TY - JOUR
T1 - A new cryo-EM system for single particle analysis
AU - Hamaguchi, Tasuku
AU - Maki-Yonekura, Saori
AU - Naitow, Hisashi
AU - Matsuura, Yoshinori
AU - Ishikawa, Tetsuya
AU - Yonekura, Koji
N1 - Funding Information:
We thank T. Ebisu, R. Fujioka, T. Kaneko, Y. Shimizu, T. Sato, T. Fukumura, J. Brink, H. Iijima, K. Kobayashi, I. Ishikawa, S. Ogiwara and S. Motoki for setting up the CRYO ARM 300 electron microscope, T. Kato and K. Tani for advice on energy filtering and useful discussions, F. Makino, T. Kato, and K. Namba for support of data collection with the Schottky emission, D. Mastronarde for implementation of new functions in SerialEM, K. N. Yoshikawa for providing the ALSV sample, L. Fairall and C. Savva for providing a plasmid of human heavy chain ferritin, Y. Kageyama for preparation of the apoferritin, T. Nakane for useful discussion for estimation of beam tilts, and D. B. McIntosh for help in improving the manuscript. A cryo-EM density map of apoferritin has been deposited in EMDataResource under the accession number EMD-9890. This work was partly supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research Grant 16H04757 (to K.Y.), Japan Society for the Promotion of Science Grant-in-Aid for Challenging Exploratory Research G rant 24657111 (to K.Y.), the Cyclic Innovation for Clinical Empowerment (CiCLE) from the Japan Agency for Medical Research and Development , AMED (to K.Y.), and the Japan Science and Technology Agency SENTAN program (to K.Y.).
Publisher Copyright:
© 2019 The Authors
PY - 2019/7/1
Y1 - 2019/7/1
N2 - A new cryo-EM system has been investigated for single particle analysis of protein structures. The system provides parallel illumination of a highly-coherent 300 kV electron beam from a cold-field emission gun, and boosts image contrast with an in-column energy filter and a hole-free phase plate. It includes motorized cryo-sample loading and automated liquid-nitrogen filling for cooling multiple samples. In this study, we describe gun and electron beam characteristics, and demonstrate the suitability of this system for single particle reconstructions. The performance of the system is tested on two examples, a spherical virus and apoferritin. GUI programs have also been developed to control and monitor the system for correct illumination, imaging with less ellipticity and steady magnification, and timing of flashing and liquid-nitrogen filling. These programs are especially useful for efficient application of the system to single particle cryo-EM.
AB - A new cryo-EM system has been investigated for single particle analysis of protein structures. The system provides parallel illumination of a highly-coherent 300 kV electron beam from a cold-field emission gun, and boosts image contrast with an in-column energy filter and a hole-free phase plate. It includes motorized cryo-sample loading and automated liquid-nitrogen filling for cooling multiple samples. In this study, we describe gun and electron beam characteristics, and demonstrate the suitability of this system for single particle reconstructions. The performance of the system is tested on two examples, a spherical virus and apoferritin. GUI programs have also been developed to control and monitor the system for correct illumination, imaging with less ellipticity and steady magnification, and timing of flashing and liquid-nitrogen filling. These programs are especially useful for efficient application of the system to single particle cryo-EM.
KW - CFEG
KW - Cold field emission
KW - CRYO ARM
KW - ParallEM
KW - Single particle cryo-EM
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U2 - 10.1016/j.jsb.2019.04.011
DO - 10.1016/j.jsb.2019.04.011
M3 - Article
C2 - 30991102
AN - SCOPUS:85064937078
SN - 1047-8477
VL - 207
SP - 40
EP - 48
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 1
ER -