REVIEW
How cryo-electron microscopy and X-ray
crystallography complement each other
Hong-Wei Wang1,2
* and Jia-Wei Wang1
1
Beijing Advanced Innovation Center for Structural Biology, Ministry of Education Key Laboratory of
Protein Sciences School of Life Sciences, Tsinghua University, Beijing, 100084
2
Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084
Received 21 July 2016; Revised 15 August 2016; Accepted 16 August 2016
DOI: 10.1002/pro.3022
Published online 20 August 2016 proteinscience.org
Abstract: With the ability to resolve structures of macromolecules at atomic resolution, X-ray crystallography
has been the most powerful tool in modern structural biology. At the same time, recent
technical improvements have triggered a resolution revolution in the single particle cryo-EM method.
While the two methods are different in many respects, from sample preparation to structure
determination, they both have the power to solve macromolecular structures at atomic resolution.
It is important to understand the unique advantages and caveats of the two methods in solving
structures and to appreciate the complementary nature of the two methods in structural biology.
In this review we provide some examples, and discuss how X-ray crystallography and cryo-EM can
be combined in deciphering structures of macromolecules for our full understanding of their
biological mechanisms.
Keywords: cryo-EM; X-ray crystallography; structural biology; structure determination methods; protein
structure determination; X-ray and electron scattering
Introduction
Since its birth in 1950s, structural biology has revolutionized
our understanding of life by revealing the
shape and structure of biological molecules in great
detail.1
Toward the aim to determine the spatial
relationship of the hundreds of thousands of atoms,
and the change of their relative locations within a
biological macromolecule, multiple methodologies
with very different physical principles were implemented
and exploited in the past half century. These
include X-ray crystallography, NMR spectroscopy,
cryo-electron microscopy (cryo-EM), X-ray solution
scattering, neutron diffraction, and various spectroscopic
techniques. Among these, X-ray crystallography
has played a dominating role in solving
molecule structures at atomic resolution. In recent
years, cryo-EM has experienced dramatic technical
advancement and is starting to become instrumental
in high-resolution structure determination of macromolecular
complexes, especially supra-assemblies.2
The past few years have seen a tendency of contrasting
X-ray crystallography and cryo-EM as being
Abbreviations: cryo-EM, cryo-electron microscopy; microED,
micro-electron diffraction; NCS, non-crystallographic symmetry;
RdRP, RNA-dependent RNA polymerase.
Grant sponsor: Ministry of Science and Technology of China;
Grant number: 2016YFA0501100; Grant sponsor: Beijing
Municipal Science & Technology Commission; Grant number:
Z161100000116034.
*Correspondence to: Hong-Wei Wang, Beijing Advanced
Innovation Center for Structural Biology, Ministry of Education
Key Laboratory of Protein Sciences School of Life Sciences,
Tsinghua University, Beijing, 100084.
E-mail: hongweiwang@tsinghua.edu.cn
32 PROTEIN SCIENCE 2017 VOL 26:32—39 Published by Wiley-Blackwell. VC 2016 The Protein Society
mutually exclusive techniques. In this review, we
would like to emphasize the complementary nature
of the two methods in structural biology.
Principles of X-Ray Crystallography and
Cryo-EM
X-ray crystallography’s foundation principle lies in
Bragg’s Law of X-ray diffraction by crystals, i.e. by
well-ordered packing of homogenous molecules in
three-dimension (3D). Illuminated by a beam of
X-ray light, the crystal can diffract the light at various
angles, some of which have stronger intensity
than others [Fig. 1(A)]. This kind of intensity variation
at different angles can be recorded on media as
a “diffraction pattern”. The diffraction pattern, normally
appearing as a series of sharp spots, reflects
the structural arrangement of atoms within the
crystal and therefore can be used to deduce the original
structure of the crystal using Bragg’s Law. In
order to solve the structure, besides the measured
intensities of the diffraction spots, additional information
called phases of the spots is required. The
phase information is normally obtained by other
experimental or computational means such as SIR/
MIR, SAD/MAD, SIRAS/MIRAS, or molecular
replacement.3
The intensity and phase information
of multiple diffraction patterns of the crystal can
then be reconstructed and Fourier transformed in a
computer to generate a virtual structure for interpretation.
