title: Direct observation of protein microcrystals in crystallization buffer by atmospheric scanning electron microscopy.
abstract: X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called "crystallization bottleneck". Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Yuusuke
authors.initials: Y
authors.lastname: Maruyama
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Ebihara
authors.affiliation: Advanced Technology Division, JEOL Ltd., Akishima, Tokyo 196-8558, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Nishiyama
authors.affiliation: Advanced Technology Division, JEOL Ltd., Akishima, Tokyo 196-8558, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Konyuba
authors.affiliation: Structure Guided Drug Development Project, JBIC Research Institute, Japan Biological Informatics Consortium (JBIC), Aomi 2-4-7, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Senda
authors.affiliation: National Institute of Environmental Health Sciences-NIH, Department of Health and Human Services, 111 TW Alexander Dr, Research Triangle Park, NC 27709, USA.
authors.forename: Takuro
authors.initials: T
authors.lastname: Numaga-Tomita
authors.affiliation: Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Aomi 2-4-7, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Senda
authors.affiliation: Advanced Technology Division, JEOL Ltd., Akishima, Tokyo 196-8558, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Suga
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Chikara
authors.initials: C
authors.lastname: Sato
mesh_terms: D000818:Animals; D002645:Chickens; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D035463:Pol1 Transcription Initiation Complex Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D035463:Pol1 Transcription Initiation Complex Proteins; C091129:TAF1B protein, human; D008049:Lipase; D009113:Muramidase