A clear path to better insights into biomolecules

Research team generates ultra-precise 3D images at the X-ray laser European XFEL

Illustration of the 3D diffraction pattern of octahedral nanoparticles

Illustration of the 3D diffraction pattern of octahedral nanoparticles, obtained by combining many snapshots after structural selection (Credit: Max Planck Institute for the Structure and Dynamics of Matter, Kartik Ayyer und Joerg Harms).

Using the world's most advanced X-ray laser, European XFEL, an international team of scientists including from DESY has obtained some of the sharpest possible 3D images of gold nanoparticles. The results lay the foundation for getting high resolution images of macromolecules, as the team led by Kartik Ayyer from the Max Planck Institute for the Structure and Dynamics of Matter reports in the journal Optica.

Examples of macromolecules include carbohydrates, lipids, proteins, and nucleic acids, all of which populate our cells and are vital for life. 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, and recorded how these diffract the X-ray flashes from European XFEL. In this manner the team measured ten million diffraction patterns and used them to generate 3D images of the nanoparticles with a record-breaking resolution of finer than three nanometres (millionths of a millimetre). Gold particles scatter much more X-rays than bio-samples and so make good test specimens. They are able to provide a 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 (XFELs) 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 crystallisation. 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.

“Our results really highlight the feasibility of single-particle imaging at X-ray free-electron lasers and chart the way forward for us to make images of macromolecules and their complexes without crystallisation or freezing,“ says DESY Lead Scientist Henry Chapman, senior author of the study. “Then, we will truly reach the long-sought dream of watching the machinery of life in action.”

Two hurdles remained in SPI: Collecting enough high-quality diffraction patterns and properly classifying the structural variability of the biomolecules. The team's work shows that both these barriers can be overcome, says Kartik Ayyer: “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. Thanks to the unique capabilities of the European XFEL facility, namely, the high number of X-ray laser pulses per second 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 will serve as a template for the future of this research field,” emphasises Ayyer.

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. The European XFEL has the fastest X-ray cameras in the world, custom built for the experiments. The team used the capabilities of the Adaptive Gain Integrating Pixel Detector (AGIPD), developed by a DESY-led consortium.

“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 European XFEL's Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument, where the experiments were carried out. “It shows that in future, European XFEL is well placed to explore the limits of ‘vision’ for uncrystallised, room-temperature biomolecules.”

(from DESY News)


3D diffractive imaging of nanoparticle ensembles using an x-ray laser; Kartik Ayyer, P. Lourdu Xavier et al.; Optica, 2021; DOI: 10.1364/OPTICA.410851