2 The Single-Particle Cryo-EM at High Resolution
The single-particle cryo-EM method for high-resolution structure determination of
proteins and protein complexes involves four major steps, viz. (i) the sample
preparation, (ii) specimen preparation, (iii) data collection, and (iv) image processing and 3D reconstruction (i.e., structure determination, which includes model
building and refinement of the protein/ligand coordinates in the EM map). Sample
preparation involves protein purification either from the source or expressed
recombinantly in a heterologous host system. The amount of sample required for
cryo-EM is very less (*1 µM) in comparison with protein crystallography or NMR
spectroscopy techniques, where typically *200 µM sample is required.
For single-particle electron microscopy (EM), there are two main ways of
specimen preparation: (a) negative stain specimen preparation and (b) solution-state
“vitrification” for cryo-EM. The former is used for quick characterization of
macromolecules and their complexes. However, this type of specimen preparation
involves inherent drawbacks (e.g., artifacts and visualizing stain rather than actual
protein), which limits the resolution of EM reconstruction map from 30 to 20 Å at
its best. Single-particle cryo-EM, the focus of this chapter, on the other hand is
synonymous to solution-state structure, and the specimen preparation does not
induce artifacts over the protein sample being studied. The vitrified specimen
preserves the resolution of the protein structure that is being studied.
Single-particle cryo-EM technique has the capability to solve protein structures
to better than 4 Å resolution nowadays. It is to be noted that, there is a consensus in
the EM community that better then 4 Å depicts high-resolution structures, while, in
the X-ray crystallography community, high resolution corresponds to better than
1.8 Å resolution, as described in the beginning of this chapter. Prior to the resolution revolution in the year 2015, most of the cryo-EM structures with resolution
4 Å or better were virus structures [20–22]. This was possible due to their large size
and high symmetry (e.g., icosahedron symmetry). Most of these data were collected
on photographic film (KODAK SO-163 FILM). However, the asymmetric particles
(i.e., particles without higher-order symmetry) were limited to sub-nanometer
(around 6–10 Å) resolution. Only 1/10th of the total number of structures in EMDB
were with resolution 4 Å or better before the resolution revolution. This has significantly increased to 1/6th of the total number of single-particle cryo-EM structures in EMDB as on July 29, 2018, clearly indicating that, currently, there are more
structures solved with resolution better than 4 Å in the database. These were
possible due to the advancement in the hardware and software and the way the
projection images are captured and processed during cryo-EM data collection and
processing. Main steps involved in single-particle cryo-EM for obtaining
high-resolution protein structure are presented in three subsections. First, we will
begin with the details of the specimen preparation in Sect. 2.1, followed by data
collection in Sect. 2.2, and finally image processing and 3D reconstruction in
Sect. 2.3, respectively.
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
379
The single-particle cryo-EM method for high-resolution structure determination of
proteins and protein complexes involves four major steps, viz. (i) the sample
preparation, (ii) specimen preparation, (iii) data collection, and (iv) image processing and 3D reconstruction (i.e., structure determination, which includes model
building and refinement of the protein/ligand coordinates in the EM map). Sample
preparation involves protein purification either from the source or expressed
recombinantly in a heterologous host system. The amount of sample required for
cryo-EM is very less (*1 µM) in comparison with protein crystallography or NMR
spectroscopy techniques, where typically *200 µM sample is required.
For single-particle electron microscopy (EM), there are two main ways of
specimen preparation: (a) negative stain specimen preparation and (b) solution-state
“vitrification” for cryo-EM. The former is used for quick characterization of
macromolecules and their complexes. However, this type of specimen preparation
involves inherent drawbacks (e.g., artifacts and visualizing stain rather than actual
protein), which limits the resolution of EM reconstruction map from 30 to 20 Å at
its best. Single-particle cryo-EM, the focus of this chapter, on the other hand is
synonymous to solution-state structure, and the specimen preparation does not
induce artifacts over the protein sample being studied. The vitrified specimen
preserves the resolution of the protein structure that is being studied.
Single-particle cryo-EM technique has the capability to solve protein structures
to better than 4 Å resolution nowadays. It is to be noted that, there is a consensus in
the EM community that better then 4 Å depicts high-resolution structures, while, in
the X-ray crystallography community, high resolution corresponds to better than
1.8 Å resolution, as described in the beginning of this chapter. Prior to the resolution revolution in the year 2015, most of the cryo-EM structures with resolution
4 Å or better were virus structures [20–22]. This was possible due to their large size
and high symmetry (e.g., icosahedron symmetry). Most of these data were collected
on photographic film (KODAK SO-163 FILM). However, the asymmetric particles
(i.e., particles without higher-order symmetry) were limited to sub-nanometer
(around 6–10 Å) resolution. Only 1/10th of the total number of structures in EMDB
were with resolution 4 Å or better before the resolution revolution. This has significantly increased to 1/6th of the total number of single-particle cryo-EM structures in EMDB as on July 29, 2018, clearly indicating that, currently, there are more
structures solved with resolution better than 4 Å in the database. These were
possible due to the advancement in the hardware and software and the way the
projection images are captured and processed during cryo-EM data collection and
processing. Main steps involved in single-particle cryo-EM for obtaining
high-resolution protein structure are presented in three subsections. First, we will
begin with the details of the specimen preparation in Sect. 2.1, followed by data
collection in Sect. 2.2, and finally image processing and 3D reconstruction in
Sect. 2.3, respectively.
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
379