The quality of the structure heavily
depends on the sharpness of the diffraction spots,
which in turn is determined by the degree of order
of the crystal. Therefore, modern X-ray crystallography’s
essential step is to obtain highly-ordered
three-dimensional crystals. In order to achieve this,
a large amount of highly purified macromolecules
may be necessary when screening a large number of
crystallization conditions. Also engineering of the
molecules, e.g. stability promotion, side-chain modification,
proteolysis, may be important to improve the
crystal quality.4
Until now, most of the atomic resolution
macromolecular structures have been solved
by X-ray crystallography.
Cryo-EM uses high energy electrons as source
to illuminate very thin specimens in a transmission
electron microscope and takes advantage of the negatively
charged electron’s trajectory bending by magnetic
field to focus the electron beam passing
through the specimen. Cryo-EM therefore uses a
magnetic objective lens to produce both the diffraction
pattern of a specimen at the back-focal plane
and the magnified image in the image plane [Fig.
1(B)]. Because of an electron’s strong interaction
with each atom’s Coulomb potential, individual molecules
within a specimen can be imaged and the
magnified image already includes the full structural
Figure 1. Technical difference between X-ray crystallography and single particle cryo-EM. A: illustrates the physics and
mathematical principles of X-ray crystallography to solve a structure. Periodic arrays of molecules in a regular threedimensional
lattice resulted in a “diffraction pattern” when illuminated by X-rays. Because of the lack of focus lens for X-rays,
only intensities can be recorded, which resulted in the phase problem in X-ray crystallography. Part (B) illustrates the physics
and mathematical principles of EM in solving a structure, because of an electron’s strong interaction with each atom’s Coulomb
potential, individual molecules within a specimen can be imaged directly. The virtue density of a biological molecule can be
reconstructed from a set of 2D projections of the molecule with common structure, imaged at various orientations by the electron
microscope. In the back-focal plane of the objective lens, the diffraction pattern of a biological specimen can also be
obtained.
Wang and Wang PROTEIN SCIENCE VOL 26:32—39 33
information of the molecule. Therefore, cryo-EM can
be used to examine non-crystalline structure. As a
matter of fact, the most recent trend of cryo-EM in
structural biology is to solve near-atomic resolution
structures from isolated macromolecules, i.e. single
particles, directly from their images. From hundreds
of thousands of single particle cryo-EM images of
one type of macromolecule, we can perform statistical
analysis to classify, align, and average the
images to finally reconstruct the common features
among all the single particle images into a 3D virtual
structure (normally called 3D cryo-EM map) in a
computer. How fine the structure can be defined
(resolution of the structure) depends on various factors
such as the microscopy performance (coherence
of illumination, detector quantum efficiency), the
similarity among the individual images (homogeneity),
the distribution of molecule orientations in the
specimen, the number of images, the robustness of
the statistical analysis, etc. Because cryo-EM reconstructs
the 3D structure directly from a lot of 2D
projections, depending on the limits imposed by
instrumental conditions, it can provide structural
information at different resolution levels from 3 A˚
to 3 nm. In contrast to X-ray crystallography,
which needs large amount of materials to optimize
the crystallization condition, cryo-EM needs much
smaller macromolecule samples to work with and
normally does not need rigorous molecular engineering.
More importantly, cryo-EM specimen is made by
fast freezing biological samples in liquid nitrogen
temperature directly from the solution, therefore
maintaining the macromolecules in their soluble
states in comparison with a state in the crystal
packing constraint. This lends cryo-EM the advantage
to reveal structures in more close-to-native
state than X-ray crystallography but also the difficulty
of dealing with intrinsic structural heterogeneity
due to the thermal fluctuation.
