Experimental phasing opportunities for macromolecular crystallography at very long wavelengths.

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

X-Ray Conformation and Structure-Activity Relationships of MA026, a Reversible Tight Junction Opener.

MA026, a cyclic lipodepsipeptide, opens the tight junction (TJ) probably via binding to claudin-1. We reported that (1) TJ-opening activity is dependent on the amino acid sequence order at Glu10-Leu11; (2) an epimer at the C3 position of the N-terminal acyl tail decreased the TJ-opening activity; and (3) the epimers D-Leu1/L-Gln6 and L-Leu1/D-Gln6 showed more potent TJ-opening activity than natural MA026, although no systematic structure-activity relationship (SAR) study was conducted. Here, we report the three-dimensional structure and systematic SAR study of MA026. X-Ray crystallography and circular dichroism analysis of MA026 revealed that MA026 forms a left-handed α-helical structure, and hydrophobic amino acids are clustered on one side. Furthermore, the SAR results clearly showed that the hydrophobic region of MA026 is important for TJ-opening activity. These results suggest that MA026 interacts with claudin-1 via the hydrophobic cluster region and provide novel structural insights toward the development of a TJ opener targeting claudin-1.

A crystal-processing machine using a deep-ultraviolet laser: application to long-wavelength native SAD experiments.

While native SAD phasing is a promising method for next-generation macromolecular crystallography, it requires the collection of high-quality diffraction data using long-wavelength X-rays. The crystal itself and the noncrystalline medium around the crystal can cause background noise during long-wavelength X-ray data collection, hampering native SAD phasing. Optimizing the crystal size and shape or removing noncrystalline sample portions have thus been considered to be effective means of improving the data quality. A crystal-processing machine that uses a deep-UV laser has been developed. The machine utilizes the pulsed UV laser soft ablation (PULSA) technique, which generates less heat than methods using infrared or visible lasers. Since protein crystals are sensitive to heat damage, PULSA is an appropriate method to process them. Integration of a high-speed Galvano scanner and a high-precision goniometer enables protein crystals to be shaped precisely and efficiently. Application of this crystal-processing machine to a long-wavelength X-ray diffraction experiment significantly improved the diffraction data quality and thereby increased the success rate in experimental phasing using anomalous diffraction from atoms.

Managing Growth and Dimensionality of Quasi 2D Perovskite Single-Crystalline Flakes for Tunable Excitons Orientation.

Hybrid perovskites are among the most promising materials for optoelectronic applications. Their 2D crystalline form is even more interesting since the alternating inorganic and organic layers naturally forge a multiple quantum-well structure, leading to the formation of stable excitonic resonances. Nevertheless, a controlled modulation of the quantum well width, which is defined by the number of inorganic layers (n) between two organic ones, is not trivial and represents the main synthetic challenge in the field. Here, a conceptually innovative approach to easily tune n in lead iodide perovskite single-crystalline flakes is presented. The judicious use of potassium iodide is found to modulate the supersaturation levels of the precursors solution without being part of the final products. This allows to obtain a fine tuning of the n value. The excellent optical quality of the as synthesized flakes guarantees an in-depth analysis by Fourier-space microscopy, revealing that the excitons orientation can be manipulated by modifying the number of inorganic layers. Excitonic out-of-plane component, indeed, is enhanced when "n" is increased. The combined advances in the synthesis and optical characterization fill in the picture of the exciton behavior in low-dimensional perovskite, paving the way to the design of materials with improved optoelectronic characteristics.

Long-wavelength native-SAD phasing: opportunities and challenges.

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 Šis demonstrated for native-SAD phasing on a 266 kDa multiprotein-ligand tubulin complex (T2R-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 Šis reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3 Šare discussed.

Improvement of Production and Isolation of Human Neuraminidase-1 in Cellulo Crystals.

In cellulo crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by in vitro crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of in cellulo crystallization because the process occurs spontaneously. Here we report a protocol to produce and extract in cellulo crystals of human lysosomal neuraminidase-1 (NEU1) in human cultured cells. Overexpression of NEU1 protein by the retransfection of cells pretransfected with neu1-overexpressing plasmid improved the efficiency of NEU1 crystallization. Microscopic analysis revealed that NEU1 proteins were not crystallized in the lysosome but in the endoplasmic reticulum (ER). Screening of the buffer conditions used to extract crystals from cells further improved the crystal yield. The optimal pH was 7.0, which corresponds to the pH in the ER. Use of a high-yield flask with a large surface area also yielded more crystals. These optimizations enabled us to execute a serial femtosecond crystallography experiment with a sufficient number of crystals to generate a complete data set. Optimization of the in cellulo crystallization method was thus shown to be possible.

