Comparison of Crystallography, NMR and EM

Inquiry

Structural biology incorporates techniques and principles of molecular biology, biochemistry and
biophysics as a means of elucidating the molecular structure and dynamics of biologically relevant
molecules. Recent progress in instrumentation has endorsed a new boost in structural biology as
complex biological molecules can now be analyzed with unprecedented ease and efficiency. The three-
dimensional structure of proteins and protein complexes provide great insights into the laws of life
activities and mechanism of diseases, and thereby allowing rational design of novel diagnostic and
therapeutic agents. There are three main research techniques for structural biology: single crystal X-ray
diffraction (SC-XRD), nuclear magnetic resonance (NMR) and cryo-electron microscopy (Cryo-EM).
However, there is no “all-purpose” method since all three of them offer unique advantages as well as
limitations.

Fig.1. Three main research techniques for structural biology. According to the statistics of PDB
(https://www.rcsb.org/ ), more than 120,000 protein structures resolved by SC-XRD, accounting for
nearly 90% of the total. And there are about 12,000 protein structures obtained by NMR. Although the
total number of protein structures resolved by Cryo-EM is not comparable to that of the first two
techniques, the explosive growth of structures from this technique is remarkable in recent years.
1. Single crystal X-ray diffraction
 Basic theory

X-ray crystallography uses X-ray to determine the position and arrangement of atoms in a crystal. The
most classical method of X-ray crystallography is single crystal X-ray diffraction, in which crystal
atoms cause the incident X-ray beam to produce scattered beams. When the scattered beams land on the
detector, these beams produce a speckle diffraction pattern. As the crystal is gradually rotated, the
angle and intensity of these diffracted beams can be measured, and then a three-dimensional image of
the electron density within the crystal is generated. Based on this electron density, the average position
of atoms in the crystal, chemical bonds, crystal barriers, and various information can be determined.
For a single crystal with sufficient purity, homogeneity and regularity, the X-ray diffraction data can
determine the average chemical bond angle and length to within a few tenths of a degree and to within
a few thousandths of an angstrom, respectively.

Fig.2. The physics and mathematical principles of X-ray crystallography to solve a structure
 History and actuality

The single crystal X-ray diffraction technique was proposed and developed in 1912, and it has become
the most important and useful tool for determining protein structure, since the protein structure of
myoglobin was first determined in 1958. Nowadays, more than 140,000 protein structures have been
deposited in protein databank (https://www.rcsb.org/), nearly 90% of which are resolved using single
crystal X-ray diffraction technique, suggesting its advantages in studying the structure of biological
macromolecule crystals.
 Procedures

The process of single crystal X-ray diffraction technique can be roughly divided into four steps. The
first step is to obtain high-quality single crystals of the target protein, which is called protein
crystallization. When the solution of the solubilized protein reaches supersaturation, it promotes
protein aggregation and nucleation. Ultimately, individual protein molecules arrange themselves in a
repeating series of unit cells by adopting a uniform orientation. Qualified crystals need to be of
sufficient size (normally larger than 50 μm in all dimension) and quality (regular structure, no cracks or
twins). Obtaining single crystals of high-quality is the limiting step to solve a structure with this
method.
After obtaining a single crystal, a diffraction experiment is required. The crystal is immobilized in an
intense X-ray beam, producing a diffraction pattern, which is recorded as the diffraction data (angle
and intensity of the diffracted X-rays). As the crystal is gradually rotated, the previous reflections
disappear and new reflections emerge. The diffraction intensity at each spot is recorded from each
direction of the crystal.
Subsequently, the diffraction data obtained from the diffraction pattern are combined with various
methods of structural analysis and data fitting to analyze the electron density distribution in the three-
dimensional space within the unit cell.
In the last step, based on the electron density map, a model of atomic arrangement in the crystal can be
produced and refined.