Nowadays, crystallographic and cryo-EM data
may complement each other for structural determination
in several ways, among which two major
ways are most widely used. In one way, a lowresolution
cryo-EM map of an entire molecule provides
the overall shape of the molecule, whose subcomponents
or their homologues may be solved by
X-ray crystallography or other methods. The crystallographic
atomic models of the components can be
docked into the cryo-EM map for interpretation of
the entire molecule. In the other way, a macromolecule
can be crystallized into well-ordered crystals
which can diffract to high resolution. The cryo-EM
reconstruction of the macromolecule at a reasonable
resolution may serve as an initial model to solve the
phasing problem of the crystals for high resolution
structural determination. The two schemes are discussed
in the following sections.
Docking of X-Ray Crystallographic Structures
Within Cryo-EM Maps
In the past decade, the most widely accepted combinatorial
approach of X-ray crystallography and cryoEM
technique has been the practice of docking Xray
crystallographic atomic models into a cryo-EM
map at low resolution.5
This practice started in
1990s when crystal structures were attempted to be
docked into low-resolution cryo-EM maps of icosahedral
viruses or cytoskeletal helical filaments.6
In the
past two decades, several dozen docking software
packages have been developed but only a few are
still being actively developed today. The docking
methods can be classified mainly into two categories:
rigid-body docking and flexible docking. Rigid-body
docking is used to find the optimal orientation and
position of sub-component atomic crystallographic
structures in the cryo-EM map. The most popular
docking software packages include Situs,7
EMfit,8
and UCSF Chimera docking utility.9
In cases where
some minor conformational differences exist between
the crystal structure of an individual component and
its counterpart in the cryo-EM map, flexible docking
algorithms, such as normal mode analysis and
molecular dynamic simulation, are used by introducing
conformational changes to the X-ray structure to
fit into the cryo-EM map while maintaining the
stereo-chemistry of the atomic models. Some flexible
docking software packages include Situs, Flex-EM,10
MDFF,11
iMODFIT12
and more recently Rosetta.13
The docking can be quite useful in defining the
protein location and protein–protein interface within
a complex and new interaction modes that are not
revealed by X-ray crystallography. If the cryo-EM
density is of good enough quality, the docking can be
quite precise even at lower resolution. In a recent
example, the crystal structure of Ryanodine receptor
1’s SPRY2 domain (50 kDa) was docked as a rigid
body into a 10 A˚ resolution cryo-EM map of the
entire complex (1.5 MDa) (Fig. 2).14
The best
scored result by the Colores program of Situs turned
out to define the domain’s location and orientation
quite precisely to agree with the same domain in the
high resolution structure of the complex in a 3.8 A˚
structure solved more recently by single particle
cryo-EM.15
The RMSD between the Ca atoms of the
docked SPRY2 model and the high resolution atomic
model of the complex was only 2.1 Angstrom. This
underscores both the quality of the low-resolution
cryo-EM map and the robustness of the docking
algorithm.
In our study, elucidating the architecture of
yeast RNA exosome complex, docking of atomic models
into low-resolution EM 3D reconstruction maps
was critical for us to uncover new mechanistic
insights of the complex’s function. We solved a 3D
reconstruction of the yeast exosome complex
34 PROTEINSCIENCE.ORG Cryo-EM and X-Ray Crystallography Complement Each Other
comprising ten subunits using single particle EM at
about 18 Angstrom resolution, in which we were
able to dock homologous atomic models of the ninesubunit
human core complex and bacterial RNase
III protein determined by X-ray crystallography.16
This allowed us to reveal the mode of interaction
between the Rrp44 subunit and the core complex. In
a later study, we docked the crystal structure of
RNA-bound yeast exosome 10-mer17
into a 3D EM
reconstruction of the complex in conformations
induced by different lengths of RNA substrates with
high fidelity.18
Alternatively, we revealed a different
conformation of the exosome bound by RNAs with
short 3’-ss tails and built atomic models by docking
available crystal structures of the core complex and
Rrp44 separately in the 3D EM reconstruction. The
two docking results allowed us to reveal distinct
pathways of RNA substrate recruitment and processing
within the complex. Interestingly, the most
recent near-atomic-resolution structure of the apoexosome
complex (PDBID: 5G06)19
verified the interfaces
between the subunits calculated from the previous
docking models in the low-resolution cryo-EM
map of the apo-exosome complex.
