23. van Heel M, Harauz G, Orlova EV et al (1996)
A new generation of the IMAGIC image processing system. J Struct Biol 116:17–24
24. van Heel M, Hollenberg J (1980) On the
stretching
of
distorted
images
of
two-dimensional crystals. In: Baumeister W,
Vogell W (eds) Electron microscopy at molecular dimensions: state of the art and strategies
for the future. Springer, Berlin, Heidelberg, pp
256–260
25. Saxton WO, Baumeister W (1982) The correlation averaging of a regularly arranged bacterial cell envelope protein. J Microsc
127:127–138
26. Henderson R, Baldwin JM, Downing KH et al
(1986) Structure of purple membrane from
halobacterium halobium: recording, measurement and evaluation of electron micrographs at
3.5
A ˚
resolution.
Ultramicroscopy
19:147–178
27. Scherer S, Arheit M, Kowal J et al (2014) Single particle 3D reconstruction for 2D crystal
images of membrane proteins. J Struct Biol
185:267–277
28. Kuang Q, Purhonen P, Pattipaka T et al (2015)
A refined single-particle reconstruction procedure to process two-dimensional crystal images
from transmission Electron microscopy.
Microsc Microanal 21:876–885
29. McMullan G, Faruqi AR, Henderson R (2016)
Direct Electron detectors. In: the resolution
revolution: recent advances in cryoEM, 1st
edn. Elsevier Inc., Amsterdam, pp 1–17
30. Sigworth FJ, Doerschuk PC, Carazo J-M,
Scheres SHW (2010) An introduction to
maximum-likelihood methods in Cryo-EM.
In: enzymology GJJBT-M in Cryo-EM, part
B: 3-D reconstruction. Academic Press,
New York, pp 263–294
31. Punjani A, Rubinstein JL, Fleet DJ, Brubaker
MA (2017) cryoSPARC: algorithms for rapid
unsupervised cryo-EM structure determination. Nat Methods 14:290–296
32. Grant T, Rohou A, Grigorieff N (2018) cisTEM, user-friendly software for single-particle
image processing. elife 7:e35383
33. Zivanov J, Nakane T, Forsberg BO et al (2018)
New tools for automated high-resolution cryoEM structure determination in RELION-3.
elife 7:e42166
34. Scheres SHW, Gao H, Valle M et al (2007)
Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nat Methods 4:27–29
35. Lyumkis D, Brilot AF, Theobald DL, Grigorieff N (2013) Likelihood-based classification of
cryo-EM images using FREALIGN. J Struct
Biol 183:377–388
36. Kowal J, Chami M, Baumgartner P et al (2014)
Ligand-induced structural changes in the cyclic
nucleotide-modulated potassium channel
MloK1. Nat Commun 5:3106
37. Kowal J, Biyani N, Chami M et al (2018) Highresolution Cryoelectron microscopy structure
of the cyclic nucleotide-modulated Potassium
Channel MloK1 in a lipid bilayer. Structure
26:20–27.e3
38. Righetto RD, Biyani N, Kowal J et al (2019)
Retrieving high-resolution information from
disordered 2D crystals by single-particle cryoEM. Nat Commun 10:1722
39. Grigorieff N (2016) Frealign: an exploratory
tool for single-particle Cryo-EM. In: enzymology BT-M in (ed). Academic Press, New York,
pp 191–226
40. Abeyrathne PD, Chami M, Pantelic RS et al
(2010) Preparation of 2D crystals of membrane proteins for high-resolution Electron
crystallography data collection. In: enzymology GJJBT-M in (ed) Cryo-EM part a sample
preparation and data collection. Academic
Press, New York, pp 25–43
41. Abeyrathne PD, Arheit M, Kebbel F et al
(2012) Analysis of 2-D crystals of membrane
proteins by Electron microscopy. In: Comprehensive biophysics. Elsevier, Amsterdam, pp
277–310
42. Goldie KN, Abeyrathne P, Kebbel F et al
(2014) Cryo-electron microscopy of membrane proteins. Humana Press, Totowa, NJ
43. Schmidt-Krey I, Cheng Y (2013) Electron
crystallography of soluble and membrane proteins: methods and protocols. Humana Press,
Totowa, NJ
44. Brilot AF, Chen JZ, Cheng A et al (2012)
Beam-induced motion of vitrified specimen
on holey carbon film. J Struct Biol
177:630–637
45. Grant T, Grigorieff N (2015) Measuring the
optimal exposure for single particle cryo-EM
using a 2.6 A ˚ reconstruction of rotavirus VP6.