Fig.3. The process of single crystal X-ray diffraction technique

 Advantages

This method may yield high atomic resolution and is not limited by the molecular weight of the
sample. It is suitable for water-soluble proteins, membrane proteins as well as macromolecular
complexes. When manipulated properly, it becomes a powerful tool to deliver reliable structural data of
biological macromolecules and determine the position and structure of the active center, and helps
understand how the protein recognizes and binds ligand molecules at the atomic level.
 Disadvantages

However, the single crystal X-ray diffraction method also has several disadvantages. First, the sample
must be crystallizable, but crystallization of biological macromolecules with large molecular weight
can be difficult, particularly, membrane proteins are more challenging to crystallize because of its large
size and poor solubilization. Second, an organized single crystal must be obtained to allow appropriate
diffraction. Finally, the obtained three-dimensional structure of biological sample only represents a
static form of the tested molecule (one of many possibilities), rather than a dynamic one.
2. Nuclear magnetic resonance (NMR)
 Basic theory

The second method is nuclear magnetic resonance (NMR). Nuclei are charged, fast spinning particles,
which are similar to outer electrons. The gyromagnetic ratios of different atomic nuclei are different
and therefore have different resonance frequencies. The movement of the nucleus is not isolated--it
interacts with the surrounding atoms both intra- and inter-molecularly. Therefore, through nuclear
magnetic resonance spectroscopy, structural information of a given molecule can be obtained. Taking
protein as an example, its secondary structure, such as α-helix, β-sheet, turn, circular, and curl, reflect
the different arrangement of the main chain atoms of protein molecules three-dimensionally. The
spacing of the atomic nuclei in different secondary domains, the interaction between nuclei, and the
dynamic characteristics of polypeptide segments all directly reflect the three-dimensional structure of
proteins. These nuclear features all contribute to spectroscopic behaviors of the analyzed sample, thus
providing characteristic NMR signals. Interpretation of these signals by computer-aided methods leads
to deciphering of the three-dimensional structure.

Fig.4. Nuclear spin

 History and actuality

Since the first observation of condensed-state NMR signals in 1946, NMR technology has experienced
a rapid development for over 70 years, and its application has been extended from the area of physics
such as nuclear magnetic moment determination to chemistry, medicine, material science, life science
and many others. Notably, in the 1980s, NMR technology was applied in the structural analysis of
protein creatively, thus promoting the application of NMR in biological field. Although the amount of
three-dimensional structure data of proteins obtained by NMR technology is not comparable to that of
single crystal X-ray diffraction, the unique advantages of NMR technology have been widely noticed:
NMR is able to provide information on a kinetic basis, such that the internal movement of proteins over
multiple time scales and their binding mechanism to ligands can therefore be solved.
 Procedures

There are four main steps in an NMR experiment: sample preparation, data acquisition, spectral
processing, and structural analysis . NMR analysis is performed on aqueous samples of protein with
high purity, high stability, and high concentration. A sample volume ranging from 300 to 600 μL with a
concentration range of 0.1-3 mM. The use of stable isotopes 15N, 13C and 2H for protein labeling can
effectively increase signal intensity and resolution. Selective labeling of certain amino acids or
chemical groups of proteins can greatly reduce signal overlap. Multidimensional NMR experiments are
utilized to acquire information about the protein. The spectral processing is then performed to
determine the atoms of the protein corresponding to each spectral peak on different NMR spectra.
Finally, a series of spatially structured information such as NOE and J coupling constants are used to
calculate the spatial structure using distance geometric or molecular dynamics methods.
 Fig.5. The process of nuclear magnetic resonance technology
 Advantages

The most important feature of the NMR method is that the three-dimensional structure of
macromolecules in the natural state can be measured directly in solution, and NMR may provide
unique information about dynamics and intermolecular interactions. The resolution of the
macromolecular three-dimensional structure can be as low as sub nanometer.
 Disadvantages

However, the NMR spectrum of biomolecules with large molecular weight is very complicated and
difficult to interpret, thereby limiting the application of NMR in analyzing large biomolecules.
Additionally, this technique requires relatively large amounts of pure samples (on the order of several
mg) to achieve a reasonable signal to noise level.
3. Cryo-electron microscopy (Cryo-EM)
 Basic theory

The third approach is the cryo-electron microscopy (Cryo-EM) technique, which includes three
different methods: single particle analysis, electron tomography and electron crystallography. The
essential mechanism of Cryo-EM is electron scattering. The basic principle is described as follows.
Samples are prepared through cryopreservation prior to analysis. The coherent electrons are used as a
light source to measure the sample. After the electron beam passes through the sample and the nearby
ice layer, the lens system converts the scattered signal into a magnified image recorded on the detector.
And signal processing is performed to obtain the three-dimensional structure of the sample.
 History and actuality

