The macromolecular crystallography (MX) beamline X06DA-PXIII at the Swiss Light Source (SLS) has undergone a significant upgrade in preparation for SLS 2.0. After 15 years of operation and >2,500 PDB depositions, this highly productive superbend magnet beamline has been completely rebuilt with new optics and experimental hutches. The new design, which aims to exploit the ×37 reduced emittance of the SLS 2.0 storage ring, consists of a toroidal mirror close to the source to harvest a large solid angle of the bending magnet beam, a horizontal 1:2 focusing concept to minimize toroidal aberration and Kirkpatrick-Baez mirrors to focus the beam down to 10 × 10 microns2 with a flux of 2 × 1012 ph/s at the sample position. The refurbished double channel-cut monochromator provides an energy range from 3 to 15 keV. While this makes the beamline highly suitable for identifying low-Z elements in macromolecular structures with data acquisition at low energy, we plan to center our main activity around high-throughput industrial applications for fragment-based drug discovery. We will perform fully autonomous and unattended experiments using the TELL sample changer, the multi-axis goniometer SmarGon, and the new Dectris PILATUS4 X 2 M detector. We also intend to automate experiments at room temperature, which will benefit from the unprecedented speed of data collection provided by the detector. In this presentation, I will present the upgrade of the X06DA-PXIII beamline and the planned experiments with the new SLS 2.0 storage ring. In addition, I will give an overview of the upgrade of the other 2 MX undulator beamlines.

Small Angle-X-ray Scattering Tensor Tomography (SAS-TT) is a relatively new but powerful technique for studying the multiscale architecture of hierarchical structures particularly relevant to life science applications. Currently, the technique is very demanding on synchrotron beamtime, which limits its applications, especially for cases requiring a statistically relevant number of samples. This study reports the first SAS-TT measurement at a macromolecular X-ray crystallography beamline, PX-I at the Swiss Light Source (SLS), with an improvement in acquisition time from 96 h/Mvoxel in the pilot experiments to 6 h/Mvoxel with comparable sampling, defining a new standard for fast SAS-TT with a micrometer beam size and allowing to record a full tomogram in 1.2 h. Measurements are performed on the long and lenticular process of the incus bone, one of the three human auditory ossicles. The main orientation and degree of alignment of the mineralised collagen fibrils are characterised, as well as the size and shape of the mineral particles which show relevant variations in different tissue locations. The study reveals three distinct regions of high fibril alignment, most likely important pathways of sound throughout the ossicular chain, and highlights the technique's potential to aid in future developments in middle ear reconstructive surgery.

Varying the chemical consistency of acoustically levitated droplets opens up an in situ study of chemical and biochemical reactions in small volumes. However, the optimization of the mixing time and the minimization of the positional instability induced by solution dispensing are necessary for practical applications such as the study of the transient state of macromolecules crystallography during the ligand binding processes. For this purpose, we study the inertial mixing in a configuration compatible with the room-temperature crystallography using the acoustic levitation diffractometer, therein solution drops ejected at high velocity collide and coalesce with droplets dispensed on acoustically levitated and rotating polymer thin-film sample holders. With the proposed method, we are able to achieve the mixing time of similar to 0.1 s for sub-micro and a few microliter droplets. The observed short mixing time is ascribed to the rapid penetration of the solution into the droplets and confirmed by a computational fluid dynamic simulation. The demonstrated accelerated solution mixing is tested in a pilot time-lapse protein crystallography experiment using the acoustic levitation diffractometer. The results indicate the detection of transient ligand binding state within 2 s after the solution dispensing, suggesting the feasibility of the proposed method for studying slow biochemical processes. The solution mixing time faster than similar to 0.1 s is achieved in a sub-micro to microliter droplet on an acoustically levitated and rotating sample holder by the collision and coalescence of fast solution drop pairs ejected by double dispensers. The finding was tested in the ligand binding reaction of protein molecules in room-temperature time-lapse X-ray crystallography experiment. The result indicates the detection of the transient state within similar to 2 s, suggesting the feasibility of the proposed method for studying slow biochemical processes. image

Acoustic levitation has attracted attention in terms of chemical and biochemical analysis in combination with various analytical methods because of its unique container-less environment for samples that is not reliant on specific material characteristics. However, loading samples with very high viscosity is difficult. To expand the scope, we propose the use of polymer thin films as sample holders, whereby the sample is dispensed on a film that is subsequently loaded onto an acoustic levitator. When applied for protein crystallography experiments, rotation controllability and positional stability are important prerequisites. We therefore study the acoustic levitation and rotation of thin films with an aspect ratio (the diameter-to-thickness ratio) of 80–240, which is an order of magnitude larger than those reported previously. For films with empirically optimized shapes, we find that it is possible to control the rotation speed in the range of 1–4 rotations per second while maintaining a positional stability of 12 ± 5 µm. The acoustic radiation force acting on the films is found to be a factor of 26–30 higher than that for same-volume water droplets. We propose use cases of the developed films for protein crystallography experiments and demonstrate data collections for large single crystal samples at room temperature.

