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Crystallography, or the science of examining crystals, allows researchers to better understand materials as well as synthetic chemistry and biological processes.
While common crystallography includes neutron and electron diffraction techniques as well as analytical techniques - such as x-ray fluorescence, spectroscopy, microscopy, and computer modeling and visualization - recent applications in biology are leaning on x-ray crystallography.
Imaging a Serotonin Transporter
Dr. Eric Gouaux, a senior scientist at the Vollum Institute of the Oregon Health & Science University used x-ray crystallography to image the serotonin transporter in 3D as it interacted with selective serotonin reuptake inhibitors (SSRIs) to better understand what might be happening to people who are resistant to the antidepressant medication. For most people, SSRIs slow the recycling process of serotonin via the serotonin transporter protein back into neurons for reuse.
Observing the transporter protein in action meant changing it first, as it would normally be unstable during any purification and crystallization processes. Gouaux prepared the protein for 3D imaging by first altering it genetically to withstand temperatures and then by adding small antibody fragments for crystallization. Once the imaging mapped the protein’s 3D structure, he could see how the different molecules needed for pumping, such as sodium and chloride ions, interacted with it.
He discovered that certain SSRIs, namely citalopram (Celexa) and paroxetine (Paxil), bind to the transporter, impeding serotonin recycling. Imaging the crystallization further allowed Gouaux to see genetic differences between transporters in a person without a certain psychiatric diagnosis and those with one, which will help to better understand what changes might need to take place to improve treatment for different groups.
Analyzing the Hantaan Virus
Others using x-ray crystallography for medical exploration are Drs. Daniel Olal and Oliver Daumke from the Max Delbrück Center for Molecular Medicine (MDC). They analyzed the 3D structure of the nucleoprotein of the Hantaan virus to assess how individual nucleoproteins oligomerize when exposed to RNA molecules, and saw how hexameric circular complexes might inhibit viral growth.
Their hypothesis is that altering the nucleoproteins, or introducing something like an RNA that alters their behavior, could stop viral growth in humans. The Hantaan virus, found in Central and Northern Europe as well as parts of East Asia, comes from rodent droppings and can kill infected humans. No treatment exists. The x-ray technique enabled Olal and Daumke to identify three binding sites on the protein that could function as disruption zones.
Working to Eliminate the Norovirus
Similarly, the Schaller Research Group at the German Cancer Research Center at Germany’s University of Heidelberg is using x-ray crystallography to inspect the norovirus, with the aim of eliminating it. The virus has a capsid composed mainly of a protein called VP1, subdivided into a shell and a protruding domain.
The shell domain is a scaffold around the viral RNA, while the protruding domain forms viral spikes on the shell domain, and it appears to govern antigenicity and host-cell interactions. With that known, it has become the crystallography target. For example, the protruding domain is where carbohydrate structures in certain antigens would bind.
It is these areas where the 3D imaging of crystallography and ELISA allow researchers to see how, once high quality norovirus protruding domains are created in high yields, antigenicity and host-cell interactions eventually interrupt viral replication and attachment.
The whole process of creating the protruding domains took less than four weeks. The protein was first cloned in an expression vector after which time it expressed in bacteria and was then purified via chromatography.
Research of X-Ray Crystallography
While these examples show just how prominent x-ray crystallography is, powder crystallography instrumentation and data analysis software is also in frequent use when the single-crystal x-ray crystallography method does not work well. such as for the evaluation of poorly crystallized materials.
So much fundamental work in understanding biology is taking place using crystallography that some in the field are wondering why more funding is not logically flowing into such research centers. In his 2015 paper, “Reviewing Biomolecular Crystallography Proposals: Time for a Paradigm Change” Bernhard Rupp, Ph.D. a Visiting Professor at The Medical University in Innsbruck, Austria writes:
The all-or-nothing nature of crystallographic structure determination projects aggravates a systemic problem caused by increasingly risk-adverse funding policies. Additionally, the unprecedented progress in targeted molecular biology and protein biochemistry methods, and crystallographic techniques, appears widely under appreciated. As I discuss here, historically rooted and outdated grant review criteria need urgent revision.
Bernhard Rupp, Ph.D., Visiting Professor, The Medical University
Rupp’s comments might need to be reiterated as evidence accrues for crystallography’s contributions at the bench.
References and Further Reading
Production of Human Norovirus Protruding Domains in E. Coli for X-Ray Crystallography
Reviewing Biomolecular Crystallography Proposals: Time for a Paradigm Change
Crystallography
Advanced Protein Crystallization Facility - Testing New Trends in Microgravity Protein Crystallization (APCF-Lysozyme)
Promising Model for Hantavirus Drug Design
Serotonin Transporter Structure Revealed
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