Electron microscopy three-dimensional reconstruction technology was first discovered in 1968. The
three-dimensional structure of T4 phage tail was reconstructed by electron micrographs. And then the
general concept and methods of three-dimensional reconstruction of electron microscopy were
proposed. For reducing the radiation damage, cryogenic electron microscopy was created in 1974.
After more than 30 years of development, Cryo-EM has become a powerful tool for studying the
structure of biological macromolecules. In recent years, the Cryo-EM technology has made
revolutionary progress, particularly in the single particle analysis. Since 2013, with the tremendous
advances in electron detector and image processing, Cryo-EM single particle analysis has progressed so
rapidly that the resolution of Cryo-EM is now comparable to single crystal X-ray diffraction. At
present, Cryo-EM is becoming a powerful tool for determining high resolution structure of biological
macromolecules.
 Procedures

Before using the Cryo-EM to observe the sample, negative staining EM can be utilized for rapid
screening of homogeneous sample. The Cryo-EM single particle analysis technique begins with the
sample vitrification. During this process, the protein solution is instantly cooled, so that the water
molecules do not crystallize, forming an amorphous solid. The frozen sample is then screened and data
is collected in the system. A series of two-dimensional images can be taken during this period. Next,
based on plenty of two-dimensional images acquired, particle alignment and classification are carried
out. In the end, the data is processed by reconstruction software to generate a three-dimensional
structural model.

Fig.6. The process of Cryo-EM single particle analysis technique

 Advantages

Compared to single crystal X-ray diffraction, the rapid freeze treatment of the sample maintains its
closer-to-native state. Moreover, this method requires only a small amount of sample (about 0.1 mg), is
more forgiven on sample purity, and does not need the protein to crystalize.
 Disadvantages

The main defect in this technique is that the particles are detected in unknown orientations. High levels
of noise, due to the use of limited electron doses to minimize radiation damage, especially at high
resolution, tends to complicate the determination of these orientations, and this is particularly a concern
for smaller particles. Hence, structure determination of biological macromolecules by Cryo-EM was
limited to large complexes or low-resolution models over the last few years.
Fig.7. Cryo-electron microscopy
4. Summary
 Basic theory

In summary, each technology has its own advantages in certain applications such that one method might be used extensively in some cases but rarely in others.
Thus, understanding the nature of the analysis is the key in method selection. Not only will the inappropriate selection of method produce compromised results,
it may also cause significant delays of the project, and result in financial losses. For more information, please see Table 1.

Advantages Disadvantages Objects Resolution

• Well developed • Difficult for crystallization • Crystallizable samples

X-ray • High resolution • Difficult for diffraction • Soluble proteins, membrane
High
Crystallography • Broad molecular weight range • Solid structure preferred proteins, ribosomes, DNA/RNA
• Easy for model building • Static crystalline state structure and protein complexes

• Need for high sample purity

• High resolution
• Difficult for sample preparation • MWs below 40–50 kDa
NMR • 3D structure in solution High
• Difficult for computational • Water soluble samples
• Good for dynamic study
simulation

• Relatively low resolution

• Easy sample preparation
Cryo-EM • Structure in native state
• Small sample size
• >150 kDa
• Applicable to samples of high Relatively Low
• Virions, membrane proteins,
molecular weights only
large proteins, ribosomes, complex (<3.5 Å)
• Highly dependent on EM techniques
compounds
• Costly EM equipment

Table 1 The comparison of X-ray crystallography, NMR and Cryo-EM

References
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2. Rankin, N.; et al. The emergence of proton nuclear magnetic resonance metabolomics in the cardiovascular arena as viewed from a clinical
perspective.Atherosclerosis 2014, 237(1): 287-300.
3. Carroni M, Saibil R. Cryo electron microscopy to determine the structure of macromolecular complexes. Methods 2016, 95: 78-85.
4. Callaway, E. The revolution will not be crystallized: a new method sweeps through structural biology. Nature News 2015 525(7568): 172.