Precision Imaging of Biological Molecules: The Marvel of Cryo-Electron Microscopy
How Cryo-Electron Microscopy (Cryo-EM) revolutionized our understanding of the molecular structure of cells and proteins, and its journey to the Nobel Prize.
In the history of biology, visualizing the three-dimensional (3D) structure of biomolecules such as proteins, viruses, and DNA has always been a major challenge. The high-energy electron beams used in traditional Transmission Electron Microscopes (TEM) would burn and destroy delicate biological samples. To provide a groundbreaking solution to this problem, the Cryogenic electron microscopy (Cryo-EM) technique was introduced in the 1970s, which has ushered in a new era in structural biology.
The journal Nature Methods selected Cryo-EM as the "Method of the Year" in 2015. Subsequently, in 2017, Jacques Dubochet, Joachim Frank, and Richard Henderson were awarded the Nobel Prize in Chemistry for the development of this technique. Through this technology, scientists can observe the structure of biological molecules in solution at near-atomic resolution.
Vitreous ice: The art of freezing molecules in their natural state
The most important aspect of Cryo-EM is the special method of freezing the sample. When biological samples are cooled normally, ice crystals form, which break the delicate structure of cells and proteins. To avoid this problem, the aqueous sample is placed extremely rapidly into liquid ethane or a mixture of liquid ethane and propane, which causes the water to become amorphous or 'vitreous ice' instead of forming crystals.
The biological solution is placed on a small sample grid mesh and flash-frozen at an extremely low temperature of -196 °C (77.1 K). Inside this vitreous ice, protein or virus molecules freeze in their natural state. Because of this, these molecules remain protected even against the extremely powerful vacuum and electron beam of the electron microscope.
- Sample preparation and flash-freezingProtein solution is placed on a sample grid and flash-frozen in liquid ethane at -196 °C to prepare vitreous ice.
- Low-dose TEM imagingThousands of 2D images are taken by applying a low electron dose to frozen molecules via transmission electron microscope.
- 2D alignment and classificationImages taken from various orientations of molecules are classified using RELION or CryoSPARC software.
- 3D model reconstructionA 3D density map and molecular model of the protein are prepared using classified images.
Working mechanism: The journey from sample to 3D model
Cryo-EM involves an extremely precise computational process for acquiring data and analyzing information. First, low-dose electron beams are passed through the frozen sample so that the sample is not damaged. This captures thousands or millions of two-dimensional (2D) images of particles scattered in the frozen ice.
Thereafter, the Single Particle Analysis (SPA) method is used. In this process, 2D images of protein particles taken from millions of different angles are classified using powerful computer software such as RELION or CryoSPARC. By averaging all these images, a 3D density map is generated, based on which an accurate 3D model of the protein can be created.
'Resolution Revolution': The miracle of direct electron detectors
In the 2010s, a major revolution occurred in the field of cryo-EM, which is known in the scientific world as the 'resolution revolution.' The primary cause of this revolution was the invention of the Direct Electron Detector (DED) camera. These detectors, developed by manufacturers such as Gatan Inc., FEI, and Direct Electron, are capable of detecting electrons directly.
Due to these new detectors and computational algorithms, the resolution limit reached below 2-3 Å (angstroms). By the year 2020, the best resolution achieved by cryo-EM was 1.22 Å. In 2018, scientists were successful in measuring even small proteins like hemoglobin (64 kDa) to a resolution of 1.8 Å.
| Feature | Cryo-EM | X-ray Crystallography |
|---|---|---|
| Sample state | In natural solution (Vitrified) | Crystallized |
| Crystallization requirement | Not required | Highly required (takes months/years) |
| Sample quantity | Very low (micrograms) | Large quantity required |
| Best resolution | 1.22 Å (2020) | 0.48 Å (highest record) |
| Molecular size limit | Larger than 50 kDa (generally) | No specific limit |
Comparison with X-ray crystallography
For decades, X-ray crystallography was used to determine the structure of biological molecules. However, X-ray crystallography requires the creation of highly pure protein crystals, a process that can take months or years, and crystals for many important proteins simply do not form. In cryo-EM, there is no need for crystallization, which makes it extremely fast and simple.
According to Protein Data Bank (PDB) statistics, the average resolution of X-ray crystallography is 2.05 Å, with a record high of 0.48 Å. However, there has been a massive surge in cryo-EM structure deposits since 2010. It is estimated that by 2035, the total number of cryo-EM structure deposits will surpass those of X-ray crystallography.
Current limitations and future direction
Cryo-EM also has some limitations. Generally, it is difficult to obtain 3D resolution for proteins smaller than 50 kDa (kilodaltons) because the signal-to-noise ratio (SNR) is low. To overcome this problem, scientists use molecular binding agents such as nanobodies and DARPins to increase the effective size of the protein.
Furthermore, modern cryo-electron microscopes like the Titan Krios cost millions of dollars and require highly specialized scientific laboratories to set up. Nevertheless, in cancer research, virus studies, and drug discovery technology, cryo-EM is proving to be an extremely important tool for humanity today.
