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.
Grasshoppers Regulate N: P Stoichiometric Homeostasis by Changing Phosphorus Contents in Their Frass
We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.
Background: Numerous studies have shown that nitrogen (N) deposition decreases biodiversity in terrestrial ecosystems. To explain the N-induced species loss, three functionally based hypotheses have been proposed: the aboveground competition hypothesis, the belowground competition hypothesis, and the total competition hypothesis. However, none of them is supported sufficiently by field experiments. A main challenge to testing these hypotheses is to ascertain the role of shoot and root competition in controlling plant responses to N enrichment. Simultaneously examining both aboveground and belowground responses in natural ecosystems is logistically complex, and has rarely been done.
Methodology/Principal Findings: In a two-year N addition experiment conducted in a natural grassland ecosystem, we investigated both above- and belowground responses of plants at the individual, species, and community levels. Plants differed significantly in their responses to N addition across the different organizational levels. The community-level species loss was mainly due to the loss of perennial grasses and forbs, while the relative abundance of plant species was dependent mainly on individual-level responses. Plasticity in biomass allocation was much smaller within a species than between species, providing a biological basis for explaining the functionally based species loss. All species increased biomass allocation to aboveground parts, but species with high belowground allocations were replaced by those with high aboveground allocations, indicating that the increased aboveground competition was the key process responsible for the observed diversity loss after N addition in this grassland ecosystem.
Conclusions/Significance: Our findings shed new light on the validity of the three competing hypotheses concerning species loss in response to N enrichment. They also have important implications for predicting the future impacts of N deposition on the structure and functioning of terrestrial ecosystems. In addition, we have developed a new technique for ascertaining the roles of aboveground and belowground competition in determining plant responses to N fertilization.
The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.