Solving the Phases of X-Ray Crystallographic
Diffraction Data Using Cryo-EM Maps
Because cryo-EM uses images to analyze the structure,
the cryo-EM reconstruction includes phase
information of the structural factors in Fourier
space. In contrast, X-ray crystallographic diffraction
data provide precise amplitude information but lack
phases. As cryo-EM reconstruction approaches
higher resolution, sufficient resolution overlap
between the cryo-EM and crystallographic data of
the same or homologous macromolecule may lead to
successful crystal structure determination. In this
approach, the low-resolution cryo-EM reconstruction
is used as the initial phasing model.20
In phasing
the high-resolution X-ray data with low-resolution
EM reconstruction by molecular replacement, once
the search EM map has been positioned, theoretical
phases can be calculated up to the EM reconstruction
resolution. This has been used in determining
the structures of some large complexes such as
viruses, ribosomes, and others.21,22
A few years ago,
in order to generate enough overlap between the low
resolution of cryo-EM map and the crystallographic
diffraction data, a greater camera length or a
smaller beam blocker had to be used to collect low
frequency signals.20
The current progress of single particle cryo-EM
reconstruction allows reconstruction of relatively
small molecules at sub-nanometer resolution.23
This
brings the opportunity to solve the phasing problem
of regular crystallographic diffraction data from molecules
with much smaller size than before. We have
applied this practice to a 280 kDa complex that is
composed of the ecto,-LRR domain of the Toll-like
receptor 13 (TLR13) and its RNA substrate.24
TLR13 recognizes a stretch of conserved nucleotides
from bacterial 23S ribosomal RNA with stringent
specificity to trigger immune response. The TLR13
ecto-LRR domain in complex with a 13-nucleotide
RNA oligomer (ssRNA13) could be crystallized and
the crystals diffracted X-rays to 2.3 A˚ . Unfortunately,
the phasing problem could not be solved by
molecular replacement with homologue structures,
Figure 2. Docking of atomic models into the 3D cryo-EM map of Ryanodine receptor 1 complex at 10 Angstrom resolution.
The 3D EM map of the Ryanodine receptor 1 (EMD-5041) is shown in semi-transparent rendering in two orthogonal views.
The atomic model of a ryanodine receptor monomer (grey) is docked in the map. The atomic model of SPRY2 domain docked
in the map with rigid docking algorithm is shown in red color. Its equivalent domain in the atomic model of the full-length protein
is shown in blue color. The atomic models of the two SPRY2 domain are in very similar orientation and location.
Wang and Wang PROTEIN SCIENCE VOL 26:32—39 35
heavy atom derivative preparations, or even selenium
substitution. We therefore performed single
particle cryo-EM reconstruction and obtained the
structure at 4.8 A˚ resolution of TLR13 bound with a
25-nucleotide RNA oligomer (ssRNA25) [Fig. 3(A)].
