How structural biologists revealed the structure of the new coronavirus so quickly

When the Wuhan, China government confirmed on December 31, 2019 that authorities there were treating dozens of cases of pneumonia of unknown origin, investigators who recalled the 2003 outbreak of severe acute respiratory syndrome (SARS) were uneasy. Was this another coronavirus, like the one that caused that incident? Gradually, concern spread throughout the scientific community, and laboratories that had already been studying coronaviruses began to prepare.

Jason S. McLellan, a structural biologist at the University of Texas at Austin, remembers receiving a call while on a ski vacation with his family. At the other end of the line was Barney S. Graham, deputy director of the Vaccine Research Center at the US National Institute of Health. USA The couple had worked together in the past, and Graham told McLellan: “It looks like it’s a coronavirus. Are you ready to work on it? McLellan says he sent a message to his team via the WhatsApp mobile service and told them that They will prepare: “We will compete as soon as we have the sequence.”

The sequence McLellan was referring to was the genomic sequence of the new coronavirus that Graham told him about. With the virus genome in hand, McLellan and his team could synthesize its most vital proteins and then determine its structures, an important first step in finding therapies to combat a pathogen. In early January, the wait was over. A team led by researchers at Fudan University had released the genome, sharing it publicly online so that labs around the world could take action before the related document was released (Nature 2020, DOI: 10.1038 / s41586-020-2008-3).

And the McLellan team really ran. The research group was one of the first to publish a cryo-electron microscopy structure of one of the proteins of the new coronavirus. Scientists determined the configuration of the virus’s spike protein, a biomolecule that decorates the virus’s outer shell and allows it to fuse and enter human cells to cause infection (Science 2020, DOI: 10.1126 / science.abb2507).

Unlike human genetic information, which is encoded in double-stranded DNA, the new coronavirus, like all coronaviruses, stores its genetic information on a single strand of RNA. The human genome contains around 3 billion well-packed base pairs within the nucleus of each of our cells. In contrast, the RNA genome of the new virus is less than 30,000 bases.

Scientists would learn that this shorter sequence encodes the 29 proteins that make up the virus, now called SARS-CoV-2. These biomolecules protect the pathogen, help it bind to host cells, and allow it to replicate.

Discovering protein structures like these helps scientists develop small molecules, antibodies, and other therapies that can alter protein function. Although the process of determining protein structure has become easier over the years due to advances in technology and knowledge, the speed at which the teams discovered the protein structures of SARS-CoV-2 was unprecedented .

“I think this is really exceptional,” says Sarah J. Butcher, a structural biologist at the University of Helsinki. “It was 5 weeks after the first cases of COVID-19 began to appear that the first structures were deposited with the PDB,” she says.

PDB is the Protein Data Bank, an internationally managed database for three-dimensional structural data of large biological molecules. According to PDB statistics, by the end of March, the service had already received more than 100 structures related to the new coronavirus. And the introductions keep coming.

The research groups that won the race to resolve the first SARS-CoV-2 protein structures were those that worked on earlier coronaviruses, says David R. Armstrong, a member of the European PDB team, based at European Bioinformatics. Institute in the UK. Those groups include McLellan in Texas, Rolf Hilgenfeld at Lübeck University, and Zihe Rao at Tsinghua University. Hilgenfeld and colleagues elucidated an early structure of the virus’s major protease, an enzyme that helps SARS-CoV-2 build its proteins (Science 2020, DOI: 10.1126 / science.abb3405), and Rao and their team determined the structures of the virus protease (Nature 2020, DOI: 10.1038 / s41586-020-2223-y) and its polymerase (Science 2020, DOI: 10.1126 / science.abb7498), which helps SARS-CoV-2 make copies of its RNA genome.

Those labs had years of shared experience working on protein structures for related viruses, such as SARS-causing coronaviruses and Middle East respiratory syndrome (MERS), a disease that was first reported in Saudi Arabia in 2012. Due Because these viruses are similar to SARS-CoV -2, the scientists were able to observe the RNA sequence of the new coronavirus and immediately find the sections that code for the proteins that interested them. The protein shapes also looked alike.

Numerous experts have told C&EN that the greatest challenge for structural biologists today is obtaining well-behaved protein samples that are stable enough to undergo structural characterization. That SARS-CoV-2 was similar to the viruses that caused the SARS and MERS outbreaks was a boon: Labs already knew how to produce and stabilize these proteins. The researchers could order pieces of DNA that encode the SARS-CoV-2 proteins and be sure they would work in processes already established in the laboratory. Once they received the DNA, the scientists were able to insert it into the cultured cells under the right conditions, and those cells were able to produce copies of the desired protein.