The recently developed acoustic levitation diffractometer, combining the ultrasonic acoustic levitation with the highly brilliant X-ray source and the fast-frame-rate Xray image detector allows for high speed data collection for protein crystallography experiments with a containerless sample support. One of the current research goals is its application to a fully automated high throughput pipeline of protein crystallography experiments at room temperature by integrating an on-demand sample delivery mechanism directly from standard crystallization plates, that is widely used to grow protein samples. We recently proposed to use the acoustic droplet ejection for the on-demand sample delivery, wherein focused acoustic radiation pulses eject samples-in-droplet from a well of the crystallization plates. However, as the sample delivery mechanism, we need to establish the stability of the droplet ejection and the reliable capture of the ejected droplets by the acoustic levitator. In this work, we study the positional distribution of the on-demand acoustic droplet ejection from a crystallization plate and its combination with air jet pulses to assist capturing the ejected droplets by the acoustic levitator. In the proposed system, we demonstrate the on-demand droplet loading with the yield reaching approximately 90%.

Ultrasonic acoustic levitation has been recently applied successfully for protein crystallography experiments. One of the development goals of such an acoustic levitation diffractometer is to realize a fully automated high-throughput crystallography pipeline at room temperature that integrates an on-demand sample delivery mechanism from crystallization plates. To study the feasibility of the on-demand droplet ejection from crystallization plates, we prepared acoustic ejectors and tested their characteristics. Acoustic droplet ejection from a vertically positioned crystallization plate and coalescence of the ejected droplet with the levitated droplet in an acoustic levitator was successfully demonstrated, indicating that feasibility of the proposed instrument.

Significance Using hybrid silica/protein templates, nature has mastered the fabrication of extremely complex macroscopic glass assemblies. Highly symmetric skeletal elements in demosponges are formed following a unique biomineralization mechanism in which polycondensation of an inherently disordered amorphous silica is guided by highly ordered proteinaceous filaments. Here we provide a comprehensive three-dimensional atomistic view of this hybrid assembly. The structure, occurring in the crystalline form in vivo, was measured in situ using the serial crystallography method. Together with a high-resolution transmission electron microscopy and energy-dispersive X-ray spectroscopy study, we provide structural, chemical, and functional information on a naturally forming hybrid mineral/organic crystal. Formation of highly symmetric skeletal elements in demosponges, called spicules, follows a unique biomineralization mechanism in which polycondensation of an inherently disordered amorphous silica is guided by a highly ordered proteinaceous scaffold, the axial filament. The enzymatically active proteins, silicateins, are assembled into a slender hybrid silica/protein crystalline superstructure that directs the morphogenesis of the spicules. Furthermore, silicateins are known to catalyze the formation of a large variety of other technologically relevant organic and inorganic materials. However, despite the biological and biotechnological importance of this macromolecule, its tertiary structure was never determined. Here we report the atomic structure of silicatein and the entire mineral/organic hybrid assembly with a resolution of 2.4 Å. In this work, the serial X-ray crystallography method was successfully adopted to probe the 2-µm-thick filaments in situ, being embedded inside the skeletal elements. In combination with imaging and chemical analysis using high-resolution transmission electron microscopy, we provide detailed information on the enzymatic activity of silicatein, its crystallization, and the emergence of a functional three-dimensional silica/protein superstructure in vivo. Ultimately, we describe a naturally occurring mineral/protein crystalline assembly at atomic resolution.

Formation of highly symmetric skeletal elements in demosponges, called spicules, follows a unique biomineralization mechanism in which polycondensation of an inherently disordered amorphous silica is guided by a highly ordered proteinaceous scaffold, the axial filament. The enzymatically active proteins, silicateins, are assembled into a slender hybrid silica/protein crystalline superstructure that directs the morphogenesis of the spicules. Furthermore, silicateins are known to catalyze the formation of a large variety of other technologically relevant organic and inorganic materials. However, despite the biological and biotechnological importance of this macromolecule, its tertiary structure was never determined. Here we report the atomic structure of silicatein and the entire mineral/organic hybrid assembly with a resolution of 2.4 Å. In this work, the serial X-ray crystallography method was successfully adopted to probe the 2-µm-thick filaments in situ, being embedded inside the skeletal elements. In combination with imaging and chemical analysis using high-resolution transmission electron microscopy, we provide detailed information on the enzymatic activity of silicatein, its crystallization, and the emergence of a functional three-dimensional silica/protein superstructure in vivo. Ultimately, we describe a naturally occurring mineral/protein crystalline assembly at atomic resolution.

Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers ( Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9 Å is demonstrated for native-SAD phasing on a 266 kDa multiprotein-ligand tubulin complex (T 2 R-TTL) and is applied in the structure determination of an 86 kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3 Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3 Å are discussed.

