“Being at a totally new class of facility we had to master many challenges that nobody had tackled before,” says DESY scientist Anton Barty from the Center for Free-Electron Laser Science (CFEL), who led the team involved in the first experiments. Jakob Andreasson and Janos Hajdu from the ELIBIO programme of the European Extreme Light Infrastructure in Prague also participated in the experiments. The studies made use of the phenomenon of “diffraction before destruction” to obtain practically unpertubed structures from tiny crystals of the biomolecule. Diffraction before destruction exploits the difference between the speed of the X-ray pulse and the speed of a shock wave, created by the interaction of the X-ray pulse with the sample. The concept was developed earlier by scientists at Uppsala University, led by Janos Hajdu, and applied subsequently to studies on nanocrystals by Henry Chapman and his colleagues, currently at CFEL in Hamburg. This method of Serial Femtosecond Crystallography (SFX) was used by Anton Barty and his team at XFEL to inject a stream of nano- and microcrystals in a liquid jet into the extremely intense beam of the European XFEL. This new type of crystallography has been transformational in obtaining practically damage-free crystal structures from sensitive proteins as well as enabling time-resolved studies at physiological temperatures on ultra-short time scales.
The European XFEL belongs to a new generation of X-ray free-electron lasers capable of megahertz pulse rates and this is orders of magnitudes higher than at any other X-ray laser, bringing structural sciences into a new era. Each exposure gives rise to a characteristic diffraction pattern on the detector. The collection of diffraction patterns reveals the spatial structure of the biomolecule. However, every crystal can only be X-rayed once since it is vaporised by the intense flash. To build up the full three-dimensional structure of the biomolecule, a new crystal has to be delivered into the beam in time for the next X-ray flash. To probe biomolecules at full speed is a challenge. Together with the sample, the water jet is also vaporised by the pulse and has to be replenished in time for the next pulse and the next crystal. “We revved up the speed of the water jet carrying the samples to 100 metres per second, that’s about as fast as the speed record in Formula 1,” explains Max Wiedorn, who took care of the sample delivery together with his colleague Dominik Oberthür, both from CFEL.
Also, nobody had ever built a detector to record X-ray diffraction patterns at this fast rate. An international consortium led by DESY scientist Heinz Graafsma designed and built one of the world’s fastest X-ray cameras, tailor-made for the European XFEL. The ‘Adaptive Gain Integrating Pixel Detector’ (AGIPD) can record images as fast as the X-ray pulses arrive, and it can also tune the sensitivity of every detector pixel for maximal sensitivity. “The requirements of the European XFEL are so unique that the detector had to be designed completely from scratch and tailored to this task,” says Graafsma. “This could only be achieved thanks to the comprehensive expertise and fruitful collaboration of the large team involved.”
Test results on a well-known sample, the enzyme lysozyme, verified the system worked as expected. “This is an excellent proof of the X-ray laser’s performance,” underscores XFEL pioneer Henry Chapman, a leading scientist at DESY and a professor at the University of Hamburg. “The European XFEL offers bright prospects for the exploration of the nanocosmos.” The striking performance of the X-ray laser is also a particular success of the DESY accelerator division that led the construction of the world’s longest and most advanced linear accelerator and also operates it.
As their main biological target, the team chose a bacterial enzyme that plays an important role in antibiotics resistance. The molecule designated CTX-M-14 β-lactamase was isolated from the bacterium Klebsiella pneumoniae whose multidrug-resistant strains are a grave concern in hospitals worldwide. A strain of the bacterium was found to be ‘pandrug-resistant’, i.e. unaffected by all 26 commonly available antibiotics.
The enzyme CTX-M-14 β-lactamase works like a molecular pair of scissors cutting up penicillin-derived antibiotics, thereby rendering them useless as medicines. To avoid this, antibiotics are often administered together with a compound called avibactam that blocks the enzyme. Unfortunately, mutations change the enzymes so that avibactam can no longer efficiently block some of them. “Some hospital strains of Klebsiella pneumoniae are already able to cleave even specifically developed third generation antibiotics,” explains Christian Betzel, co-author of the paper and also a professor at the University of Hamburg. “If we understand how this happens, it might help us to design antibiotics that avoid this problem.”
The scientists investigated a complex of the ‘wild type’ enzyme with avibactam bound to the active centre of the enzyme. “The results show with 0.17 nanometres precision how the drug fits snugly into a sort of canyon on the enzyme’s surface that marks its active centre,” says Markus Perbandt from the University of Hamburg, also a co-author of the paper.
These first experiments show that it is possible to record high quality structural information at very high repetition rates, which is the first step towards recording snapshots of the biochemical reaction between enzymes and substrates at different stages with the European XFEL. Together with the research group of co-author Martin Aepfelbacher and Holger Rohde, professors at the University Hospital UKE in Hamburg, the team plans to use the X-ray laser as a movie camera to assemble those snapshots into movies of the molecular dynamics of avibactam and this β-lactamase. “Such movies would give us crucial insights into the biochemical process that could one day help us to design better inhibitors, reducing antibiotics resistance,” says Betzel.
Movies of chemical and biochemical reactions are just one example of a whole new spectrum of scientific experiments enabled by the European XFEL. A key factor is the speed at which data can be collected. “This opens up new avenues of structural discovery,“ underscores European XFEL scientist Adrian Mancuso who heads the SPB/SFX instrument (Single Particles, Clusters and Biomolecules & Serial Femtosecond Crystallography) where the pioneering experiments were done. “The impact is potentially enormous.”
This first ‘beamtime’ for experiments at the European XFEL was open to all scientists from the community to participate, contribute, learn, and gain experience in how to carry out such measurements at this facility. “The success of this ‘open science’ policy is illustrated by – among other things – the rapid dissemination of results from later campaigns by participating groups,” explains Chapman. “Additionally, the large concentration of effort by the community addressed previously unsolved challenges of managing and visualising data – crucial to conducting all serial crystallography experiments at the European XFEL.”
Reference
“Megahertz serial crystallography”; Max O. Wiedorn et al.; „Nature Communications“, 2018; DOI: 10.1038/s41467-018-06156-7
Picture
#Enzyme_20_09_2018:
Artistic rendering of the experiment.
Credit: DESY/Lucid Berlin
