Electron Microscopy: Assembling a biological puzzle

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Imagine trying to assemble a three-dimensional jigsaw consisting of hundreds of thousands of pieces, but without any sense of the final picture.

That’s the challenge facing researchers at the University of Wollongong’s cryogenic electron microscopy unit, who are using one of Australia’s most powerful microscopes to determine the structure of proteins down to the level of the individual amino acids or even to the level of its individual atoms. Cryo-electron microscopy is a fundamental tool in structural biology, as it enables researchers to work out the function of a protein or protein complex by studying its 3D structure at the molecular level.

Unlike X-ray crystallography, which requires a protein to be grown into a crystal form to determine its molecular structure, cryo-electron microscopy involves vitrifying the protein, or freezing it in its hydrated form without generating ice crystals. This protects its structure from the vacuum of the electron microscopy chamber, and keeps the protein close to its native state as much as possible. “Freezing it also immobilises it, because you don’t want things to move around when you’re imaging them,” says Dr Gökhan Tolun, senior lecturer in the School of Chemistry and Molecular Bioscience at the University of Wollongong.That protein is then imaged using a cryo-electron microscope. But this generates only two-dimensional images of a three-dimensional structure—like an X-ray of the human body.

For the three-dimensional structure to be reconstructed, hundreds of thousands, even millions of protein particles have to be imaged to generate tens of thousands of quality images of the ‘good’ particles.

There are also a lot of artefacts in the images, which can sometimes make it difficult to clearly see the protein particles amid the noise or contamination. To make matters more complicated, the protein itself could be in any position; upside-down, side- ways, or tumbling.

Putting together this three-dimensional structure therefore requires huge amounts of computing power, which is provided by MASSIVE. “To be able to back-project the two-dimensional images into three dimensions to reconstruct the three-dimensional structures of the proteins, protein complexes or nucleoprotein complexes, we have to know exactly at what angle they froze, which is called orientational assignment,” Tolun says. “That’s where we need computational power, because we are analysing hundreds of thousands of particles.”

Wojtek James Goscinski