Serial Femtosecond Crystallography (SFX) is a promising technique that enables the high-resolution protein structure determination using micron- and submicron-sized crystals at room temperature with minimal radiation damage1-4. Important proof-of-principle results have demonstrated that SFX, when applied to protein microcrystals grown in lipidic cubic phase (LCP), is capable of solving structures of membrane proteins5-7. This novel LCP-SFX technique has been successfully applied to many challenging membrane proteins within the last few years 8-11, including the protein complex containing rhodopsin bound to arrestin, a high-impact study recently published on Nature (https://www.bioxfel.org/news/142-bioxfel-journal-article-most-read-in-nature). However, due to the difficulties associated with sample handling and limited availability of high-throughput LCP crystallization platform, the LCP-SFX is not readily accessible for structure determination of membrane proteins in the field. To address this issue, here we propose to establish a Nanocrystallization-and-Characterization-in-LCP (NCIL) platform as an integral component of BioXFEL STC at Arizona State University (ASU), which will be initially available to all STC members and gradually open to non-STC users. We have demonstrated that combining LCP with SFX allows us to (i) reduce protein amount to below 0.1 - 0.3 mg for the collection of high-quality diffraction data, and to (ii) use LCP as an alternative delivery medium for soluble proteins. Moreover, the LCP-SFX platform will enable us to achieve high-throughput screening of optimal conditions for protein complex microcrystal formation in LCP, and thus advance our understandings on the signaling and function of important membrane protein complexes, such as the G-protein-coupled receptor (GPCR)-G protein signaling machinery. Dr. Lius lab is focusing on GPCR/Gi complex structural determination, which will reveal the structural bases for the activation of G-proteins by GPCRs. As part of this effort, full proposal application to beam time for Run 14 at Linac Coherent Light Source (LCLS) has be submitted. Once NCIL is established, Dr. Liu and his group expect to speed up scientific discoveries and the collaborations within BioXFEL STC. In addition, national workshops will be hosted by NCIL at ASU to educate more students and researchers interested in learning the LCP-SFX technology.
|Effective start/end date||5/1/16 → 12/31/18|
- National Science Foundation (NSF): $120,000.00
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