30. As long as magnification-dependent zoom is disabled, changes
in magnification will not alter the size of the electron beam at
the specimen level. Changing magnification will alter how the
camera samples the electron beam, with an increase in magnification resulting in a decrease in observed exposure rate (e
À /
pixel/s). However, the flux, as measured in e
À
/A ˚ 2 /s, should,
in theory, remain unchanged.
31. Exposure rates greater than 10 eÀ/pixel/s will result in
increased coincidence loss and decreased K2 camera recording
capability of spatial frequencies lower than half Nyquist
[19]. For other direct electron detectors, please refer to the
user manual for optimal exposure rates and dose fractionation.
32. The condenser lenses of the Talos Arctica use a zoom system
and thus if properly aligned, changing spot size number will
change the C2 intensity value without affecting parallel illumination. If the spot size-dependent zoom alignment is off, then
changing spot size will alter parallel illumination and steps 1–
10 (Subheading 3.3) should be repeated.
Acknowledgments
I would like thank Gabe Lander, Mengyu Wu, and Mr. Bill Anderson at The Scripps Research Institute (TSRI) and Matthijn Vos for
helpful advice and discussion regarding TEM alignments and data
acquisition. I would like to thank Mr. Bill Anderson (TSRI), Kevin
Corbett (UCSD), Gabe Lander (TSRI), Andres Leschziner
(UCSD), Sergey Suslov (UCSD), and Mengyu Wu (TSRI) for
critical reading of the chapter and helpful discussions.
References
1. Eyidi D, Hebert C, Schattschneider P (2006)
Short note on parallel illumination in the TEM.
Ultramicroscopy 106:1144–1149
2. Glaeser RM, Typke D, Tiemeijer PC,
Pulokas J, Cheng A (2011) Precise beam-tilt
alignment and collimation are required to minimize the phase error associated with coma in
high-resolution cryo-EM. J Struct Biol
174:1–10
3. Christenson KK, Eades JA (1988) Skew
thoughts on parallelism. Ultramicroscopy
26:113–132
4. Zivanov J et al (2018) New tools for automated high-resolution cryo-EM structure
determination in RELION-3. elife 7:e42166
5. Zhang K (2016) Gctf: Real-time CTF determination and correction. J Struct Biol 193:1–12
6. Punjani A, Rubinstein JL, Fleet DJ, Brubaker
M (2017) A. cryoSPARC: algorithms for rapid
unsupervised cryo-EM structure determination. Nat Methods 14:290–296
7. Noble AJ et al (2018) Routine single particle
CryoEM sample and grid characterization by
tomography. elife 7:e34257
8. Danev R, Yanagisawa H, Kikkawa M (2019)
Cryo-electron microscopy methodology: current aspects and future directions. Trends Biochem Sci 44:837–848. https://doi.org/10.
1016/j.tibs.2019.04.008
9. Tan YZ et al (2017) Addressing preferred specimen orientation in single-particle cryo-EM
through tilting. Nat Methods 14:793–796
10. Avila-Sakar A, Li X, Zheng SQ, Cheng Y
(2013) Recording High-Resolution Images of
Parallel Illumination on the Talos Arctica
143
in magnification will not alter the size of the electron beam at
the specimen level. Changing magnification will alter how the
camera samples the electron beam, with an increase in magnification resulting in a decrease in observed exposure rate (e
À /
pixel/s). However, the flux, as measured in e
À
/A ˚ 2 /s, should,
in theory, remain unchanged.
31. Exposure rates greater than 10 eÀ/pixel/s will result in
increased coincidence loss and decreased K2 camera recording
capability of spatial frequencies lower than half Nyquist
[19]. For other direct electron detectors, please refer to the
user manual for optimal exposure rates and dose fractionation.
32. The condenser lenses of the Talos Arctica use a zoom system
and thus if properly aligned, changing spot size number will
change the C2 intensity value without affecting parallel illumination. If the spot size-dependent zoom alignment is off, then
changing spot size will alter parallel illumination and steps 1–
10 (Subheading 3.3) should be repeated.
Acknowledgments
I would like thank Gabe Lander, Mengyu Wu, and Mr. Bill Anderson at The Scripps Research Institute (TSRI) and Matthijn Vos for
helpful advice and discussion regarding TEM alignments and data
acquisition. I would like to thank Mr. Bill Anderson (TSRI), Kevin
Corbett (UCSD), Gabe Lander (TSRI), Andres Leschziner
(UCSD), Sergey Suslov (UCSD), and Mengyu Wu (TSRI) for
critical reading of the chapter and helpful discussions.
References
1. Eyidi D, Hebert C, Schattschneider P (2006)
Short note on parallel illumination in the TEM.
Ultramicroscopy 106:1144–1149
2. Glaeser RM, Typke D, Tiemeijer PC,
Pulokas J, Cheng A (2011) Precise beam-tilt
alignment and collimation are required to minimize the phase error associated with coma in
high-resolution cryo-EM. J Struct Biol
174:1–10
3. Christenson KK, Eades JA (1988) Skew
thoughts on parallelism. Ultramicroscopy
26:113–132
4. Zivanov J et al (2018) New tools for automated high-resolution cryo-EM structure
determination in RELION-3. elife 7:e42166
5. Zhang K (2016) Gctf: Real-time CTF determination and correction. J Struct Biol 193:1–12
6. Punjani A, Rubinstein JL, Fleet DJ, Brubaker
M (2017) A. cryoSPARC: algorithms for rapid
unsupervised cryo-EM structure determination. Nat Methods 14:290–296
7. Noble AJ et al (2018) Routine single particle
CryoEM sample and grid characterization by
tomography. elife 7:e34257
8. Danev R, Yanagisawa H, Kikkawa M (2019)
Cryo-electron microscopy methodology: current aspects and future directions. Trends Biochem Sci 44:837–848. https://doi.org/10.
1016/j.tibs.2019.04.008
9. Tan YZ et al (2017) Addressing preferred specimen orientation in single-particle cryo-EM
through tilting. Nat Methods 14:793–796
10. Avila-Sakar A, Li X, Zheng SQ, Cheng Y
(2013) Recording High-Resolution Images of
Parallel Illumination on the Talos Arctica
143