A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers.

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Time-resolved crystallography captures light-driven DNA repair.

Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.

Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography.

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we capture the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.

Crystallization Systems for the High-Resolution Structural Analysis of Tubulin-Ligand Complexes.

Since the first moderate resolution, structural description of Taxol bound to tubulin by electron crystallography in 1998, several tubulin crystal systems have been developed and optimized for the high-resolution analysis of tubulin-ligand complexes by X-ray crystallography. Here we describe three tubulin crystal systems that have allowed investigating the molecular mechanisms of action of a large number of diverse anti-tubulin agents.

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

Structural basis of the radical pair state in photolyases and cryptochromes.

We present the structure of a photoactivated animal (6-4) photolyase in its radical pair state, captured by serial crystallography. We observe how a conserved asparigine moves towards the semiquinone FAD chromophore and stabilizes it by hydrogen bonding. Several amino acids around the final tryptophan radical rearrange, opening it up to the solvent. The structure explains how the protein environment stabilizes the radical pair state, which is crucial for function of (6-4) photolyases and cryptochromes.

Dynamics and mechanism of a light-driven chloride pump.

Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump.

Versatile microporous polymer-based supports for serial macromolecular crystallography.

Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.

A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton.

Optically controlled chemical reagents, termed "photopharmaceuticals," are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biological use. We rationally designed a photoswitchable "SBT" scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal "SBTub" photopharmaceutical tubulin inhibitors. Lead compound SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biology, and their robustness and druglikeness are favorable for intracellular biological control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biological targets.

Femtosecond-to-millisecond structural changes in a light-driven sodium pump.

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.