In recounting the first few days of the outbreak of COVID-19, the disease caused by SARS-CoV-2, McLellan displays a timeline on his computer: “January. 30 was a great day, “he says. That was the day that graduate student Daniel Wrapp harvested the team’s spike protein and purified it. The team had previously inserted a DNA sequence, in the form of a circular construct called a plasmid, into some mammalian cells, reprogramming its machinery to synthesize the spike protein. “Later that night,” says McLellan, Wrapp “began to freeze the networks, and we started the initial rounds of data collection.”

Grids are part of a cryo-electron microscope, one of the two main tools that structural biologists use to determine protein structures.

“Oh yeah,” says Cynthia Wolberger, an expert in structural biology at Johns Hopkins University School of Medicine, giggling when she hears that all of those steps occurred in 1 day. “Once you have a well-behaved, purified protein, you can toss it directly onto the grill.”

“I used to say that in structural biology, a third of the work was sample preparation, a third of the work was microscopy, and a third of the work was image processing,” says the butcher at the University of Helsinki. Due to countless technological advances, today “it can be 80% sample preparation, 10% imaging, and 10% image processing,” he says. “Both image and image processing have improved tremendously.”

In 1912, William Lawrence Bragg demonstrated that X-rays could be used to reveal the atomic structures of crystalline salts. In the 1950s, that same technology began to probe DNA and protein structures. The field of structural biology was born. Since then, diffuse photographic plates of X-ray diffraction patterns and low-resolution biological molecule models have given way to near-atomic resolution images.

For years, the workhorse of structural biology has been X-ray crystallography. If researchers can produce enough protein and cause that protein to crystallize in a highly ordered network, the technique can reveal a three-dimensional structure of the protein. The crystal rotates through an X-ray beam, and the resulting diffraction patterns are transformed into an electron density map that reveals the protein’s structural secrets. Crystallographers combine that map with the amino acid sequence to build a model of how the protein is folded into sheets and helices.

Today, much of the work of protein crystallography is automated. Liquid handling robots can perform miniature crystallization experiments to find the right solution conditions for protein crystal growth. Synchrotron X-ray sources equipped with micro-focus ray lines and cryo-cooling instruments protect protein crystals from radiation damage, allowing for analysis. Data can now be quickly collected from crystals that would have been dismissed as too fragile or small 10 years ago. Today, hybrid solid-state pixel detectors on X-ray instruments can capture more than 100 images of diffraction patterns per second. “If the crystals are good,” says Manfred Weiss, head of macromolecular crystallography at the Helmholtz-Zentrum Berlin für Materialien und Energie (HZB), “you can collect a good set of data in 10-15 minutes or so.” Fifteen years ago, the same data collection could have taken a few hours.

Weiss has spent years optimizing protein crystallography work at the BESSY II synchrotron in Berlin. In late January, he received a phone call from Hilgenfeld of the University of Lübeck, the leader of one of the teams that had been studying coronavirus proteins for a long time. Weiss recalls that Hilgenfeld told him: “We have crystals of this coronavirus protease, and we need really urgent beam time.” Recognizing the importance of the request, Weiss complied: “I basically identified a free space and gave them a hotline 3 days later.” Synchrotron lightline time requests are generally made in writing weeks or even months in advance.

Fortunately, the enzyme that Hilgenfeld’s team was trying to obtain a structure had a configuration similar to that of the virus protease involved in the MERS outbreak in 2012-13, so scientists could take some shortcuts to speed up the process. for determining the structure. Linlin Zhang, a postdoc from Hilgenfeld’s lab, took the samples to Berlin on Saturday that Weiss had assigned the team’s broadcast time. In mid-February, the researchers had a crystalline structure from the protease of the new virus. That map of the protein’s curves and cracks meant that the team could optimize an existing α-ketoamide inhibitor as a possible drug candidate.

Another technology that has been key during the COVID-19 crisis is crystallographic detection. In the past 8 years or so, says Weiss of HZB, the technology has advanced to the point where scientists can quickly crystallize a selection of small molecules with protein samples to find out if one of the compounds binds to a target protein. , a step towards the design of a therapeutic. The industry has been using detection techniques for a long time, Weiss says, although crystallography has not been a primary detection technique for companies. More than just a yes or no answer as to whether a compound binds, he explains, crystallography can give information about how the molecule binds.

Weiss is testing compounds against the SARS-CoV-2 protease in the synchrotron in Berlin. At Diamond Light Source, a national synchrotron facility in Oxfordshire, England, a group led by Frank von Delft is also testing compounds against the new protease.

