mScarlet3: a brilliant and fast-maturing red fluorescent protein.

We report the evolution of mScarlet3, a cysteine-free monomeric red fluorescent protein with fast and complete maturation, as well as record brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The mScarlet3 crystal structure reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues. mScarlet3 behaves well as a fusion tag, displays no apparent cytotoxicity and it surpasses existing red fluorescent proteins as a Förster resonance energy transfer acceptor and as a reporter in transient expression systems.

SDU - software for high-throughput automated data collection at the Swiss Light Source.

Recent advances in automation have fostered the development of unattended data collection services at a handful of synchrotron facilities worldwide. At the Swiss Light Source, the installation of new high-throughput sample changers at all three macromolecular crystallography beamlines and the commissioning of the Fast Fragment and Compound Screening pipeline created a unique opportunity to automate data acquisition. Here, the DA+ microservice software stack upgrades, implementation of an automatic loop-centering service and deployment of the Smart Digital User (SDU) software for unattended data collection are reported. The SDU software is the decision-making software responsible for communications between services, sample and device safety, sample centering, sample alignment with grid based X-ray diffraction and, finally, data collection.

Riboflavin-binding proteins for singlet oxygen production.

miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen.

mScarlet: a bright monomeric red fluorescent protein for cellular imaging.

We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.

Cryo2RT: a high-throughput method for room-temperature macromolecular crystallography from cryocooled crystals.

Advances in structural biology have relied heavily on synchrotron cryo-crystallography and cryogenic electron microscopy to elucidate biological processes and for drug discovery. However, disparities between cryogenic and room-temperature (RT) crystal structures pose challenges. Here, Cryo2RT, a high-throughput RT data-collection method from frozen crystals that leverages the cryo-crystallography workflow, is introduced. Tested on endothiapepsin crystals with four soaked fragments, thaumatin and SARS-CoV-2 3CLpro, Cryo2RT reveals unique ligand-binding poses, offers a comparable throughput to cryo-crystallography and eases the exploration of structural dynamics at various temperatures.

Identification of the SARS-unique domain of SARS-CoV-2 as an antiviral target.

SARS-CoV-2 nsp3 is essential for viral replication and host responses. The SARS-unique domain (SUD) of nsp3 exerts its function through binding to viral and host proteins and RNAs. Herein, we show that SARS-CoV-2 SUD is highly flexible in solution. The intramolecular disulfide bond of SARS-CoV SUD is absent in SARS-CoV-2 SUD. Incorporating this bond in SARS-CoV-2 SUD allowed crystal structure determination to 1.35 Å resolution. However, introducing this bond in SARS-CoV-2 genome was lethal for the virus. Using biolayer interferometry, we screened compounds directly binding to SARS-CoV-2 SUD and identified theaflavin 3,3'-digallate (TF3) as a potent binder, Kd 2.8 µM. TF3 disrupted the SUD-guanine quadruplex interactions and exhibited anti-SARS-CoV-2 activity in Vero E6-TMPRSS2 cells with an EC50 of 5.9 µM and CC50 of 98.5 µM. In this work, we provide evidence that SARS-CoV-2 SUD harbors druggable sites for antiviral development.

Slow protein dynamics probed by time-resolved oscillation crystallography at room temperature.

The development of serial crystallography over the last decade at XFELs and synchrotrons has produced a renaissance in room-temperature macromolecular crystallography (RT-MX), and fostered many technical and methodological breakthroughs designed to study phenomena occurring in proteins on the picosecond-to-second timescale. However, there are components of protein dynamics that occur in much slower regimes, of which the study could readily benefit from state-of-the-art RT-MX. Here, the room-temperature structural study of the relaxation of a reaction intermediate at a synchrotron, exploiting a handful of single crystals, is described. The intermediate in question is formed in microseconds during the photoreaction of the LOV2 domain of phototropin 2 from Arabidopsis thaliana, which then decays in minutes. This work monitored its relaxation in the dark using a fast-readout EIGER X 4M detector to record several complete oscillation X-ray diffraction datasets, each of 1.2 s total exposure time, at different time points in the relaxation process. Coupled with in crystallo UV-Vis absorption spectroscopy, this RT-MX approach allowed the authors to follow the relaxation of the photoadduct, a thio-ether covalent bond between the chromophore and a cysteine residue. Unexpectedly, the return of the chromophore to its spectroscopic ground state is followed by medium-scale protein rearrangements that trigger a crystal phase transition and hinder the full recovery of the structural ground state of the protein. In addition to suggesting a hitherto unexpected role of a conserved tryptophan residue in the regulation of the photocycle of LOV2, this work provides a basis for performing routine time-resolved protein crystallography experiments at synchrotrons for phenomena occurring on the second-to-hour timescale.

Probing ligand binding of endothiapepsin by `temperature-resolved' macromolecular crystallography.

Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.

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.

Millisecond time-resolved serial oscillation crystallography of a blue-light photoreceptor at a synchrotron.

The recent development of serial crystallography has popularized time-resolved crystallography as a technique to determine the structure of protein-reaction intermediate states. However, most approaches rely on the availability of thousands to millions of microcrystals. A method is reported here, using monochromatic synchrotron radiation, for the room-temperature collection, processing and merging of X-ray oscillation diffraction data from <100 samples in order to observe the build up of a photoreaction intermediate species. Using this method, we monitored with a time resolution of 63 ms how the population of a blue-light photoreceptor domain in a crystal progressively photoconverts from the dark to the light state. The series of resulting snapshots allows us to visualize in detail the gradual rearrangement of both the protein and chromophore during this process.

Specific radiation damage is a lesser concern at room temperature.

Carrying out macromolecular crystallography (MX) experiments at cryogenic temperatures significantly slows the rate of global radiation damage, thus facilitating the solution of high-resolution crystal structures of macromolecules. However, cryo-MX experiments suffer from the early onset of so-called specific radiation damage that affects certain amino-acid residues and, in particular, the active sites of many proteins. Here, a series of MX experiments are described which suggest that specific and global radiation damage are much less decoupled at room temperature than they are at cryogenic temperatures. The results reported here demonstrate the interest in reviving the practice of collecting MX diffraction data at room temperature and allow structural biologists to favourably envisage the development of time-resolved MX experiments at synchrotron sources.

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect.

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

Tailing miniSOG: structural bases of the complex photophysics of a flavin-binding singlet oxygen photosensitizing protein.

miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving as a genetically-encodable light-induced source of singlet oxygen (1O2). We have determined its 1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization using an integrated approach combining spectroscopic and structural methods. Our results provide a structural framework to explain the ability of miniSOG to produce 1O2 as a competition between oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway has been identified that is pivotal for the performance of miniSOG as 1O2 photosensitizer, namely the photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1O2 quantum yield by one order of magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich residues that are progressively photo-oxidized, further contributing to facilitate the production of 1O2. Our results help reconcile the apparent poor level of 1O2 generation by miniSOG and its excellent performance in correlative light and electron microscopy experiments.