Quick shear-flow alignment of biological filaments for X-ray fiber diffraction facilitated by methylcellulose.
X-ray fiber diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish fiber alignment within seconds before diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe fiber diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that fibers were aligned within 5 s by giving high shear flow (1000-5000 s(-1)) to the medium and that methylcellulose contained in the medium (approximately 1%) was essential to the accomplishment of uniform orientation with a small angular deviation (<5 degrees). The new alignment method enabled us to execute structure analyses of axonemes by small-angle x-ray diffraction. Since this method was also useful for the quick alignment of purified microtubules, as well as tobacco mosaic tobamovirus, we expect that we can apply it to the structural analysis of many other biological filaments.
Sugiyama
Takaaki
T
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
Miyashiro
Daisuke
D
Takao
Daisuke
D
Iwamoto
Hiroyuki
H
Sugimoto
Yasunobu
Y
Wakabayashi
Katsuzo
K
Kamimura
Shinji
S
D000818:Animals; D054468:Axoneme / Q000737:chemistry / Q000378:metabolism; D001704:Biopolymers / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008747:Methylcellulose; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012212:Rheology / Q000295:instrumentation* / Q000379:methods; D012399:Rotation; D053838:Scattering, Small Angle; D017898:Tobamovirus / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction / Q000379:methods*
D001704:Biopolymers; D008747:Methylcellulose