(25):17474-17481
16621801
Virus Res. 2004 Dec;106(2):199-209
15567498
Cold Spring Harb Symp Quant Biol. 1962;27:1-24
14019094
J Virol. 1996 Jul;70(7):4269-74
8676448
Infect Immun. 2005 Jun;73(6):3587-97
15908388
J Virol. 2003 Jul;77(13):7673-6
12805468
J Virol. 2000 Oct;74(19):9099-105
10982356
Nature. 2001 Oct 18;413(6857):748-52
11607035
J Struct Biol. 1996 Jan-Feb;116(1):120-30
8742733
Q Rev Biophys. 2004 Feb;37(1):3-13
17390603
J Virol. 2004 Dec;78(24):13653-68
15564475
J Virol. 2002 Jul;76(13):6510-7
12050364
Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15821-6
16247012