Cryo2RT: a high-throughput method for room-temperature macromolecular crystallography from cryo-cooled 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 cryo-cooled crystals that leverages the cryo-crystallography workflow, is introduced.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 (O). 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.

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.