The first crystal structure of a family 129 glycoside hydrolase from a probiotic bacterium reveals critical residues and metal cofactors.

The α-N-acetylgalactosaminidase from the probiotic bacterium Bifidobacterium bifidum (NagBb) belongs to the glycoside hydrolase family 129 and hydrolyzes the glycosidic bond of Tn-antigen (GalNAcα1-Ser/Thr). NagBb is involved in assimilation of O-glycans on mucin glycoproteins by B. bifidum in the human gastrointestinal tract, but its catalytic mechanism has remained elusive because of a lack of sequence homology around putative catalytic residues and of other structural information. Here we report the X-ray crystal structure of NagBb, representing the first GH129 family structure, solved by the single-wavelength anomalous dispersion method based on sulfur atoms of the native protein. We determined ligand-free, GalNAc, and inhibitor complex forms of NagBb and found that Asp-435 and Glu-478 are located in the catalytic domain at appropriate positions for direct nucleophilic attack at the anomeric carbon and proton donation for the glycosidic bond oxygen, respectively. A highly conserved Asp-330 forms a hydrogen bond with the O4 hydroxyl of GalNAc in the -1 subsite, and Trp-398 provides a stacking platform for the GalNAc pyranose ring. Interestingly, a metal ion, presumably Ca2+, is involved in the recognition of the GalNAc N-acetyl group. Mutations at Asp-435, Glu-478, Asp-330, and Trp-398 and residues involved in metal coordination (including an all-Ala quadruple mutant) significantly reduced the activity, indicating that these residues and the metal ion play important roles in substrate recognition and catalysis. Interestingly, NagBb exhibited some structural similarities to the GH101 endo-α-N-acetylgalactosaminidases, but several critical differences in substrate recognition and reaction mechanism account for the different activities of these two enzymes.

3D structural analysis of protein O-mannosyl kinase, POMK, a causative gene product of dystroglycanopathy.

Orchestration of the multiple enzymes engaged in O-mannose glycan synthesis provides a matriglycan on α-dystroglycan (α-DG) which attracts extracellular matrix (ECM) proteins such as laminin. Aberrant O-mannosylation of α-DG leads to severe congenital muscular dystrophies due to detachment of ECM proteins from the basal membrane. Phosphorylation at C6-position of O-mannose catalyzed by protein O-mannosyl kinase (POMK) is a crucial step in the biosynthetic pathway of O-mannose glycan. Several mis-sense mutations of the POMK catalytic domain are known to cause a severe congenital muscular dystrophy, Walker-Warburg syndrome. Due to the low sequence similarity with other typical kinases, structure-activity relationships of this enzyme remain unclear. Here, we report the crystal structures of the POMK catalytic domain in the absence and presence of an ATP analogue and O-mannosylated glycopeptide. The POMK catalytic domain shows a typical protein kinase fold consisting of N- and C-lobes. Mannose residue binds to POMK mainly via the hydroxyl group at C2-position, differentiating from other monosaccharide residues. Intriguingly, the two amino acid residues K92 and D228, interacting with the triphosphate group of ATP, are donated from atypical positions in the primary structure. Mutations in this protein causing muscular dystrophies can now be rationalized.

Crystallographic analysis of murine p24γ2 Golgi dynamics domain.

The p24 family proteins form homo- and hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. It consists of four subfamilies (p24α, p24ß, p24γ, and p24δ). p24γ2 plays crucial roles in the selective transport of glycosylphosphatidylinositol-anchored proteins. Here, we determined the crystal structure of mouse p24γ2 Golgi dynamics (GOLD) domain at 2.8 Å resolution by the single anomalous diffraction method using intrinsic sulfur atoms. In spite of low sequence identity among p24 family proteins, p24γ2 GOLD domain assumes a ß-sandwich fold, similar to that of p24ß1 or p24δ1. An additional short α-helix is observed at the C-terminus of the p24γ2 GOLD domain. Intriguingly, p24γ2 GOLD domains crystallize as dimers, and dimer formation seems assisted by the short α-helix. Dimerization modes of GOLD domains are compared among p24 family proteins. Proteins 2017; 85:764-770. © 2016 Wiley Periodicals, Inc.

Protein crystallography beamline BL2S1 at the Aichi synchrotron.

