Acknowledgments
Thank you to Yifan Cheng, Zanlin Yu, and Kaihua Zhang for
providing micrographs used in Figs. 1d and 3 and to Tamir
Gonen for micrographs used in Figs. 1c and 2b.
References
1. Cheng Y (2018) Single-particle cryo-EM—
how did it get here and where will it
go. Science 361:876–880. https://doi.org/
10.1126/science.aat4346
2. Glaeser RM, Hall RJ (2011) Reaching the
information limit in Cryo-EM of biological
macromolecules: experimental aspects. Biophys J 100:2331–2337. https://doi.org/10.
1016/j.bpj.2011.04.018
3. Scheres SH (2016) Processing of structurally
heterogeneous cryo-EM data in relion. Methods Enzymol 579:125–157. https://doi.org/
10.1016/bs.mie.2016.04.012
4. Li X et al (2013) Electron counting and beaminduced motion correction enable nearatomic-resolution single-particle cryo-EM.
Nat Methods 10:584–590. https://doi.org/
10.1038/nmeth.2472
5. Schorb M, Haberbosch I, Hagen WJH,
Schwab Y, Mastronarde DN (2019) Software
tools for automated transmission electron
microscopy. Nat Methods 16:471–477.
https://doi.org/10.1038/s41592-019-0396-9
6. Ohi M, Li Y, Cheng Y, Walz T (2004) Negative
staining and image classification—powerful
tools in modern electron microscopy. Biol
Proced Online 6:23–34. https://doi.org/10.
1251/bpo70
7. Kim LY et al (2018) Benchmarking cryo-EM
single particle analysis workflow. Front Mol
Biosci 5:50. https://doi.org/10.3389/fmolb.
2018.00050
8. Gonen S et al (2012) The structure of purified
kinetochores reveals multiple microtubuleattachment sites. Nat Struct Mol Biol
19:925–929.
https://doi.org/10.1038/
nsmb.2358
9. Passmore LA, Russo CJ (2016) Specimen
preparation for high-resolution Cryo-EM.
Methods Enzymol 579:51–86. https://doi.
org/10.1016/bs.mie.2016.04.011
10. Cheng Y, Grigorieff N, Penczek PA, Walz T
(2015) A primer to single-particle cryo-electron microscopy. Cell 161:438–449. https://
doi.org/10.1016/j.cell.2015.03.050
11. Wu S et al (2012) Fabs enable single particle
cryoEM studies of small proteins. Structure
20:582–592. https://doi.org/10.1016/j.str.
2012.02.017
12. De Carlo S, Harris JR (2011) Negative staining
and cryo-negative staining of macromolecules
and viruses for TEM. Micron 42:117–131.
https://doi.org/10.1016/j.micron.2010.06.
003
13. D’Imprima E et al (2019) Protein denaturation
at the air-water interface and how to prevent
it. elife 8:e42747. https://doi.org/10.7554/
eLife.42747
14. Coscia F et al (2016) Fusion to a homooligomeric scaffold allows cryo-EM analysis of
a small protein. Sci Rep 6:30909. https://doi.
org/10.1038/srep30909
15. Booth DS, Avila-Sakar A, Cheng Y (2011)
Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition. J Vis Exp 22
(58):3227. https://doi.org/10.3791/3227
Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples
123
Thank you to Yifan Cheng, Zanlin Yu, and Kaihua Zhang for
providing micrographs used in Figs. 1d and 3 and to Tamir
Gonen for micrographs used in Figs. 1c and 2b.
References
1. Cheng Y (2018) Single-particle cryo-EM—
how did it get here and where will it
go. Science 361:876–880. https://doi.org/
10.1126/science.aat4346
2. Glaeser RM, Hall RJ (2011) Reaching the
information limit in Cryo-EM of biological
macromolecules: experimental aspects. Biophys J 100:2331–2337. https://doi.org/10.
1016/j.bpj.2011.04.018
3. Scheres SH (2016) Processing of structurally
heterogeneous cryo-EM data in relion. Methods Enzymol 579:125–157. https://doi.org/
10.1016/bs.mie.2016.04.012
4. Li X et al (2013) Electron counting and beaminduced motion correction enable nearatomic-resolution single-particle cryo-EM.
Nat Methods 10:584–590. https://doi.org/
10.1038/nmeth.2472
5. Schorb M, Haberbosch I, Hagen WJH,
Schwab Y, Mastronarde DN (2019) Software
tools for automated transmission electron
microscopy. Nat Methods 16:471–477.
https://doi.org/10.1038/s41592-019-0396-9
6. Ohi M, Li Y, Cheng Y, Walz T (2004) Negative
staining and image classification—powerful
tools in modern electron microscopy. Biol
Proced Online 6:23–34. https://doi.org/10.
1251/bpo70
7. Kim LY et al (2018) Benchmarking cryo-EM
single particle analysis workflow. Front Mol
Biosci 5:50. https://doi.org/10.3389/fmolb.
2018.00050
8. Gonen S et al (2012) The structure of purified
kinetochores reveals multiple microtubuleattachment sites. Nat Struct Mol Biol
19:925–929.
https://doi.org/10.1038/
nsmb.2358
9. Passmore LA, Russo CJ (2016) Specimen
preparation for high-resolution Cryo-EM.
Methods Enzymol 579:51–86. https://doi.
org/10.1016/bs.mie.2016.04.011
10. Cheng Y, Grigorieff N, Penczek PA, Walz T
(2015) A primer to single-particle cryo-electron microscopy. Cell 161:438–449. https://
doi.org/10.1016/j.cell.2015.03.050
11. Wu S et al (2012) Fabs enable single particle
cryoEM studies of small proteins. Structure
20:582–592. https://doi.org/10.1016/j.str.
2012.02.017
12. De Carlo S, Harris JR (2011) Negative staining
and cryo-negative staining of macromolecules
and viruses for TEM. Micron 42:117–131.
https://doi.org/10.1016/j.micron.2010.06.
003
13. D’Imprima E et al (2019) Protein denaturation
at the air-water interface and how to prevent
it. elife 8:e42747. https://doi.org/10.7554/
eLife.42747
14. Coscia F et al (2016) Fusion to a homooligomeric scaffold allows cryo-EM analysis of
a small protein. Sci Rep 6:30909. https://doi.
org/10.1038/srep30909
15. Booth DS, Avila-Sakar A, Cheng Y (2011)
Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition. J Vis Exp 22
(58):3227. https://doi.org/10.3791/3227
Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples
123