SPring-8, the large synchrotron radiation facility

Release Date

29 Jun, 2005

June 29, 2005
JASRI/SPring-8

1. Background
      The X-ray beam from the third-generation synchrotron radiation source such as SPring-8 is so intense that it has enough flux for diffraction studies even after its diameter is limited by a micrometer-sized aperture. By using this, the same research team has previously recorded diffraction patterns from single myofibrils (see the topic on Aug. 6, 2002). However, there are a number of technical problems to be solved before diffraction patterns can be recorded from hydrated biological specimens much smaller than a whole myofibril, besides the use of more intense X-ray beams from the High Flux Beamline, BL40XU. The major problem is radiation damage; minute specimens require long exposure times even if intense microbeams are used, and the specimens will be quickly damaged. Here this problem was overcome by quick-freezing the specimens and recording diffraction patterns while keeping the specimens at a liquid nitrogen temperature (74 K). By doing so, the specimens become far more resistant to radiation damage. The specimens are quick-frozen to prevent the formation of ice crystals that would destroy the fine structures to be studied.

2. Methods
      A suspension of myofibrils was prepared by homogenizing the flight muscle of a bumblebee by using a blender⁽¹⁾. The suspension was dropped on a grid for electron microscopy covered with a thin plastic film, to adsorb the myofibrils on its surface. The grid was then quick-frozen by plunging it into liquid propane (93K), and was transferred to a cryochamber⁽²⁾ installed at the beamline, where the specimen was kept at 74K until the end of X-ray recording. X-ray microbeams were generated by using a 2-µm pinhole as described in the previous Topics.

3. Results
      To catch a myofibril in the beam, the whole grid was scanned in x and y directions. The grid was expected to have adsorbed a large number of myofibrils, but diffraction patterns were rarely observed. This is probably because only a limited number of adsorbed myofibrils met the Bragg condition⁽³⁾ to generate reflections. Figure 2 shows one of examples in which reflections were clearly recorded. Observed in the figure are the strongest 1,0 and 2,0 equatorial reflections, which are indexable to the hexagonal lattice of myofilaments. The size of the diffracting object was estimated by scanning the grid across the long axis and recording the change of reflection intensities. The analysis revealed that there were two objects lying next to each other, and their diameters were 2.7 and 3.5 µm, respectively, after correction for the beam size. These values coincide well with the known diameter of a single myofibril. Because the beam size was also ~2 µm (i.e., less than the length of a single sarcomere), this means that diffraction patterns were taken from the volume equivalent to a single sarcomere within a myofibril.
      Figure 3 shows the intensity profile of the reflections measured along the equator (black curve). There are two reflection peaks, i.e., the 1,0 and 2,0 reflections. If a muscle fiber is irradiated with a larger beam, the 1,1 reflection is expected to appear between these two reflections. In Fig. 3., an intensity profile of an end-on diffraction pattern recorded with a 50-µm diameter beam (gray curve). When compared with this, it is evident that the 1,1 reflection is completely missing from the profile from the myofibril (black). This is because a single sarcomere contains only one hexagonal lattice, and when the Bragg condition is met for the 1,0 (and 2,0) lattice plane, the Bragg condition is never met for the 1,1 plane. This is another piece of evidence that the reflections came from a single lattice of a sarcomere.

4. Perspectives
      A cell contains a variety of organelles, in which various proteins, lipids and other biological molecules are functioning by interacting with each other. There will be increasing demands for the in-situ observation of the structure of these organelles. The success of the diffraction recording from a single sarcomere will lead to the X-ray structural analyses of similarly-sized organelles. The combination of the quick-freezing technique and X-ray diffraction studies will also be useful in applications with conventionally-sized X-ray beams, in reducing radiation damage and making it possible to record diffraction patterns from smaller specimens.


<Notes>

(1) Blender: a device for homogenizing specimens with a fast-rotating blade.

(2) Cryochamber: a specimen chamber designed to keep the specimen stably at a liquid nitrogen or liquid helium temperature. For thermal insulation, the specimens are usually kept in vacuum.

(3) Bragg condition: The X-rays scattered by the lattice planes of a crystal positively interfere with each other to generate reflections only when the incident X-ray beam makes a specific angle with respect to the lattice plane. The condition of the incident angle that produces reflections is called the Bragg condition. The range of angles which meet the Bragg condition is very narrow, and is of the order of 10 seconds in perfect inorganic crystals.

<Figures>

Fig. 1    Structure of vertebrate skeletal muscle (striated muscle).
Fig. 2   A series of diffraction patterns recorded from a single sarcomere.
Fig. 3   Intensity profile of the diffraction pattern recorded from a single sarcomere