elife 4:e06980
46. Rohou A, Grigorieff N (2015) CTFFIND4:
fast and accurate defocus estimation from electron micrographs. J Struct Biol 192:216–221
47. Zhang K (2016) Gctf: real-time CTF determination and correction. J Struct Biol 193:1–12
48. Biyani N, Scherer S, Righetto RD et al (2018)
Image processing techniques for highresolution structure determination from badly
ordered 2D crystals. J Struct Biol
203:120–134
Single Particle Analysis for High-Resolution 2D Electron Crystallography
283
A new generation of the IMAGIC image processing system. J Struct Biol 116:17–24
24. van Heel M, Hollenberg J (1980) On the
stretching
of
distorted
images
of
two-dimensional crystals. In: Baumeister W,
Vogell W (eds) Electron microscopy at molecular dimensions: state of the art and strategies
for the future. Springer, Berlin, Heidelberg, pp
256–260
25. Saxton WO, Baumeister W (1982) The correlation averaging of a regularly arranged bacterial cell envelope protein. J Microsc
127:127–138
26. Henderson R, Baldwin JM, Downing KH et al
(1986) Structure of purple membrane from
halobacterium halobium: recording, measurement and evaluation of electron micrographs at
3.5
A ˚
resolution.
Ultramicroscopy
19:147–178
27. Scherer S, Arheit M, Kowal J et al (2014) Single particle 3D reconstruction for 2D crystal
images of membrane proteins. J Struct Biol
185:267–277
28. Kuang Q, Purhonen P, Pattipaka T et al (2015)
A refined single-particle reconstruction procedure to process two-dimensional crystal images
from transmission Electron microscopy.
Microsc Microanal 21:876–885
29. McMullan G, Faruqi AR, Henderson R (2016)
Direct Electron detectors. In: the resolution
revolution: recent advances in cryoEM, 1st
edn. Elsevier Inc., Amsterdam, pp 1–17
30. Sigworth FJ, Doerschuk PC, Carazo J-M,
Scheres SHW (2010) An introduction to
maximum-likelihood methods in Cryo-EM.
In: enzymology GJJBT-M in Cryo-EM, part
B: 3-D reconstruction. Academic Press,
New York, pp 263–294
31. Punjani A, Rubinstein JL, Fleet DJ, Brubaker
MA (2017) cryoSPARC: algorithms for rapid
unsupervised cryo-EM structure determination. Nat Methods 14:290–296
32. Grant T, Rohou A, Grigorieff N (2018) cisTEM, user-friendly software for single-particle
image processing. elife 7:e35383
33. Zivanov J, Nakane T, Forsberg BO et al (2018)
New tools for automated high-resolution cryoEM structure determination in RELION-3.
elife 7:e42166
34. Scheres SHW, Gao H, Valle M et al (2007)
Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nat Methods 4:27–29
35. Lyumkis D, Brilot AF, Theobald DL, Grigorieff N (2013) Likelihood-based classification of
cryo-EM images using FREALIGN. J Struct
Biol 183:377–388
36. Kowal J, Chami M, Baumgartner P et al (2014)
Ligand-induced structural changes in the cyclic
nucleotide-modulated potassium channel
MloK1. Nat Commun 5:3106
37. Kowal J, Biyani N, Chami M et al (2018) Highresolution Cryoelectron microscopy structure
of the cyclic nucleotide-modulated Potassium
Channel MloK1 in a lipid bilayer. Structure
26:20–27.e3
38. Righetto RD, Biyani N, Kowal J et al (2019)
Retrieving high-resolution information from
disordered 2D crystals by single-particle cryoEM. Nat Commun 10:1722
39. Grigorieff N (2016) Frealign: an exploratory
tool for single-particle Cryo-EM. In: enzymology BT-M in (ed). Academic Press, New York,
pp 191–226
40. Abeyrathne PD, Chami M, Pantelic RS et al
(2010) Preparation of 2D crystals of membrane proteins for high-resolution Electron
crystallography data collection. In: enzymology GJJBT-M in (ed) Cryo-EM part a sample
preparation and data collection. Academic
Press, New York, pp 25–43
41. Abeyrathne PD, Arheit M, Kebbel F et al
(2012) Analysis of 2-D crystals of membrane
proteins by Electron microscopy. In: Comprehensive biophysics. Elsevier, Amsterdam, pp
277–310
42. Goldie KN, Abeyrathne P, Kebbel F et al
(2014) Cryo-electron microscopy of membrane proteins. Humana Press, Totowa, NJ
43. Schmidt-Krey I, Cheng Y (2013) Electron
crystallography of soluble and membrane proteins: methods and protocols. Humana Press,
Totowa, NJ
44. Brilot AF, Chen JZ, Cheng A et al (2012)
Beam-induced motion of vitrified specimen
on holey carbon film. J Struct Biol
177:630–637
45. Grant T, Grigorieff N (2015) Measuring the
optimal exposure for single particle cryo-EM
using a 2.6 A ˚ reconstruction of rotavirus VP6.
elife 4:e06980
46. Rohou A, Grigorieff N (2015) CTFFIND4:
fast and accurate defocus estimation from electron micrographs. J Struct Biol 192:216–221
47. Zhang K (2016) Gctf: real-time CTF determination and correction. J Struct Biol 193:1–12
48. Biyani N, Scherer S, Righetto RD et al (2018)
Image processing techniques for highresolution structure determination from badly
ordered 2D crystals. J Struct Biol
203:120–134
Single Particle Analysis for High-Resolution 2D Electron Crystallography
283