Increasing XFEL beamtime success through enhanced protein crystal screening
The Imaging CoE is committed to bringing our experts together to work on and solve some of the biggest questions in advanced molecular imaging.
What do you do with protein crystals that are too small to image at the synchrotron?
This is a central question that CoE physics node researchers are trying to answer.
The Imaging CoE is unique in its ability to link experts from Australian physics with experts from Australian biology; a paper published in May is a shining example of this synergy in action.
CoE researcher Connie Darmanin, La Trobe University, along with CoE AI Bostjan Kobe, University of Queensland, worked closely together to develop a deeper understanding of how to handle, characterise and produce nanocrystals.
Why is this a big deal?
It’s because beamtime at an XFEL is a scarce and precious commodity. Being able to determine the quality of nanocrystals for Serial Femtosecond Nanocrystallography (SFX) experiments using local resources is critical in developing successful bids for access to these cutting-edge facilities.
In an article published in Nature Scientific Reports, Connie and Bostjan outline a new protocol for testing nanocrystals prior to applying for XFEL beamtime.
“Nanocrystallography and XFELs are at the cutting edge of X-ray scientific research and enormous efforts are being devoted to translating these technologies into a useful tool for structural biologists,” Connie tells us. “Whenever there are fundamental advancements in X-ray sources, such as the development of the first synchrotrons, there are a lot of unknowns to be solved and a lot of research needs to be conducted into what works and what doesn’t work.”
Connie heard that Bostjan had obtained beautiful crystals which were, unfortunately, too thick for electron diffraction and too small for conventional synchrotron crystallography, so she thought they could pool their expertise, characterise Bostjan’s crystals, determine whether or not they were sufficiently crystalline for X-ray analysis and then see if they were suitable for SFX measurement.
“There are two main challenges my lab faces: handling and characterising nanocrystals, which are too small to be viewed in optical microscopes and producing a sufficient quantity of nanocrystals for use with SFX,” Connie explains.
People the globe over are facing the same problems. And with high demand for access to the two XFELs in current operation, anything that is able to increase the odds of capturing high resolution data is potentially of enormous benefit to the entire community.
The collaboration between the two CoE nodes combined data from several biophysical characterisation techniques including light microscopy, transmission electron microscopy, dynamic light scattering and synchrotron imaging. They looked at the size, the quality and the ordering of the crystals – these are the three most significant factors that influence the success of XFEL experiments.
Connie hopes the work will help other researchers wanting to use nanocrystallography —within the Centre and in the wider community — to carry out experiments for protein structure determination.
“In developing the protocols we published, I drew upon a range of biophysical techniques and my experience over the past 10 years as a protein crystallographer,” she says.
“We have since been able to leverage the work presented in this publication into beamtime at the Stanford XFEL (the Linac Coherence Light Source), where we were able to collect preliminary data out to three angstrom resolution. We are currently working on developing this into a full proposal to collect a complete dataset and are also using this project to help bring in the support of our collaborators from our partner centre at DESY in Hamburg.”
Connie says the collaboration between the Univeristy of Queensland and La Trobe Univeristy nodes brings us a step closer to understanding the health issues associated with inflammatory disease.
“By combining my expertise in nanocrystallography with Bostjan’s passion for understanding the fundamental biochemical and biophysical origins of inflammatory disease, we have developed new techniques for molecular imaging – that’s what drives me,” Connie concludes.
Read the article online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853777/