Using this medium-resolution reconstruction, we
successfully solved the phases of the TLR13ssRNA13
X-ray diffraction data to 2.3 A˚ resolution
[Fig. 3(B)].24
Phase extension to higher resolution provided
by the X-ray diffraction data was usually achieved
by iterated density-modification procedures, especially
non-crystallographic symmetry (NCS) map
averaging. This strategy has been successfully
applied to the study of macromolecular particles
with a high-degree of internal symmetry, e.g. virus
crystallography, which results in a good-quality electron
density map for interpretation even at a resolution
of around 3 A˚ because of the power of the NCS
averaging. However, in the case of TLR13, there are
only two copies of TLR13 molecules in the asymmetric
unit of the crystal, which degrades the power of
the usual phase extension method. To solve this
problem, we have developed an improved method for
phasing crystal structures with no or low noncrystallographic
symmetry using cryo-EM data,
which iterates the phase improvements through the
combination of a reciprocal-space bias-removal method
and a real-space fragment extraction method.25
Additional benefits would be available with the cryoEM
phasing X-ray method if the Bijvoet differences
were measured for some weak anomalous scattering
atoms, which could help determine the correct absolute
hand of the cryo-EM map.26
The fast-developing micro-electron diffraction
(microED) technique has shown the power to collect
high quality diffraction data from micro crystals
using an electron beam to solve protein or polypeptide
structures at high resolution.27
The phasing
problem of microED is currently solved mainly by
molecular replacement from available homologous
structures,28
or directly from intensities by so-called
direct methods if the data are measured to atomic
resolution. It is predictable in the future to use single
particle cryo-EM reconstructions as initial models to
solve the phasing problem of the microED data.
Figure 3. Cryo-EM 3D reconstruction helps phasing in X-ray crystallography. A: is the single particle cryo-EM 3D reconstruction
of the TLR13-ssRNA at 4.8 A˚ resolution. Part (B) is the electron density map (blue mesh) of the TLR13-ssRNA after
phase extension from the initial cryo-EM map to X-ray crystallographic data at 2.3 Angstrom resolution. The main chain of the
atomic model of TLR13 protein in the density is shown in green sticks. A few glycosylated sites with the sugar molecules are
shown in stick model. Both (A) and (B) are shown in stereo pairs.
36 PROTEINSCIENCE.ORG Cryo-EM and X-Ray Crystallography Complement Each Other
Cryo-EM and X-Ray Crystallographic Data
Complement Each Other in Deciphering
Structures
The most recent technical breakthroughs in cryo-EM
have resulted in a major leap in solving structure of
macromolecules at high resolution. Because cryo-EM
does not need crystallization of the target molecules,
many molecules, especially the super-complexes that
are either very hard to produce in large quantity or
almost impossible to crystallize, are now possible to
be determined at reasonably high resolution. A
major hurdle for single particle reconstruction at
present is the presence of conformational heterogeneity
in the sample because of the flexible nature of
the molecule. Therefore, most of the single particle
reconstructions suffer from uneven distribution of
resolutions within the 3D EM maps, with the molecular
core region normally having the highest resolution
whereas the molecule periphery being worse
resolved. Algorithms dealing with heterogeneity by
stringent classification and local masking have been
developed but are still not powerful enough to solve
all the flexible domains in atomic detail.29
In contrast,
crystallization of molecules or their partial
components, such as a subunit or a domain, locks
the molecules in the highly ordered crystal
lattice and thus enforces them to stay in a rigid conformation.
For instance, the single particle cryo-EM
reconstruction of influenza RNA-dependent RNA
polymerase (RdRP)30
revealed the near atomic model
of the PB1 subunit and C-terminal domain of
PA subunit but could not solve the structure of the
N-terminal domain of the PA subunit due to its
flexibility in the tetrameric single particle complex
[Fig. 4(A)]. The same N-terminal domain of the PA
subunit, however, is well characterized in the crystal
structure of a monomeric complex of the influenza
RdRP.31
It is clear that the lattice packing in the
crystal caused the N-terminal domain of PA subunit
in one RdRP to interact with the PB1 domain of an
adjacent RdRP, thus stabilizing the complex in a rigid
conformation [Fig. 4(B)]. Therefore, crystallography
has the advantage to freeze macromolecules in
ordered states. This works even better for welldefined
domains that are normally easy to be crystallized
and solved by crystallography possibly with
the initial phases provided by the corresponding
part of cryo-EM map.