When Tsinghua University’s Rao released a crystal structure of the main protease SARS-CoV-2 bound to an inhibitor in January, its installation was already beginning a scheduled shutdown. Rao relayed what he had learned to the Diamond Light Source team so the work could continue. Von Delft’s team focused their efforts and within 2 weeks completed the first inhibitor test for the SARS-CoV-2 protease. The result was 66 small molecules capable of covalently and non-covalently binding to the active site of the protein. The researchers submitted their data to the PDB and quickly published the results on their website instead of writing a journal article. The achievement was, according to von Delft, the result of many “very smart, very smart and focused people working together as a collective.”

However, X-ray crystallography does not work for all proteins. Some cannot crystallize. For these, scientists often turn to cryo-electron microscopy, or cryo-EM. In cryo-EM, a protein instantly freezes on a metal grid in a thin layer, ideally not much thicker than the diameter of the protein itself. Radiating that shell with low-energy electrons instead of X-rays produces 2-D images of individual proteins. Thousands or even hundreds of thousands of these noisy images are computationally classified and reconstructed to build a three-dimensional image. Although X-rays produce cleaner images and can show how proteins bind to small molecules, electron beams are less damaging to proteins. Cryo-EM also generates images of multiple copies of a protein, so an added benefit is that researchers can see how proteins can move or wobble in different conformations.

Over the years, the computational power and quality of the microscope have gradually improved for cryo-EM, producing higher and higher resolution biomolecule structures. In 2011, a breakthrough occurred when direct electron detectors became widely available. These devices provided great gains in the signal-to-noise ratio compared to previous indirect detectors, such as load-coupled devices. The advance caused an avalanche of high-resolution cryo-EM structures determined by the scientific community. In addition to all of these improvements, Butcher says, “image processing methods have also taken leaps and bounds.”

Once ridiculed as “blobology” for its blurry images, cryo-EM, whose pioneers won the 2017 Nobel Prize in Chemistry, is now producing high-resolution structures of anything biologists can freeze into a grid. During the race to discover the structure of SARS-CoV-2 proteins, it has been highlighted.

The fact that Rao and von Delft’s teams were able to share data on the SARS-CoV-2 protease to keep the experiments running highlights another reason why structural biology research has advanced so rapidly during the COVID pandemic- 19. Even groups that were once competitors are working more closely with each other, according to the scientists C&EN spoke with. One project promoting this type of collaboration is the COVID Moonshot crowdsourcing initiative, in which scientists around the world are asked to help find inhibitors for the main protease of the new coronavirus. The initiative took off after contributions from the Rao and von Delft teams. Von Delft’s group is a key contributor to the project.

And Rao and von Delft are not the only researchers who share their work for the common good. When C&EN spoke to McLellan in early April, he was still responding to hundreds of emails a day from researchers around the world, some wanting more details of its cryo-EM structure or asking him to share samples of the spike protein or code. Protein DNA.

A partnership that emerged between McLellan and others recently paid off in the form of a publication. Collaborators led by Xavier Saelens at the VIB-UGent Center for Medical Biotechnology immunized llamas with viruses that cause SARS and MERS to generate antibodies against pathogens. Fusion of a flame SARS antibody with a fragment of a human antibody produced a hybrid that neutralized the virus responsible for COVID-19. The data suggests that such hybrid antibodies could be useful in fighting coronavirus epidemics (Cell 2020, DOI: 10.1016 / j.cell.2020.04.031). Flame antibodies are much smaller than human antibodies and are sometimes called nanobodies. Scientists have been trying to make them therapeutics for more than 20 years, but it took until 2019 for the first nanobody-based therapeutic treatment (caplacizumab) to be approved by the US Food and Drug Administration. USA

“Clearly, everyone is looking for new entities right now,” says Ian A. Wilson of Scripps Research in California, who is using X-ray crystallography to look for antibodies that bind to the spike protein of SARS-CoV- 2. Wilson’s approach since the 1980s has been to use structural biology to develop universal influenza and HIV vaccines.

The first labs to determine the protein structures of SARS-CoV-2 may have had a history of coronaviruses, but other structural biology groups are now rolling up their sleeves to get involved as well. To observe patterns of social distancing while advancing science, many structural biology facilities are now open only to coronavirus-related projects and researchers.

“I’m not really surprised that there is so much activity and that so many structures come out,” says Weiss, noting that SARS-CoV-2 gives scientists a common enemy against which to unite. “I think the first time this happened was the race for HIV protease in the 1980s and 1990s.”

With so many joining the fight, it’s hard to keep track of the structural research being done on SARS-CoV-2 proteins, the researchers say. Almost every day, new preprints are released, documents that have not yet been peer reviewed, and most are posted on the bioRxiv server. And the scientists C&EN spoke to say they now trust Twitter or their colleagues to alert them to new developments in the field.

“We all want it to move as fast as we can,” says Wilson. “And I think things are moving at a very, very fast rate.” But developing any type of drug or vaccine from these efforts and taking it through all the steps necessary for regulatory approval will take time.