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Quick shear-flow alignment of biological filaments for X-ray fiber diffraction facilitated by methylcellulose.

ABSTRACT: 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.

SUBMITTER: Sugiyama T

PROVIDER: S-EPMC2794338 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Quick shear-flow alignment of biological filaments for X-ray fiber diffraction facilitated by methylcellulose.

Sugiyama Takaaki T Miyashiro Daisuke D Takao Daisuke D Iwamoto Hiroyuki H Sugimoto Yasunobu Y Wakabayashi Katsuzo K Kamimura Shinji S

Biophysical journal 20091201 12

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 ...[more]

PMID: 20006950

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Project description:The high homology of its axonemal components with humans and a large repertoire of axonemal mutants make Chlamydomonas a useful model system for experiments on the structure and function of eukaryotic cilia and flagella. Using this organism, we explored the spatial arrangement of axonemal components under physiological conditions by small-angle x-ray fiber diffraction. Axonemes were oriented in physiological solution by continuous shear flow and exposed to intense and stable x rays generated in the synchrotron radiation facility SPring-8, BL45XU. We compared diffraction patterns from axonemes isolated from wild-type and mutant strains lacking the whole outer arm (oda1), radial spoke (pf14), central apparatus (pf18), or the ?-chain of the outer arm dynein (oda11). Diffraction of the axonemes showed a series of well-defined meridional/layer-line and equatorial reflections. Diffraction patterns from mutant axonemes exhibited a systematic loss/attenuation of meridional/layer-line reflections, making it possible to determine the origin of various reflections. The 1/24 and 1/12 nm(-1) meridional reflections of oda1 and oda11 were much weaker than those of the wild-type, suggesting that the outer dynein arms are the main contributor to these reflections. The weaker 1/32 and 1/13.7 nm(-1) meridional reflections from pf14 compared with the wild-type suggest that these reflections come mainly from the radial spokes. The limited contribution of the central pair apparatus to the diffraction patterns was confirmed by the similarity between the patterns of the wild-type and pf18. The equatorial reflections were complex, but a comparison with electron micrograph-based models allowed the density of each axonemal component to be estimated. Addition of ATP to rigor-state axonemes also resulted in subtle changes in equatorial intensity profiles, which could report nucleotide-dependent structural changes of the dynein arms. The first detailed description of axonemal reflections presented here serves as a landmark for further x-ray diffraction studies to monitor the action of constituent proteins in functional axonemes.

Dataset Information

Quick shear-flow alignment of biological filaments for X-ray fiber diffraction facilitated by methylcellulose.

Publications

Quick shear-flow alignment of biological filaments for X-ray fiber diffraction facilitated by methylcellulose.

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