33. Schaffer M et al (2015) Cryo-focused ion beam
sample preparation for imaging vitreous cells
by cryo-electron tomography. Bio Protoc 5:
e1575
34. Nicolas W, Bayer E, Brocard L (2018) Electron
tomography to study the three-dimensional
structure of plasmodesmata in plant tissues–from high pressure freezing preparation to
ultrathin section collection. Bio Protocol 7:1
35. Ladinsky MS (2010) Micromanipulatorassisted vitreous cryosectioning and sample
preparation by high-pressure freezing. Methods Enzymol 481:165–194
36. Ladinsky MS, Pierson JM, McIntosh JR
(2006) Vitreous cryo-sectioning of cells facilitated by a micromanipulator. J Microsc
224:129–134
37. Iancu CV et al (2007) The structure of isolated
Synechococcus strain WH8102 carboxysomes
as revealed by electron cryotomography. J Mol
Biol 372:764–773
38. Kovtun O et al (2018) Structure of the
membrane-assembled retromer coat determined by cryo-electron tomography. Nature
561:561–564
39. Metskas LA, Briggs JAG (2019) Fluorescencebased detection of membrane fusion state on a
cryo-EM grid using correlated cryofluorescence and cryo-electron microscopy.
Microsc Microanal 25:942–949
40. Resch GP (2019) Software for automated
acquisition of electron tomography tilt series.
Methods Cell Biol 152:135–178
41. Mastronarde DN (2005) Automated electron
microscope tomography using robust prediction of specimen movements. J Struct Biol
152:36–51
42. Hutchings J, Stancheva V, Miller EA, Zanetti G
(2018) Subtomogram averaging of COPII
assemblies reveals how coat organization dictates membrane shape. Nat Commun 9:4154
43. Kremer JR, Mastronarde DN, McIntosh JR
(1996) Computer visualization of threedimensional image data using IMOD. J Struct
Biol 116:71–76
44. Mastronarde DN (2008) Correction for
non-perpendicularity of beam and tilt axis in
tomographic reconstructions with the IMOD
package. J Microsc 230:212–217
45. Grant T, Grigorieff N (2015) Measuring the
optimal exposure for single particle cryo-EM
using a 2.6 Å reconstruction of rotavirus VP6.
elife 4:e06980
46. Turon ˇova ´ B, Schur FKM, Wan W, Briggs JAG
(2017) Efficient 3D-CTF correction for cryoelectron
tomography
using
NovaCTF
improves subtomogram averaging resolution
to 3.4 Å. J Struct Biol 199:187–195
47. Nicastro D (2006) The molecular architecture
of axonemes revealed by cryoelectron tomography. Science 313:944–948
48. Hrabe T et al (2012) PyTom: a python-based
toolbox for localization of macromolecules in
cryo-electron tomograms and subtomogram
analysis. J Struct Biol 178:177–188
49. Bharat TAM, Scheres SHW (2016) Resolving
macromolecular structures from electron cryotomography data using subtomogram averaging in RELION. Nat Protoc 11:2054–2065
50. Castan ˜o-Dı ´ez D, Kudryashev M, Arheit M,
Stahlberg H (2012) Dynamo: a flexible, userfriendly development tool for subtomogram
averaging of cryo-EM data in highperformance computing environments. J
Struct Biol 178:139–151
51. Tang G et al (2007) EMAN2: an extensible
image processing suite for electron microscopy.
J Struct Biol 157:38–46
52. Galaz-Montoya JG, Flanagan J, Schmid MF,
Ludtke SJ (2015) Single particle tomography
in EMAN2. J Struct Biol 190:279–290
53. Carter SD et al (2018) Distinguishing signal
from autofluorescence in cryogenic correlated
light and electron microscopy of mammalian
cells. J Struct Biol 201:15–25
Methods in Cryo-Electron Tomography
111
sample preparation for imaging vitreous cells
by cryo-electron tomography. Bio Protoc 5:
e1575
34. Nicolas W, Bayer E, Brocard L (2018) Electron
tomography to study the three-dimensional
structure of plasmodesmata in plant tissues–from high pressure freezing preparation to
ultrathin section collection. Bio Protocol 7:1
35. Ladinsky MS (2010) Micromanipulatorassisted vitreous cryosectioning and sample
preparation by high-pressure freezing. Methods Enzymol 481:165–194
36. Ladinsky MS, Pierson JM, McIntosh JR
(2006) Vitreous cryo-sectioning of cells facilitated by a micromanipulator. J Microsc
224:129–134
37. Iancu CV et al (2007) The structure of isolated
Synechococcus strain WH8102 carboxysomes
as revealed by electron cryotomography. J Mol
Biol 372:764–773
38. Kovtun O et al (2018) Structure of the
membrane-assembled retromer coat determined by cryo-electron tomography. Nature
561:561–564
39. Metskas LA, Briggs JAG (2019) Fluorescencebased detection of membrane fusion state on a
cryo-EM grid using correlated cryofluorescence and cryo-electron microscopy.
Microsc Microanal 25:942–949
40. Resch GP (2019) Software for automated
acquisition of electron tomography tilt series.
Methods Cell Biol 152:135–178
41. Mastronarde DN (2005) Automated electron
microscope tomography using robust prediction of specimen movements. J Struct Biol
152:36–51
42. Hutchings J, Stancheva V, Miller EA, Zanetti G
(2018) Subtomogram averaging of COPII
assemblies reveals how coat organization dictates membrane shape. Nat Commun 9:4154
43. Kremer JR, Mastronarde DN, McIntosh JR
(1996) Computer visualization of threedimensional image data using IMOD. J Struct
Biol 116:71–76
44. Mastronarde DN (2008) Correction for
non-perpendicularity of beam and tilt axis in
tomographic reconstructions with the IMOD
package. J Microsc 230:212–217
45. Grant T, Grigorieff N (2015) Measuring the
optimal exposure for single particle cryo-EM
using a 2.6 Å reconstruction of rotavirus VP6.
elife 4:e06980
46. Turon ˇova ´ B, Schur FKM, Wan W, Briggs JAG
(2017) Efficient 3D-CTF correction for cryoelectron
tomography
using
NovaCTF
improves subtomogram averaging resolution
to 3.4 Å. J Struct Biol 199:187–195
47. Nicastro D (2006) The molecular architecture
of axonemes revealed by cryoelectron tomography. Science 313:944–948
48. Hrabe T et al (2012) PyTom: a python-based
toolbox for localization of macromolecules in
cryo-electron tomograms and subtomogram
analysis. J Struct Biol 178:177–188
49. Bharat TAM, Scheres SHW (2016) Resolving
macromolecular structures from electron cryotomography data using subtomogram averaging in RELION. Nat Protoc 11:2054–2065
50. Castan ˜o-Dı ´ez D, Kudryashev M, Arheit M,
Stahlberg H (2012) Dynamo: a flexible, userfriendly development tool for subtomogram
averaging of cryo-EM data in highperformance computing environments. J
Struct Biol 178:139–151
51. Tang G et al (2007) EMAN2: an extensible
image processing suite for electron microscopy.
J Struct Biol 157:38–46
52. Galaz-Montoya JG, Flanagan J, Schmid MF,
Ludtke SJ (2015) Single particle tomography
in EMAN2. J Struct Biol 190:279–290
53. Carter SD et al (2018) Distinguishing signal
from autofluorescence in cryogenic correlated
light and electron microscopy of mammalian
cells. J Struct Biol 201:15–25
Methods in Cryo-Electron Tomography
111