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How amazing images of the Zika virus were made with a Nobel Prize winning technique

Pretty Scientific is a new Gizmodo series where we explore how the best images were created in science and why.

"Form follows function" is a cliché frequently repeated in biology, if you know how something looks, then perhaps you can discover how it works. But inevitably, some of the most intricate and impressive forms will take some of the most devilish functions imaginable. Such is the case of the Zika virus pandemic that has terrorized the planet in recent years.

A cryo-EM reconstruction of the Zika virus showing the structure of its component proteins.
Image: Devika Sirohi (2016)

The Nobel Prize in Chemistry 2017 was for three scientists, not for a specific discovery, but for the advancement of a technology called Cryo-Electron Microscopy, or cryo-EM. This method of freezing a sample to create precise molecular images has revolutionized the understanding of biologists to almost atomic levels. An image that perhaps best summarizes the power of the technique is that of the Zika virus, whose resolution has been resolved to such an extent that the details of the virus can be appreciated during the height of the epidemic.

"Many people use this image because it puts a face to the Zika virus," said Devika Sirohi, a postdoctoral researcher at Purdue University who co-authored a 2016 document detailing the structure of the virus. "It was a kind of poster to highlight the expanding possibilities of cryo-EM."

An animation demonstrating the component parts of the Zika virus through cryo-EM reconstruction (Video: Purdue University)

When the Zika virus began to spread and with its link to confirmed microcephaly, the scientists flooded with questions. What happens to the structure caused the symptoms? How is it different from other viruses in the same family, such as dengue and West Nile? The competition began in January 2016, as several laboratories worked quickly to publish the structure of the virus. Sirohi revealed the results only three months later along with the rest of his team: Zhenguo Chen, Lei Sun Thomas Klose, Michael Rossmann and Richard Kuhn at Purdue, and Theodore Pierson at the NIH National Institute of Allergy and Infectious Diseases. [19659011] A single two-dimensional cryo-EM micrograph

Image: Devika Sirohi

Scientists have long used a method called X-ray crystallography for imaging viruses, in which X-rays are taken as a sample , whose structure is mapped by how the X-rays bounce. The softer structure of the virus makes that method less than optimal. With cryo-EM, the researchers instead freeze the cells rapidly in a grid with liquid ethane, which does not alter the structure as much. Then they hit the cells with electrons, tiny subatomic particles and use a detector to produce many two-dimensional projections of the virus.

The difficulty is in the details. Sirohi's team needed to take some 3,000 microscopic images to obtain sufficient data, so they needed a high-purity, high-concentration virus sample. "We were working all day, purifying, collecting and processing the data, purifying more viruses and collecting more data," said Sirohi. "It was a condensed period of hyperactivity."

Rebuilding a 3D image from a group of 2D images is not an easy task. Once they had enough individual images, they combined them using various computer programs, including Relion and jspr, to analyze and construct the view, averaging the data on many images and correcting relics that the microscope might have added. Each image is noisy: the electrons shoot with relative lightness so as not to distort the sample.

Each 2D image corresponds to a different orientation of the Zika 3D virus, rotated in space. The programs mathematically convert these images into abstract shapes that are easy to manipulate using something called "Fourier transform". Any pair of these images transformed in 2D would share a common line. Think of slices taken from a ball, one from a vertical slice and the other from a horizontal slice. Each sector would look like a disk, and the two discs would intersect in a single line. The software can build these lines based on certain previous assumptions and turn them back into the 3D figure of the virus. In this case, the construction required the assumption that Zika would have icosahedral symmetry (in other words, it would be quite typical of spherical viruses).

So, in reality, the results are being understood. This requires even more analysis and several other programs, including Coot, Phenix and CNS, to delve into the molecular components of the structure: the individual proteins and their amino acids. Different colors are applied to specific structures, typically proteins or protein domains (such as those that form the capsule or membrane of the virus).

Close-up: Representation of ZIKV in the shadow of the surface showing five times the characteristic of the capsule in the center.
Image: Devika Sirohi

All this creates the intricate, three-dimensional and colorful image of the Zika virus, which shows how the components combine to form the capsule.

Although cryo-EM has existed for a few decades, structures have been launched so clearly in the last five years alone. This is what Melissa Chambers, cryo-EM specialist at Harvard's Center for Cryo-Electron Microscopy for Structural Biology, and others call "the resolution revolution." Chambers sets the revolution in a combination of many factors, including improved electron detectors, better analysis software and algorithms, better freezing networks and more precise tools and methods. The Zika document is one of many, many new articles that use cryo-EM to discover the structure and function of the smallest pieces of life.

A diagram showing the "resolution revolution" of cryo-EM.
Graphic: Martin Högbom / The Royal Swedish Academy of Sciences

It is becoming easier to take high-resolution images. Taking pictures like these often requires you to be an expert in using the tools, Chambers said. The newer microscopes and facilities are more accessible, automated and easy to use. "Instead of having to become an electronic microscopist … this opens the door to more people who may not have time to learn all this."

Since then, Sirohi and his group, as well as others, have taken important advances in understanding the Zika virus, specifically on how antibodies combine with it so that the body's immune system can attack and neutralize the threat. Maybe the antibodies could be used to help treat the disease.

Three images showing structures collected through cryo-EM, including the Zika virus on the right.
Image: The Royal Swedish Academy of Sciences

Cryo-EM continue to be an important resource for biologists who hope to understand the structure of the most important molecules that cause and cure the problems we face as humans. But working on something as threatening and compelling as Zika has been particularly rewarding.

"I could not have asked for a better laboratory or have been in a better situation," Sirohi said. "Doing it fast enough, and having an experience working with a pathogen that is an imminent threat, was very rewarding."

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