In 2022, we're celebrating our fifth anniversary. This is part of a series of articles about some of the biggest changes we're seeing in biology since the Crick opened.
In 2017 the final research groups moved into the Crick and the building became fully operational. That same year, the Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank and Richard Henderson for developing cryogenic electron microscopy (cryoEM) as an imaging technique for high-resolution structure determination.
Cryogenic refers to anything that has been cooled below -150°C, while electron microscopy involves a type of microscope that uses beams of electrons instead of light beams, which are able to resolve much smaller things in greater detail.
Researchers at the Crick use cryoEM to capture 3D images of proteins and biological processes at the atomic level in a way that was never before possible.
Until recently, a technique called X-ray crystallography was the most common way to create 3D images of biological structures. This technique involves arranging your sample into a crystal and shining X-rays through it. The resulting pattern can be used to determine the structure of the individual molecules in your sample. While X-ray crystallography is a good way to determine the structures of specific molecules, it doesn’t capture important information about how these molecules actually behave in and around cells.
CryoEM has changed the game, allowing researchers to capture images of biological molecules without the need to artificially crystallise them. In recent years, advances in computing power have massively increased the resolution and efficiency of cryoEM, and it’s quickly becoming the preferred method of structural imaging for many biologists.
As Peter Rosenthal, a principal group leader at the Crick, puts it, “The whole world has just switched over the last few years, since the Crick began really, to studying biological structures with cryoEM. It’s suddenly become the number one method for structure determination.”
To start the cryoEM process, researchers prepare small plates or ‘grids‘ containing many samples of the molecule they want to get a closer look at, which are suspended in water. Getting these samples just right can sometimes be the most time-consuming part of the entire process, depending on what particle or process the researchers are hoping to capture. In some cases, it can take years of trial and error to perfect the sample before it can be imaged in the microscope.
Once the samples are ready for the microscope, the grids are flash-frozen by plunging them in liquid ethane, which drops the samples to around -190°C. “The freezing is so fast that the water molecules don’t have time to freeze in an array of crystals” points out Andrea Nans, who manages the cryoEM facilities at the Crick. Instead, it leads to the formation of what’s known as amorphous ice, where the molecules all end up randomly oriented. This is different from the way ice typically freezes, where the molecules align into regular patterns, forming a crystal. This amorphous ice has two major benefits over crystallised ice; it prevents the samples from getting damaged and leads to a clearer image.
“It’s like a glass,” explains Peter, “It’s the most uniform background possible.” There is a team at the Crick who are trying to find better ways of freezing samples to make the resulting images even more clear.
One of the Crick's cryo-electron microscopes
The frozen grid is then loaded into the microscope, which fires beams of electrons through the sample. The electrons are picked up by a camera on the other side of the sample. This is similar to the way light is used in conventional light microscopes, but using electrons allows for a much more detailed set of images.
Data collections can sometimes take up to a week, depending on the number of images needed, and the end result is a set that might contain millions of 2D images showing the same protein from every possible angle. A computer combines all these images and produces a detailed 3D representation of the structure.
The cryoEM facility at the Crick is located 20 meters below ground. Because of how sensitive they are, the microscopes are kept in rooms that mitigate interference from external vibrations and magnetic fields. These rooms were purpose-built from the very beginning and are fitted with additional systems to safeguard against potential future disturbances such as building works. The Crick’s cryoEM facilities are part of the Structural Biology team headed by Phil Walker, and are in use almost constantly by teams around the building and from our partner institutions.
Peter’s team, in collaboration with other groups at the institute and the Structural Biology team, now uses cryoEM to study the structures of different viruses so they can better understand the molecular machinery that controls how the viruses interact with cells.
The team is currently using cryoEM to capture structural changes of proteins found in the membranes of flu viruses that allow them to fuse with and infect cells. Understanding the structural changes involved in this process could help develop new therapies that interfere with these processes to prevent infections.
This is just one example of how cryoEM is being used to make new discoveries at the Crick. Researchers in the Structural Biology of Disease Processes Laboratory have used cryoEM to study structural aspects of COVID-19 variants, and in the Chromosome Replication Laboratory they’ve used it to uncover how the double helix structure of DNA is opened to allow replication. Researchers have also used cryoEM to figure out the mechanism behind how HIV can develop resistance to a certain widely-prescribed group of drugs.
Peter and Andrea agree that there is more we can accomplish with cryoEM. Peter believes that developments in software will continue to change the field: “Computing is a huge aspect of it, and in the future there’s the idea that artificial intelligence will be an even more important tool.”
One active and developing frontier in the field is cryogenic electron tomography, or cryoET. This is a similar process to cryoEM, except it involves capturing a single biological sample from multiple different angles rather than using multiple samples in different orientations. This allows for high-resolution imaging of biological systems directly in cells.
The instruments used for cryoEM are also improving. “We’re currently in the middle of upgrading our machines with new sensors and energy filters, which will allow us to produce even higher resolution structures with fewer images,” explains Andrea. “The final structures have less noise, they’re sharper, and they require fewer images to achieve a certain resolution.” These improvements will make cryoEM an even more powerful tool for researchers at the Crick, helping them see details they may never have seen before.
