Co-flow injection for serial crystallography at X-ray free-electron lasers.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

The structure of photosystem I from a high-light-tolerant cyanobacteria.

Photosynthetic organisms have adapted to survive a myriad of extreme environments from the earth's deserts to its poles, yet the proteins that carry out the light reactions of photosynthesis are highly conserved from the cyanobacteria to modern day crops. To investigate adaptations of the photosynthetic machinery in cyanobacteria to excessive light stress, we isolated a new strain of cyanobacteria, Cyanobacterium aponinum 0216, from the extreme light environment of the Sonoran Desert. Here we report the biochemical characterization and the 2.7 Å resolution structure of trimeric photosystem I from this high-light-tolerant cyanobacterium. The structure shows a new conformation of the PsaL C-terminus that supports trimer formation of cyanobacterial photosystem I. The spectroscopic analysis of this photosystem I revealed a decrease in far-red absorption, which is attributed to a decrease in the number of long- wavelength chlorophylls. Using these findings, we constructed two chimeric PSIs in Synechocystis sp. PCC 6803 demonstrating how unique structural features in photosynthetic complexes can change spectroscopic properties, allowing organisms to thrive under different environmental stresses.

Author Correction: The structure of a red-shifted photosystem I reveals a red site in the core antenna.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19953-w.

The structure of a red-shifted photosystem I reveals a red site in the core antenna.

Photosystem I coordinates more than 90 chlorophylls in its core antenna while achieving near perfect quantum efficiency. Low energy chlorophylls (also known as red chlorophylls) residing in the antenna are important for energy transfer dynamics and yield, however, their precise location remained elusive. Here, we construct a chimeric Photosystem I complex in Synechocystis PCC 6803 that shows enhanced absorption in the red spectral region. We combine Cryo-EM and spectroscopy to determine the structure-function relationship in this red-shifted Photosystem I complex. Determining the structure of this complex reveals the precise architecture of the low energy site as well as large scale structural heterogeneity which is probably universal to all trimeric Photosystem I complexes. Identifying the structural elements that constitute red sites can expand the absorption spectrum of oxygenic photosynthetic and potentially modulate light harvesting efficiency.

Author Correction: Membrane protein megahertz crystallography at the European XFEL.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Membrane protein megahertz crystallography at the European XFEL.

The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.

High-viscosity injector-based pink-beam serial crystallography of microcrystals at a synchrotron radiation source.

Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth ('pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A2A adenosine receptor (A2AAR) were determined to resolutions of 1.8 and 4.2 Šusing 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A2AAR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.