Molecular imaging of protein dynamics using megahertz X-ray sources: The illustration in the foreground is of a microfluidic mixing device developed at the imaging CoE that enable capture of sub-millisecond protein dynamics at the XFEL. The background image shows the structure of the first ever membrane protein to be solved at the European XFEL. The goal is to create molecular movies of the light-induced changes to the electron density that occur during photosynthesis.
“The recent availability of high-repetition rate ultra-bright X-ray sources is creating new opportunities for creating atomic scale molecular movies and single-particle imaging.”
Capturing the real-time, atomic scale dynamics of molecules in crystals and in solution offers significant opportunities to investigate the motion and interaction of molecules under realistic physiological conditions. Historically, one of the biggest challenges facing researchers has been collecting a sufficient amount of data to enable a complete picture of both the dynamics and structure of molecules to be formed. With a repetition rate that is orders of magnitude higher than first-generation XFEL sources, megahertz pulsed X-ray sources such as the European XFEL have driven some major advancements in 2020, allowing us to capture images of single particles with unprecedented time and spatial resolution. Imaging CoE researchers have played a pivotal role in these breakthroughs, helping to develop the necessary technology to deliver samples to the X-ray beam and to analyse single-particle imaging data. So far, researchers from the Centre are the only scientists from Australia who have had the opportunity to collaborate on these groundbreaking experiments.
PROF. BRIAN ABBEY
PROF. KEITH NUGENT
AT A GLANCE
The advent of ultra-bright, X-ray Free Electron Laser (XFEL) sources is driving fundamental advances in our ability to study dynamic changes in molecular structure at the atomic scale. However, as the field of time-resolved molecular movies at the XFEL has progressed, it has become increasingly clear that some of the biggest challenges faced when imaging atomic scale molecular dynamics are related to building up sufficient statistics to be able to resolve individual atoms. Due to the random orientation of the crystal (or particle) as it arrives at the XFEL beam, it typically takes thousands of diffraction patterns to image the 3D structure of a static molecule using serial crystallography. This number increases dramatically for molecular movies which typically involve imaging hundreds if not thousands of individual structures. For single particles the problem is even more acute due to the lower single-to-noise ratio for non-crystalline samples.
Creating atomic scale molecular movies at the XFEL thus presents a range of statistical and computational challenges to tackle this inherently 4D problem. To make this problem more tractable, the simplest solution is to collect much larger datasets but until the advent of megahertz XFEL sources this was not feasible practically. This is why the Centre’s collaboration with the European XFEL (the world’s first megahertz XFEL source) has been so pivotal to Australia being a key part of the global effort to create the first-atomic scale molecular movies from single particles in solution.
In 2020, the Imaging Centre built on its formative work at the EuXFEL and published the first single-particle imaging experimental results at a megahertz XFEL source (Sobolev et al, Communication Physics, 2020), where scientists at La Trobe University played a key role in sample injection of the single particles. The Imaging CoE has also played a leading role in developing solutions for triggering chemical reactions at sub-millisecond timescales and delivering the sample to the XFEL beam. Work by Centre researchers to develop so-called ‘mix-and-inject’ microfluidic devices led to the first commissioning of this new technology at the European XFEL in 2020.
As part of the joint PhD partnership formed between the La Trobe University node and the European XFEL in 2020, two new PhD students, Jaydeep Patel and Trey Guest, began their PhDs with the Centre and plan to spend up to 18 months based in Hamburg in 2022. The work of these students is vitally important in driving international capability for controlling and imaging the structure and dynamics of molecules at the atomic scale.
- Four joint ARC serial crystallography training workshops were staged at Australia’s first serial crystallography facility, designed and developed by the Imaging CoE in partnership with ANSTO.
- Data analysis pipeline for serial crystallography diffraction data was delivered to the Australian Synchrotron MX beamline.
- La Trobe University, collaborating with RMIT and the University of Melbourne (UoM), led experiments applying fluctuation microscopy,
- for the first time, to the study protein-protein interactions at the Australian Synchrotron.
- Researchers at the La Trobe node played a key role in the first-ever single-particle imaging experiments at megahertz XFELs.
In 2020 La Trobe University was granted five full patents related to a new imaging platform developed by Imaging CoE researchers, and is currently commercialising its molecular imaging technology with support from both university and industry partners.
Workshops on the new serial crystallography platform at the Australian Synchrotron will be organised; results of our XFEL structure determination of an immunological target using serial femtosecond crystallography methods published in a high-impact journal; and collaborative experiments will be led into the role of ionic liquids and high viscous materials in protein stability for suitable injector delivery.