Gearing up for challenging proteins at MicroMAX

Gearing up for challenging proteins at MicroMAX

A group of researchers from Sweden and Germany provides proof of concept for implementing serial crystallography at MAX IV in a recent study. The researchers have studied three different proteins using high-viscosity extrusion and fixed-target methods. The acquired data is promising and shows a clear path towards confidently starting serial crystallography experiments at the BioMAX beamline and the new beamline MicroMAX, which is under construction.

Proteins are large, complex molecules responsible for carrying out many of the functions in our bodies. They are also important targets for pharmaceutical drugs. One fundamental class, in which we find around half of all the drug targets today, are membrane proteins. They sit in our cell membranes and perform tasks such as regulating substances travelling in or out of the cell. Membrane proteins are thought to play a role in, for example, heart disease or Alzheimer’s disease. To develop a drug for a specific target protein, we need to know what it looks like.

Essential proteins are hard to study
Membranes proteins are, however, notoriously hard to study. Crystallography studies typically start by letting a droplet of the protein in a buffer solution slowly dry. As the buffer solution evaporates away, the concentration of the protein gets higher and higher. The solution’s protein molecules eventually bind to each other to form an ordered arrangement, a crystal. Typically, the crystal is then put in the X-ray beam while being held at liquid nitrogen temperature, -196°C, to delay radiation damage. The crystal diffracts the X-rays, and a diffraction pattern appears on the X-ray detector as the sample is rotated relative to the X-ray beam. By interpreting the pattern, the researchers can make a model of what the protein looks like. Membrane proteins are challenging because they tend to form smaller crystals that diffract weakly and are damaged before giving enough data.

Serial crystallography is a complement
With the rise of X-ray free-electron lasers (XFELs), the X-ray pulses were so bright that most protein crystals could only be used for collecting a single diffraction image. The serial crystallography method was developed over the last decade to handle such data, made up of many single diffraction images from independent crystals. Since the proof-of-principle experiments at XFELs and developments there, the method has been taken up by synchrotron light sources and become a complementary tool at modern synchrotron beamlines for protein crystallography.

“Serial crystallography is a complementary technique to “ordinary” (rotation method) crystallography. We use it when crystallisation of high quality or larger size protein crystals isn’t possible. It also allows for studies of dynamics of proteins since it is done at room temperature,” explain Anastasya Shilova, first author of the study, from Lund University.

With the serial crystallography methods, many crystals are passed through the X-ray beam, each receiving only a short burst of X-rays and often pass the X-ray beam with a random orientation. One crystal would, under these circumstances, not give enough data to determine a model, but with each giving its small contribution, using many crystals, enough data can be collected.

“Data collection using serial crystallography spreads the absorbed energy over many crystals, thereby reducing radiation damage,” Shilova continues.

The methods of serial crystallography
In high-viscosity extrusion, the crystals are mixed with a thick goo which carries the crystals and is pushed through a capillary and into the beam at a controlled speed. The beam will hit the encapsulated crystals as they pass through in a random orientation. The viscous media used to carry the crystals makes it suitable for stabilizing membrane protein crystals and has therefore been a method of choice when using serial crystallography on challenging membrane protein crystals.

Another method is to spread a liquid droplet containing crystals over a solid support material and to scan the support plate through the beam. This way of setting up the experiment is called the fixed-target method. One benefit of the fixed-target method at a synchrotron is that it can make it easier to collect many “mini rotation-wedges”. This is due to the crystals being fixed on a surface that one can control the motion of. Such experiments thereby act as a bridging point between traditional rotation-method experiments and the one-crystal, one-shot type of serial crystallography.

Room temperature measurements are important
Liquid nitrogen temperature is quite far from the temperature in the body where proteins usually function. The possibility of conducting the experiment at room temperature offered by serial crystallography is vital to understand the actual protein dynamics.

“Many irreversible reactions become possible to follow with serial crystallography at room temperature,” says Oskar Aurelius, another author of the study, from Lund University. “Instead of trying to cryo-trap reaction intermediates with single-crystal crystallography, serial crystallography can provide tools such as laser excitation or substrate mixing to initiate reactions of interest at room temperature and structurally probe them with a variable time delay between reaction initiation and X-ray probing.”

Bright future for serial crystallography at MAX IV
One of the study’s aims is to provide proof of concept for the methods that are going to be offered to users of the beamline BioMAX and the coming beamline MicroMAX.

“The paper showcases data collected both with fixed-target supports, silicon nitride membranes onto which the sample is pipetted that are raster-scanned through the X-ray beam with a motorized translation stage and a high-viscosity extrusion injector based on extruding a viscous matrix. The samples studied in this paper were already characterized with X-ray diffraction before and could thereby act as model systems,” says Aurelius. “An important prerequisite is the collaborative user community and that support from both academic and industrial parties, including Saromics Biostructures, Gothenburg & Stockholm University and the Max Planck Institute for Medical Research, made these experiments possible. These results are used to keep on improving serial crystallography capabilities at MAX IV.”

The experiments continue at BioMAX while the MicroMAX beamline is being constructed.

“At BioMAX, optimisation for improved procedures is ongoing, and PhD student Monika Bjelčić leads the experimental work. The development of serial crystallography at BioMAX is directly coupled to the development of the new macromolecular crystallography beamline, MicroMAX, at MAX IV,” concludes Aurelius. “MicroMAX will have serial crystallography as one of its main experiment forms.”

 

Publication
A. ShilovaH. LebretteO. AureliusJ. NanM. WelinR. KovacicS. GhoshC. SafariR. J. FrielM. MilasZ. MatejM. HögbomG. BrändénM. KloosR. L. ShoemanB. DoakT. UrsbyM. HåkanssonD. T. Logan and U. Mueller, Current status and future opportunities for serial crystallography at MAX IV Laboratory, J. Synchrotron Rad., 27, 1095 (2020), DOI: 10.1107/S1600577520008735