Articles by Robin Schubert in JoVE
Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography Yannig Gicquel*1,2, Robin Schubert*3,4,5, Svetlana Kapis3, Gleb Bourenkov6, Thomas Schneider6, Markus Perbandt3,4, Christian Betzel3,4,5, Henry N. Chapman1,2,4, Michael Heymann1,7 1Center for Free Electron Laser Science, DESY, 2Department of Physics, University of Hamburg, 3Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, 4The Hamburg Center for Ultrafast Imaging, University of Hamburg, 5Integrated Biology Infrastructure Life-Science Facility at the European XFEL (XBI), 6European Molecular Biology Laboratory, EMBL c/o DESY, 7Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.
Other articles by Robin Schubert on PubMed
Exosomal Cellular Prion Protein Drives Fibrillization of Amyloid Beta and Counteracts Amyloid Beta-mediated Neurotoxicity Journal of Neurochemistry. | Pubmed ID: 26710111 Alzheimer's disease is a common neurodegenerative, progressive, and fatal disorder. Generation and deposition of amyloid beta (Aβ) peptides associate with its pathogenesis and small soluble Aβ oligomers show the most pronounced neurotoxic effects and correlate with disease initiation and progression. Recent findings showed that Aβ oligomers bind to the cellular prion protein (PrP(C) ) eliciting neurotoxic effects. The role of exosomes, small extracellular vesicles of endosomal origin, in Alzheimer's disease is only poorly understood. Besides serving as disease biomarkers they may promote Aβ plaque formation, decrease Aβ-mediated synaptotoxicity, and enhance Aβ clearance. Here, we explore how exosomal PrP(C) connects to protective functions attributed to exosomes in Alzheimer's disease. To achieve this, we generated a mouse neuroblastoma PrP(C) knockout cell line using transcription activator-like effector nucleases. Using these, as well as SH-SY5Y human neuroblastoma cells, we show that PrP(C) is highly enriched on exosomes and that exosomes bind amyloid beta via PrP(C) . Exosomes showed highest binding affinity for dimeric, pentameric, and oligomeric Aβ species. Thioflavin T assays revealed that exosomal PrP(C) accelerates fibrillization of amyloid beta, thereby reducing neurotoxic effects imparted by oligomeric Aβ. Our study provides further evidence for a protective role of exosomes in Aβ-mediated neurodegeneration and highlights the importance of exosomal PrP(C) in molecular mechanisms of Alzheimer's disease. We show that the prion protein (PrP(C) ) on exosomes captures neurotoxic species of amyloid beta (Aβ) promoting its fibrillization. Our study provides evidence for a protective role of exosomes in Alzheimer`s disease and suggests that, depending on its membrane topology, PrP(C) holds a dual function: when expressed at the neuronal surface it acts as receptor for Aβ leading to neurotoxic signaling, whereas it detoxifies Aβ when present on exosomes. This provides further support for key roles of PrP(C) in Alzheimer's disease.
A Multicrystal Diffraction Data-collection Approach for Studying Structural Dynamics with Millisecond Temporal Resolution IUCrJ. | Pubmed ID: 27840678 Many biochemical processes take place on timescales ranging from femto-seconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach for crystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.
X-ray and UV Radiation-damage-induced Phasing Using Synchrotron Serial Crystallography Acta Crystallographica. Section D, Structural Biology. | Pubmed ID: 29652263 Specific radiation damage can be used to determine phases de novo from macromolecular crystals. This method is known as radiation-damage-induced phasing (RIP). One limitation of the method is that the dose of individual data sets must be minimized, which in turn leads to data sets with low multiplicity. A solution to this problem is to use data from multiple crystals. However, the resulting signal can be degraded by a lack of isomorphism between crystals. Here, it is shown that serial synchrotron crystallography in combination with selective merging of data sets can be used to determine high-quality phases for insulin and thaumatin, and that the increased multiplicity can greatly enhance the success rate of the experiment.