Type of presentation: Poster

IT-6-P-1960 Time-resolved Observations of Single Protein's Motions Using Diffracted Electron Tracking (DET) with Wet Cell SEM

Ishikawa A.¹, Ogawa N.^1,2, Hirohata Y.¹, Yohda M.², Sekiguchi H.³, Sasaki Y. C.⁴

¹Nihon University, Tokyo, JAPAN, ²Tokyo University of Agriculture and Technology, Tokyo, JAPAN, ³SPring-8/JASRI, Hyogo, JAPAN, ⁴The University of Tokyo, Chiba, JAPAN

ishikawa@phys.chs.nihon-u.ac.jp

Diffracted electron tracking (DET) method has been developed for obtaining the information about the dynamics of a single protein molecule[1,2]. DET can be performed using a Scanning Electron Microscope (SEM) equipped with a highly sensitive detector for electron backscattered diffraction (EBSD). DET can trace the rotating motion of individual nanocrystals linked to the specific site in the molecule. Fig.1 shows the principle of the DET and the parameters to be measured. (a) When the electron beam irradiates a nanocrystal, inelastically scattered primary electrons form a band-like EBSD pattern (EBSP) and the 3D motion of nanocrystals can be traced from the shifts of the pattern. (b) shows the rotation angle ω around a single axis and the rotation angles α, β, and γ of the principal lattice vectors , a, b and c of the nanocrystal, respectively, between each time step. For tracing the motion of protein molecule, we have developed the wet cell sealed with the very thin carbon film for SEM observation[1,2]. The EBSP can be obtained from the colloidal gold linked to chaperonin protein in water under the carbon sealing film of the wet cell. In DET, radiation damage of the specimen is the biggest problem. To reduce the damage, specimen supporting was improved, as shown in Fig.2. (a) When the chaperonin protein is fixed to the carbon sealing film, although the motion of the colloidal gold could be traced, no directional motion could be observed in both conditions with and without adenosine tri-phosphate (ATP) which causes the rotation of the chaperonin protein. With this supporting, the chaperonin was irradiated by both the incident electron beam and EBSD electrons, and so damaged it could not move. Therefore the chaperonin supporting system was changed. The molecules are fixed to thin tri-acetyl-cellulose film to the opposite side of the sealing film as shown in (b). With this supporting, the chaperonin is covered with the “shadow” of the colloidal gold and hardly irradiated by the electrons. With this supporting system, the motion of the colloidal gold was traced by DET. The mean square of displacement (MSD) of the rotation angles of the colloidal gold particles, in both conditions with and without ATP, are shown in Fig.3. (a) Without ATP, each MSD of the α, β, and γ is almost same, and no directional motion is observed. (b) On the other hand, with ATP, the magnitude of the γ is clearly decreasing compared to other angles. This means that the chaperonin, linked to the colloidal gold, increases rotational motion around the ND axis as shown in (c). These results correspond with other single protein observations using other techniques.

References [1] N. Ogawa et al., Scientific Reports, 3, 2201 (2013) 1-7 [2] N. Ogawa et al., Ultramicroscopy, 140 (2014) 1-8

This research was supported by the Japan Science and Technology Agency under the Core Research for Evolutional Science and Technology (CREST) program.

Fig. 1: Principle of DET and parameters to be measured. (a) Inelastically scattered electrons in the crystal form a band pattern and crystal motion can be traced from the shifts of the EBSP. (b) The rotation angle ω around a single axis and the rotation angles α, β, and γ of the principal lattice vectors a, b and c are measured.	Fig. 2: Improvement of specimen supporting system for DET to reduce the radiation damage for chaperonin protein. (a) The chaperonin protein is fixed to the carbon sealing film of the wet cell. (b) The chaperonin protein is fixed to thin tri-acetyl-cellulose film opposed to the sealing film and is covered from the electron beam by the colloidal gold.
Fig. 3: MSD of the rotation angles of chaperonin molecules measured by DET. (a) Without ATP, almost no directional motion is observed. (b) With ATP, the g was clearly decreasing compared to other angular. This means that the many motions are the rotations around ND axis (γ = 0) corresponding to the motion of each chaperonin protein (c).