Because macromolecules may be prepared under
different biochemical conditions for cryo-EM analysis
or crystallization, the corresponding structures
solved thus can represent different biological states,
providing a more complete structural picture of the
molecules. The crystal structure of a macromolecule
can serve as a good starting model to fit into heterogeneous
conformations solved by cryo-EM. Cryo-EM
reconstructions can in turn provide important guidance
to engineer the macromolecule for crystallization
at high order. Currently, X-ray crystallography
is the major tool in elucidating the detail mechanisms
at chemistry level such as the enzymatic catalytic
reaction or ion transportation. Summarizing
the advantages of both cryo-EM and X-ray crystallography
methods, a future structural biology study
of a macromolecular complex may be a scenario combining
the two technologies along the following lines.
Figure 4. Domain structure locked in rigid state by crystal packing. Part (A) is a partial map from the single particle cryoEM
3D reconstruction of the influenza RdRP tetramer at 4.3 A˚ resolution in semi-transparent rendering, with the crystal structure
of an RdRP monomer docked in the map. The N-terminal domain of PA subunit is not resolved in the 3D map so the atomic
model protruding out from the map. Part (B) shows two adjacent RdRP monomers packed in the crystal lattice for X-ray
crystallography. The N-terminal domain of PA subunit is in close contact with the PB subunit of the adjacent monomer. In both
(A) and (B), PA subunit is in blue color, PB subunit is in cyan color, and the RNA is in yellow color.
Wang and Wang PROTEIN SCIENCE VOL 26:32—39 37
First, the structure of the whole complex as an entity
is solved using single particle cryo-EM at near
atomic resolution with the overall architecture as
well as subunit and domain boundaries defined.
Some peripheral portion or flexible part of the complex
may not be defined well but have a rough shape
and location in the molecule. The cryo-EM map and
architecture can guide purification and crystallization
of sub-complexes or domains that can be solved
at atomic resolution by crystallography. The atomic
models can then be fit back to the cryo-EM map to
envision more details of the entire complex.
As cryo-EM is approaching higher and higher
resolution, will X-ray crystallography finally be
replaced by cryo-EM to reveal the fine structure of
biological molecules? Our discussions in the previous
sections already argue against this statement.
More importantly, we should keep in mind that Xrays
and electron beams interact with substances in
different ways. X-ray photons interact with the electrons
around an atom while a high energy electron
beam deviates by the Coulomb potential around an
atom. The mathematical function describing a
structure solved by X-ray crystallography reflects
the electron cloud distribution within the molecule
and is called “electron density”. In contrast, the
structural mathematical function solved by cryo-EM
reflects the Coulomb potential distribution within
the molecule and is called “Coulomb potential
density”. The two types of density represent different
physical properties of a molecule. At a resolution
below 2 A˚ , the electron density and Coulomb
potential density of a molecule have very similar
shape and features. This might be the reason that
“electron density” was misused in some literatures
describing a 3D cryo-EM reconstruction. When the
resolution is higher than 1 A˚ , the electron density
of a molecule will appear different from its Coulomb
potential. In that range, the X-ray and electron will
give distinct outcomes to a molecule’s structure
reflecting different physical properties. This is
underlined by the most recent work of ultra-high
resolution X-ray crystallography of an iron–sulfur
protein, in which the electron orbitals can be clearly
identified by X-ray.32
We speculate that structural
biology will move in at least two directions. In one
direction, the structures can be solved with high
precision in larger scale due to the fast development
of cryo-EM.33
In the other direction, the structures
can be solved at ultra-high resolution by both cryoEM
and X-ray diffraction, therefore revealing more
in-depth chemical and physical mechanisms behind
the principle of life.34
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