Filtering by
- Status: Published
Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.
This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
Mof4 family associated protein 1 (MRFAP1) is a 14 kDa nuclear protein, which involves in maintaining normal histone modification levels by negatively regulating recruitment of the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase complex to chromatin. MRFAP1 has been identified as one of the most up-regulated proteins after NEDD8 (neural precursor cell expressed developmentally down- regulated 8) inhibition in multiple human cell lines. However, the biological function of MRFAP1 and the E3 ligase that targets MRFAP1 for destruction remain mysterious. Here we show, by using an immunoprecipitation-based proteomics screen, that MRFAP1 is an interactor of the F-box protein FBXW8. MRFAP1 is degraded by means of the ubiquitin ligase Cul7/FBXW8 during mitotic anaphase-telophase transition and accumulated in mitotic metaphase. Overexpression of FBXW8 increased the polyubiquitination and decreased the stability of MRFAP1, whereas knockdown of FBXW8 prolonged the half-life of MRFAP1. Moreover, forced expression of MRFAP1 in HeLa cells caused growth retardation and genomic instability, leading to severe mitotic cell death. Thus, Cul7/FBXW8-mediated destruction of MRFAP1 is a regulatory component monitoring the anaphase-telophase transition and preventing genomic instability.