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.

Figure 1

A schematic drawing and photographs showing the apparatus used for shear-flow alignment of biological filaments. (a) The incident x-ray beam (1) was passed through an area 6 mm away from the center (2) of a pair of disks (3 and 4) that were glued to stainless steel tubes (Fig. S1). Suspensions of axonemes (2–5 mg/mL), TMV (5 mg/mL), or microtubules (2–5 mg/mL), were placed between the two parallel disks (^∗). X-ray diffraction was measured on the downstream side (5). One of the disks (3) was rotated by a DC motor (6) and a rubber belt (7). (b and c) The apparatus was placed between two crossed polarizing plates and the specimens (axonemes) were observed through a disk window from the upstream side of the incident x-ray beam before applying shear flow (b) and under shear flow during rotation of the disk (c). A cross-shaped pattern can be clearly observed, indicating the birefringence of the specimen formed by shear flow. Occasionally, air bubbles were formed during disk rotation, but they stayed consistently near the center area of the disk. Scale bar, 10 mm.

Figure 2

Examples of observed x-ray diffraction patterns (λ = 0.15 nm; exposure time, 30 s). The vertical axis in each diffraction pattern corresponds to the circumferential direction of the parallel disks. (a–c) Diffraction patterns (reciprocal spacing up to 0.142 nm⁻¹) observed with the suspension of axonemes isolated from sea urchin (A. crassispina) sperm flagella before applying shear flow (a), during application of shear flow in a medium without methylcellulose (b), and during application of shear flow in a medium containing 1% methylcellulose (c). Obvious differences between diffraction patterns in the meridional and equatorial directions are shown.

Figure 3

Detailed analysis of the diffraction pattern (<0.18 nm⁻¹; λ = 0.09 nm) of axonemes isolated from sea urchin (H. pulcherrimus) sperm flagella. Profiles of diffraction intensity on the meridian (a) and on the equator (b) are shown. The meridional peaks are indexed as orders of 96 nm. The same scale was used for the equator.

Figure 4

Diffraction patterns (<0.09 nm⁻¹, λ = 0.09 nm) of TMV suspended in a medium that contained 1% methylcellulose. (a) The pattern before applying shear flow. (b) The pattern during application of shear flow to the specimen. (c) The pattern during application of shear flow, but with a higher range of diffraction angle (<1.1 nm⁻¹). The latter pattern was recorded after an exposure time of 10 min on an image plate (BAS-IP MS2025, Fuji Film) placed 390 mm from the specimen.

Figure 5

Diffraction pattern (<0.67 nm⁻¹, λ = 0.09 nm) from taxol-stabilized microtubules in a medium containing 1% methylcellulose, averaged from 50 images recorded with 2-s exposures. (a) The pattern before applying shear flow (averaged from five images at 2 s exposure). (b) The pattern during application of shear flow to the specimen (averaged from 50 images). The spacings of two layer lines, indicated by the single and double arrowheads, were obtained as 4.05 and 2.03 nm, respectively, after the x-ray optics was calibrated for diffraction from Ag-behenate.