Diatoms are abundant photosynthetic organisms in aquatic environments and contribute 40% of its primary productivity. An important factor that contributes to the success of diatoms is their fucoxanthin chlorophyll a/c-binding proteins (FCPs), which have exceptional light-harvesting and photoprotection capabilities. Here, we report the crystal structure of an FCP from the marine diatom Phaeodactylum tricornutum, which reveals the binding of seven chlorophylls (Chls) a, two Chls c, seven fucoxanthins (Fxs), and probably one diadinoxanthin within the protein scaffold. Efficient energy transfer pathways can be found between Chl a and c, and each Fx is surrounded by Chls, enabling the energy transfer and quenching via Fx highly efficient. The structure provides a basis for elucidating the mechanisms of blue-green light harvesting, energy transfer, and dissipation in diatoms.

We are developing the acoustic levitation diffractometer, a new container-free diffractometer at the Swiss Light Source, Paul Scherrer Institut. By rotating single crystals in an acoustically levitated droplet and collecting the diffraction images by a fast-frame-rate X-ray image detector, the data collection can be completed within a few hundred milliseconds or shorter at room temperature. Here we report time-lapse measurements of ligand soaking using a single crystal. This was achieved by collecting a series of datasets after soaking ligand solution into single lysozyme crystals in a levitated droplet in the acoustic cavity of the diffractometer. Electron density maps of the lysozyme crystals obtained every 30 seconds after the ligand soaking showed meaningful conformational changes around the binding site of the ligand and the radiation damage for 300 seconds after the ligand soaking.

Swiss Light Source, Paul Scherrer Institut, Villigen PSI, 5232, Switzerland, Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization, Tsukuba, 305-0801, Japan, Advanced Photon Technology Division, RIKEN SPring-8 Center, Hyogo 679-5148, Japan, Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, Villigen, PSI 5232, Switzerland, Department of Biochemistry, Max Planck Institute of Biochemistry, Munich, Germany, and Department of Biology, University of Konstanz, Konstanz, 78457, Germany. *Correspondence e-mail: meitian.wang@psi.ch

The positional stability of acoustically levitated droplets in air and the settling time of droplet positions on loading are important for precision applications of acoustic levitation. We therefore study their dependence on ultrasound pressure for droplets with diameters 0.01–0.2 times the acoustic wavelength in a single-axis acoustic levitator operating at 39 kHz. We find that the observed resonance frequencies agree well with theory. However, the damping coefficients of the oscillations exhibit large anisotropy, and their dependence on the droplet size deviates greatly from the behavior expected from the Stokes coefficient. These results suggest that acoustic streaming plays an important role in the motion of acoustically levitated droplets.

Common to all macromolecular crystallographic (MX) structure determination is the phase problem which is primarily addressed by multi-and single-wavelength anomalous diffraction (MAD and SAD) for novel structures.While those experiments typically involved heavy atom derivatization or selenomethionine labeling, more structures are nowadays solved directly from native crystals using the intrinsic anomalous scattering from light scatterers (S essentially).Such signal increases as the X-ray energy is lowered but low energy presents technical challenges caused by the increased absorption and scattering from both the air and the sample, as well as larger diffraction angles.Native-SAD is therefore often practiced at a compromise energy of ~6 keV at conventional synchrotron beamlines with high-multiplicity measurements, obtained from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose [1-3].Dedicated low-energy beamlines, namely I23 at Diamond, UK, and BL-1A at the Photon Factory, Japan offer specific sample environments (vacuum or helium) and special detector configurations (a curved detector or a V-shaped arrangement).In addition, the use of laser ablation technology to control sample shape and thickness [4], as well as the detector performance are of crucial importance when collecting at energies below 6 keV.Here, I will present our recent native-SAD experiments on laser-shaped crystals at 3.75 keV at BL-1A using a JUNGFRAU 4M detector.The JUNGFRAU, a hybrid pixel charge integrating detector currently being developed at the Paul Scherrer Institut, is particularly well suited for long-wavelength native-SAD application [5].I will show that JUNGFRAU provided very accurate measurement of reflections intensity from both test and real-life crystals at 3.75 keV in total experiment time below a minute.The potential and challenges of using even lower energy will be discussed.

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

Single-wavelength Anomalous Dispersion (SAD) is the most popular experimental phasing technique to determine X-ray structure in the field of structural biology.The data collection, in this method, is performed at the absorption edge of anomalous scatterers, which are either introduced in the crystal or natively present in the macromolecules.In case of native SAD phasing, which uses the weak anomalous scattering signals from light elements -such as sulfur, phosphorous, or any ions, naturally present in the macromolecules, the most suitable energy would be around 2.5 keV (or λ = 5 Å), above the sulfur K-edge.However, such low X-ray energy is not attainable at most of the current operational macromolecular crystallography beamlines.In addition, native SAD at such low energy comes with more challenges, caused by x-ray absorption due to crystal thickness, cryo-loop, solvent around the crystal, air, as well as detector efficiency.Thereby, an Xray energy around 6 keV is considered as a good "compromise" between anomalous diffraction signal and absorption effect [1, 2, 3].Here, we present the promises and challenges associated with native-SAD data collection at X-ray energy below 6 keV, in particular for X-ray absorption effects and optimum crystal size, using both test proteins and real-life examples.

Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H...H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.