Novel nozzle saves crystals—double flow concept widens spectrum for protein crystallography

March 16, 2017, Deutsches Elektronen-Synchrotron
Radiograph of the working nozzle, showing the inner protein stream surrounded by the ethanol jet. Credit: Dominik Oberthuer, DESY

Scientists are interested in the spatial structure of proteins to learn about the workings of these biomolecules. This knowledge can lead to a better understanding of the functions of biomolecules and to tailored medicines. X-ray crystallography is the prime tool to solve protein structures. However, it requires growing crystals of the proteins under investigation. When X-rays hit these crystals, they are diffracted from the atoms to form a characteristic pattern from which the spatial structure of the crystal—and hence the protein molecules—can be calculated.

However, many proteins do not like being squeezed into as it contradicts their natural state. "Growing protein crystals is complex. The amount of protein that can be produced is often limited to few millionths of a gram, and often, only very tiny crystals can be obtained," says Dominik Oberthür from DESY, main author of the report. With the extremely bright flashes of X-ray free-electron lasers, even those micro crystals can be analysed, but usually thousands of diffraction patterns are needed to solve the protein structure. Since the delicate micro crystals are completely vaporised by the intense X-ray flash after delivering their diffraction pattern, a stream of fresh micro crystals is sent through the laser beam. This concept is known as serial X-ray crystallography, and has enabled the analysis of many previously inaccessible proteins.

Still, even those micro-crystals are hard to obtain, and only a fraction are actually hit by the X-ray flash, depending on the geometry of the crystal stream and the technical parameters of the X-ray laser. "The less crystals, the less protein material you need, the more feasible is the analysis," says Oberthür. Bajt's team conceived a new concept for a so-called double flow-focusing nozzle (DFFN) that greatly reduces protein crystal consumption. Usually, the protein crystals are injected with some carrier liquid buffer into the X-ray beam using a special nozzle. To form a thin jet, the carrier liquid is accelerated by a fast stream of gas surrounding the liquid. But to form a stable jet, a minimum flow rate is needed, usually wasting most of the crystals in the jet.

Concept of the novel nozzle: a fast jet of gas (white) accelerates a stream of ethanol (blue) in which the protein crystals carrying buffer (green) is injected. Credit: Anton Barty, DESY, and Juraj Knoška, Universität Hamburg

To overcome these difficulties, the team added ethanol as a secondary "sheath" liquid between the gas and the buffer. This leads to the sheath liquid being accelerated by the gas. The crystals in their buffer can then be injected as a very thin stream into the centre of the ethanol jet. "Before, the buffer with the crystals had to do two jobs: form a stable jet and carry ," explained Juraj Knoška, a Ph.D. student at CFEL and the University of Hamburg, who developed the nozzles. "Our approach separates these roles and uses the liquids that are best for the job." Ethanol has ideal characteristics to form a very stable jet, which flows with just a fine stream of the crystal carrying buffer in the centre. This way, the flow rate of the buffer could be reduced from about 40 micro litres (millionths of a litre) to just two micro litres per minute. Also, the fine, stable stream of nano crystals can be kept precisely overlapping with the small beam of the X-ray laser. In addition the reduction in overall flow-rate enhances the quality of the diffraction patterns and the rate at which crystals are actually hit by the X-ray flashes.

"Not only do we reduce crystal consumption, but our double flow-focusing nozzle also makes the use of the X-ray source more efficient by increasing the rate at which we collect high-quality ," says Bajt. "Moreover, using the sheath liquid allows us to investigate proteins in buffers that couldn't be injected before. Our concept widens the spectrum of biomolecules that can be analysed." Her team tested the new nozzle at the X-ray laser LCLS of the SLAC National Accelerator Laboratory in the US. The scientists teamed up with different groups to solve the structures of various proteins.

"Together with the group of Nobel laureate Roger Kornberg from Stanford University, we could solve the structure of the enzyme RNA polymerase II at room temperature for the first time," explains Oberthür. "Since crystallography at room temperature is a prerequisite to study structural dynamics in detail, this opens the door for future time-resolved studies or 'molecular movies' with this important system." The new device was also used to analyse two other enzymes, a membrane bound hydrogenase and a dioxygenase as well as naturally occurring protein nano crystals, from the protective cocoon of a specialised virus (Cydia pomonella granulovirus, CpGV).

The first room-temperature structure of the enzyme RNA polymerase II (here shown as cartoon plot) could be solved by serial femtosecond crystallography using a double flow-focused nozzle. Credit: Dave Bushnell, Stanford University, and Dominik Oberthür, DESY

The double flow-focusing nozzle also does away with another practical problem of this form of jet injection: Usually, at the edge of conventional nozzles, buffer material, protein and water ice crystals aggregate over time to form dripstone-like features. The same frequently happens at the bottom of the catch tank below the nozzle. If these protein-ice stalactites and stalagmites grow into the X-ray beam, they do not only render the diffraction pattern useless, their reflections can be so strong that they destroy the detector. So, every now and then, experiments need to be suspended to remove the protein-ice dripstones. "The sheath liquid in our nozzle prevents formation of such unwanted structures. The double flow-focusing nozzle enabled stable experimental conditions for many hours," explains Oberthür.

"In all experiments the nozzle worked extremely well," summarises Bajt. "We could reduce the number of interruptions from ten to zero in a shift, and we expect that experimental stations at other X-ray lasers and at synchrotron light sources like DESY's PETRA III can also benefit from the advantages of our device."

Explore further: X-ray pulses reveal structure of viral cocoon: Scientists analyze smallest ever protein crystals

More information: Double-flow focused liquid injector for efficient serial femtosecond crystallography; Dominik Oberthür et al. Scientific Reports, 2017; DOI: 10.1038/srep44628

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