Cornell Chronicle. By Tom Fleischman
Figure 1: A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo. (a) Schematic of the native E. coli disulfide-bond-formation pathway, which involves the endogenous transmembrane enzyme DsbB. DsbB is located in the inner membrane and interacts with its soluble periplasmic partner DsbA, which is localized to the periplasmic compartment by an N-terminal signal peptide specific for the cotranslational signal recognition particle (SRP) pathway. Electron transport is represented by the black arrows. DsbB obtains its electrons directly from quinones (Q). (b) Expression of DsbB as a soluble biocatalyst in the E. coli cytoplasm is accomplished by using the SIMPLEx technology, which renders IMPs water soluble by introduction of a 'decoy' domain (cMBP) and a 'shield' domain (ApoAI*). DsbA is redirected to the cytoplasm through removal of its native signal peptide. After coexpression, solubilized SxDsbB and export-defective DsbA (cDsbA) effectively transform the cytoplasm into a disulfide-bond-formation compartment. If needed, cDsbA expression can be improved by fusion to E. coli GST, a resident cytoplasmic protein that promotes solubility of its fusion partners.
BioSAXS Essentials 7 participants. Lectures were held atop the new Physical Sciences Building overlooking the Cornell Campus and Cayuga Lake. All but 4 participants were from outside Cornell.
As in past years, we configured both G1 and F1 as essentially identical BioSAXS stations to give more students a chance at hands-on experience during the course. Many students were able to bring their own samples to test for the first time. This year, we were fortunate to have two running size exclusion chromatography (SEC) systems attached directly to the beamlines. These “SEC-SAXS” configurations greatly expand the number of samples that can be successfully analyzed by allowing researchers to separate complex mixtures in real time. MacCHESS is unique in the United States in running two such setups simultaneously for SEC-SAXS. Students were introduced to our RAW software, a very easy to use application for collecting and analyzing SAXS and SEC-SAXS data. RAW is available free for Mac, Windows, and Linux, so students were able to install the program on their laptops to take home (https://sourceforge.net/projects/bioxtasraw/). Steve Meisburger (Ando Group, Princeton) recently published a powerful new algorithm for analyzing difficult-to-separate species in SEC-SAXS, the Evolving Factor Analysis (EFA). Jesse Hopkins (MacCHESS) has created the first publicly available implementation of EFA in our RAW software and students got to learn how to use it on their data. Kushol Gupta (UPENN, Perelman School of Medicine) talked about how he used RAW and EFA to solve a difficult separation problem in his own research.
The course started with Richard Gillilan (MacCHESS) giving the introductory lectures on basic SAXS theory and practice. Since proper sample preparation is essential to success in BioSAXS, Kushol Gupta gave a lecture devoted exclusively to that topic. Thomas Grant (HWI) continued the development of SAXS theory and practice with emphasis on shape reconstruction and other more advanced concepts. The three final lectures of the first day were devoted to advanced methods. Steve Meisburger lectured on SEC-SAXS technique, introducing his EFA method. Kushol Gupta discussed how to deal with mixtures, and described how to use contrast variation to locate polynucleotides within protein complexes. Finally, Thomas Grant showed some preliminary results from a very exciting new method to reconstruct actual electron density from SAXS data.
The Wednesday morning session, led by Jesse Hopkins and Steve Meisburger, was devoted to software tutorials. Students installed our RAW software on their laptops and learned how to process data. In addition to basic quality assessment and common processing practice, they learned how to analyse SEC-SAXS data including the novel EFA method.
The tutorial was followed by two days of intensive around-the-clock data collection by students in small groups. Students were able to choose between regular robotic BioSAXS and SEC-SAXS. Both stations were virtually identical in operation, so students trained on F1 find themselves fully competent to operate G1 when they return for normal research beamtime in the future.
Many thanks to all who helped make this training workshop a success!
BioSAXS Essentials students learn data collection skills from Jesse Hopkins on G1 station (top center). F1 station, normally used for crystallography, was configured as an operationally identical BioSAXS station for the course (Richard Gillilan, bottom left). Both stations supported inline size exclusion chromatography (SEC-SAXS), a popular new technique for separating complex mixtures of biomolecules. During the course, CHESS was the only facility in the nation running two simultaenous SEC-SAXS systems. Also shown: Pedro De La Torre, Michael Durney, and Balasubramnian Harish.
Submitted by: Richard Gillilan, MacCHESS, Cornell University 06/11/2017
Cornell Chronicle. By Elodie Gazave.
Cartoon representation of a 'dolphin-like' single subunit of the apo pdP2X7 structure. Fourteen beta strands are labeled as ß1-14. Each domain is colored consistent with the previous studies for better comparison
Akira Karasawa*, Toshimitsu Kawate*. "Structural basis for subtype-specific inhibition of the P2X7 receptor". eLife, 2016.
*Cornell University, United States