Germany: Designing a Crystallization Chamber
The Crystallization Chamber
Marialucia Longo and Tobias Schrader at the Jülich Centre for Neutron Science (JCNS) based at FRM II in Garching, Germany have been designing and testing a crystallization chamber to grow large protein crystals.
The chamber consists of two round stainless steel holders that incorporate Peltier heating elements, to control the temperature conditions, and a glass window to allow crystal growth to be monitored. The circular design facilitates an even temperature distribution to hopefully provide uniform temperature control in all directions.
An example of the spacer
Between the holders is placed a Teflon “spacer” that forms the crystallization chamber itself. This is where all the action takes place. The spacer module is interchangeable to allow different configurations to be designed and provide a choice of crystallization methods (currently vapour diffusion and batch crystallization spacers are available). As well as having a compartment for the crystal to grow in, these spacers also have pipe inlets and outlets for transporting protein solutions in and out of it. The spacers were designed and 3D printed with the help of engineers at the Forschungszentrum Jülich in western Germany.
Post-doctoral researcher Marialucia Longo worked on the design and production of the apparatus for over a year, with expert help from Neils Lumma at Jülich, and is now in the testing phase. She has started with hen-egg white lysoyzme as it is a well-known protein and forms big crystals quickly and easily. Other proteins to try are thermolysin and streptavidin as so far, large crystals of these have been elusive. Streptavidin would be a particularly interesting molecule to study with neutrons as not a lot is known about the hydrogen bonds to the biotin ligand within the structure. Making a crystal big enough to use neutron techniques on could shed light on this.
Tobias Schrader and Marialucia Longo
However, Marialucia has had many obstacles to overcome and still has plenty of problems to solve. Not least because, with a background in DNA and inelastic scattering, she has first had to learn about proteins and elastic scattering.
Then there have been issues in the apparatus itself: unwanted bubbles in the chamber, inadequate performance of the sealing and unreliable control of the temperature. Particularly frustrating has been the user-“unfriendly” heating elements. Tweaking the temperature using knobs and waiting 2 minutes every time for the temperature controller to resume normal operation has been very time-consuming and difficult. It is anticipated that a computer link to the temperature controller may enable the temperature to be stepped-down gradually, e.g. by 1 degree a day. This requires development but could aid the quest for growing bigger crystals.
Ultimately, the team’s ambition is to use this apparatus to produce crystals that they, and their users, can use on their instrument BIODIFF, which although it is a sophisticated instrument ideally requires a crystal volume of at least 0.1 mm3. BIODIFF is a monochromatic single crystal diffractometer – a joint project of the FRM II (TUM) and JCNS (Forschungszentrum Jülich) run by Tobias Schrader and Andreas Ostermann, who has also been a big help on this project.
So far the biggest crystals they have grown are 0.2 mm3 using the model protein lysozyme. As SINE2020 reaches its end, fortunately this project will continue with extra funding provided by Forschungszentrum Jülich.
Acknowledgements: Marialucia Longo and Tobias Schrader, JCNS