A clear path to better insights into biomolecules
An international team of scientists, led by Kartik Ayyer from the Max Planck Institute for the Structure and Dynamics of Matter, Germany, has obtained some of the sharpest possible 3D images of gold nanoparticles, and the results lay the foundation for getting high resolution images of macromolecules. The study was carried out at European XFEL's Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument and the results have been published in Optica.
Carbohydrates, lipids, proteins, and nucleic acids, all of which populate our cells and are vital for life, are macromolecules. A key to understanding how these macromolecules work lies in learning the details about their structure. The team used gold nanoparticles, which acted as a substitute for biomolecules, measured 10 million diffraction patterns and used them to generate 3D images with record-breaking resolution. Gold particles scatter much more X-rays than bio-samples and so make good test specimens. They are able to provide lot more data and this is good for fine-tuning methods that can then be used on biomolecules.
"Techniques used to obtain high-resolution images of biomolecules include X-ray crystallography, which requires the biomolecules to be crystallized and this is not an easy process, or cryo-electron microscopy, which works with frozen molecules," says Ayyer. The advent of X-ray free electron lasers opened the doors to single particle imaging (SPI), a technique that has the potential to deliver high resolution images of biomolecules at room temperature and without crystallization. This meant that the biomolecules can be studied closer to their native state leading, for example, to better insights into their structure and function in our bodies.
"But two hurdles remained in SPI: collecting enough high-quality diffraction patterns and properly classifying structural variability of the biomolecules. Our work has shown that both these barriers can be overcome," he adds. "Previous SPI experiments only produced around tens of thousands of diffraction patterns, even in best-case scenarios. However, to get resolutions relevant for structural biology, researchers need 10 to 100 times more diffraction patterns," explains Ayyer. Because of the unique capabilities of the European XFEL facility, namely, the high number of X-ray laser pulses per second (also known as the repetition rate) and high pulse energy, the team were able to collect 10 million diffraction patterns in a single 5-day experiment. "This amount of data is unprecedented and we believe our experiment sets a template for the future of the field," he says.
To overcome the hurdle of structural variability of biomolecules, that is, dealing with a snapshot from each particle that is slightly different from each other, the team used a special algorithm that they developed. The diffraction patterns are collected by a two-dimensional detector-much like a fast X-ray camera. An algorithm then sorts the data and allows the researchers to reconstruct the image of the biomolecule. "We used the capabilities of the Adaptive Gain Integrating Pixel Detector (AGIPD), which allowed us to capture patterns at that high rate. We then collected and analysed the data with customized algorithms to obtain images with record-breaking resolutions." says Ayyer.
"This study truly exploited the unique property of the high repletion rate of our facility, the fast-framing detector, and effective sample delivery," says Adrian Mancuso, leading scientist of the SPB/SFX group. "It shows that in future, European XFEL is well placed to explore the limits of 'vision' for uncrystallised, room-temperature biomolecules."
Source: European XFEL GmbH