a proviso. That is, we need diffractable protein crystals of reasonable 10–100s of
micron size, in order to obtain a high-resolution X-ray crystallography protein
structure. Also, as the unit cell parameter of the protein crystals increase, the
resolution of diffraction data drops as the cube of unit cell parameter [1]. Moreover,
many proteins, in particular membrane proteins and fibrous proteins, are recalcitrant
to crystallization. An analysis of deposited protein structures in PDB by Kozma and
co-workers in 2017 [2] showed that the majority of the solved structures (97.6%)
are globular proteins and only *2.4% of them are membrane protein structures.
This is primarily because obtaining good diffraction quality 3D crystals for membrane proteins is challenging. As a result, single-particle cryo-EM has gained
popularity nowadays for solving membrane protein structures as well along with
globular proteins. Also, in cases where single-particle cryo-EM cannot give
high-resolution maps, protein crystallography and cryo-EM can be used as hybrid
method to visualize macromolecular assemblies at pseudo-atomic resolution as
described in Natesh [3] and references cited therein.
SBDD is among one of the most important stages for drug discovery in industrial
drug discovery pipelines [4]. It requires the best possible resolution protein structures, preferably better than 2.5 Å resolution. Until 2015, single-particle cryo-EM
could not achieve the resolution comparable to resolution of structures in protein
crystallography [5, 6]. Recently, Danev and co-workers have solved a structure of
Mus musculus apo ferritin at 1.62 Å (EMD-9599). Others have solved the structures
of proteins with bound ligands at resolution 2.5 Å or better [7–10], presented in
Table 1. The foundation for this was laid 36 years ago in December 1981 when
Jacques Dubochet (along with AW Mc Dowall) published the paper on vitrification
(amorphous ice) of pure water for electron microscopy [11]. Jacques was excited
about the prospects of making electron microscopy water friendly. Five years after
that, they got the first cryo-EM virus structure at 35 Å resolution [12]. However,
before that the first EM structure came from Henderson and Unwin [13] of purple
membrane protein by electron crystallography, but however not using cryo, and
hence, the resolution was bit low at 7 Å. This encouraged Joachim Frank to develop
image processing algorithms for solving protein structures by building 3D reconstruction from fussy cryo-EM projection images of proteins [14–16]. These
developments led to the first cryo-EM atomic model of the protein bacteriorhodopsin 15 years later in the year 1990 [17]. In recent years, other developments
like field emission gun electron source, direct electron detectors, and movie-based
cryo-EM imaging methods have led to an avalanche of high-resolution
single-particle cryo-EM protein structures [5, 6, 18]. Thus, the full potential of
cryo-EM in obtaining high-resolution structure of proteins was realized in 2015,
which led to the Noble Prize in Chemistry in the year 2017 for “developing
cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution.” The predictions made by Henderson in 1995 [19] that
single-particle cryo-EM can be used for atomic-resolution structure determination
of protein and protein complexes has become a reality today. Thus, single-particle
cryo-EM technique can be used as a pipeline for obtaining atomic structures of
druggable targets in preclinical SBDD.
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
377
micron size, in order to obtain a high-resolution X-ray crystallography protein
structure. Also, as the unit cell parameter of the protein crystals increase, the
resolution of diffraction data drops as the cube of unit cell parameter [1]. Moreover,
many proteins, in particular membrane proteins and fibrous proteins, are recalcitrant
to crystallization. An analysis of deposited protein structures in PDB by Kozma and
co-workers in 2017 [2] showed that the majority of the solved structures (97.6%)
are globular proteins and only *2.4% of them are membrane protein structures.
This is primarily because obtaining good diffraction quality 3D crystals for membrane proteins is challenging. As a result, single-particle cryo-EM has gained
popularity nowadays for solving membrane protein structures as well along with
globular proteins. Also, in cases where single-particle cryo-EM cannot give
high-resolution maps, protein crystallography and cryo-EM can be used as hybrid
method to visualize macromolecular assemblies at pseudo-atomic resolution as
described in Natesh [3] and references cited therein.
SBDD is among one of the most important stages for drug discovery in industrial
drug discovery pipelines [4]. It requires the best possible resolution protein structures, preferably better than 2.5 Å resolution. Until 2015, single-particle cryo-EM
could not achieve the resolution comparable to resolution of structures in protein
crystallography [5, 6]. Recently, Danev and co-workers have solved a structure of
Mus musculus apo ferritin at 1.62 Å (EMD-9599). Others have solved the structures
of proteins with bound ligands at resolution 2.5 Å or better [7–10], presented in
Table 1. The foundation for this was laid 36 years ago in December 1981 when
Jacques Dubochet (along with AW Mc Dowall) published the paper on vitrification
(amorphous ice) of pure water for electron microscopy [11]. Jacques was excited
about the prospects of making electron microscopy water friendly. Five years after
that, they got the first cryo-EM virus structure at 35 Å resolution [12]. However,
before that the first EM structure came from Henderson and Unwin [13] of purple
membrane protein by electron crystallography, but however not using cryo, and
hence, the resolution was bit low at 7 Å. This encouraged Joachim Frank to develop
image processing algorithms for solving protein structures by building 3D reconstruction from fussy cryo-EM projection images of proteins [14–16]. These
developments led to the first cryo-EM atomic model of the protein bacteriorhodopsin 15 years later in the year 1990 [17]. In recent years, other developments
like field emission gun electron source, direct electron detectors, and movie-based
cryo-EM imaging methods have led to an avalanche of high-resolution
single-particle cryo-EM protein structures [5, 6, 18]. Thus, the full potential of
cryo-EM in obtaining high-resolution structure of proteins was realized in 2015,
which led to the Noble Prize in Chemistry in the year 2017 for “developing
cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution.” The predictions made by Henderson in 1995 [19] that
single-particle cryo-EM can be used for atomic-resolution structure determination
of protein and protein complexes has become a reality today. Thus, single-particle
cryo-EM technique can be used as a pipeline for obtaining atomic structures of
druggable targets in preclinical SBDD.
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
377