The protein crystallography beamline BL2S1, constructed at one of the 5 T superconducting bending-magnet ports of the Aichi synchrotron, is available to users associated with academic and industrial organizations. The beamline is mainly intended for use in X-ray diffraction measurements of single-crystals of macromolecules such as proteins and nucleic acids. Diffraction measurements for crystals of other materials are also possible, such as inorganic and organic compounds. BL2S1 covers the energy range 7-17 keV (1.8-0.7 Å) with an asymmetric-cut curved single-crystal monochromator [Ge(111) or Ge(220)], and a platinum-coated Si mirror is used for vertical focusing and as a higher-order cutoff filter. The beamline is equipped with a single-axis goniometer, a CCD detector, and an open-flow cryogenic sample cooler. High-pressure protein crystallography with a diamond anvil cell can also be performed using this beamline.

On the influence of crystal size and wavelength on native SAD phasing.

Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur. However, the absorption edge of sulfur is at low energy (2.472 keV = 5.016 Å), which makes it challenging to carry out native SAD phasing experiments as most synchrotron beamlines are optimized for shorter wavelength ranges where the anomalous signal of sulfur is weak; for longer wavelengths, which produce larger anomalous differences, the absorption of X-rays by the sample, solvent, loop and surrounding medium (e.g. air) increases tremendously. Therefore, a compromise has to be found between measuring strong anomalous signal and minimizing absorption. It was thus hypothesized that shorter wavelengths should be used for large crystals and longer wavelengths for small crystals, but no thorough experimental analyses have been reported to date. To study the influence of crystal size and wavelength, native SAD experiments were carried out at different wavelengths (1.9 and 2.7 Šwith a helium cone; 3.0 and 3.3 Šwith a helium chamber) using lysozyme and ferredoxin reductase crystals of various sizes. For the tested crystals, the results suggest that larger sample sizes do not have a detrimental effect on native SAD data and that long wavelengths give a clear advantage with small samples compared with short wavelengths. The resolution dependency of substructure determination was analyzed and showed that high-symmetry crystals with small unit cells require higher resolution for the successful placement of heavy atoms.

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography.

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.

Improvement of an automated protein crystal exchange system PAM for high-throughput data collection.

Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable.

Improvements toward highly accurate diffraction experiments at the macromolecular micro-crystallography beamline BL-17A.

BL-17A is a macromolecular crystallography beamline dedicated to diffraction experiments conducted using micro-crystals and structure determination studies using a lower energy X-ray beam. In these experiments, highly accurate diffraction intensity measurements are definitively important. Since this beamline was constructed, the beamline apparatus has been improved in several ways to enable the collection of accurate diffraction data. The stability of the beam intensities at the sample position was recently improved by modifying the monochromator. The diffractometer has also been improved. A new detector table was installed to prevent distortions in the diffractometer's base during the repositioning of the diffractometer detector. A new pinhole system and an on-axis viewing system were installed to improve the X-ray beam profile at the sample position and the centering of tiny crystal samples.

New methodologies at PF AR-NW12A: the implementation of high-pressure macromolecular crystallography.

The macromolecular crystallography (MX) beamline AR-NW12A is evolving from its original design of high-throughput crystallography to a multi-purpose end-station. Among the various options to be implemented, great efforts were made in making available high-pressure MX (HPMX) at the beamline. High-pressure molecular biophysics is a developing field that attracts the interest of a constantly growing scientific community. A plethora of activities can benefit from high pressure, and investigations have been performed on its applicability to study multimeric complex assemblies, compressibility of proteins and their crystals, macromolecules originating from extremophiles, or even the trapping of higher-energy conformers for molecules of biological interest. Recent studies using HPMX showed structural hydrostatic-pressure-induced changes in proteins. The conformational modifications could explain the enzymatic mechanism differences between proteins of the same family, living at different environmental pressures, as well as the initial steps in the pressure-denaturation process that have been attributed to water penetration into the protein interior. To facilitate further HPMX, while allowing access to various individualized set-ups and experiments, the AR-NW12A sample environment has been revisited. Altogether, the newly added implementations will bring a fresh breath of life to AR-NW12A and allow the MX community to experiment in a larger set of fields related to structural biology.

S-SAD phasing study of death receptor 6 and its solution conformation revealed by SAXS.

A subset of tumour necrosis factor receptor (TNFR) superfamily members contain death domains in their cytoplasmic tails. Death receptor 6 (DR6) is one such member and can trigger apoptosis upon the binding of a ligand by its cysteine-rich domains (CRDs). The crystal structure of the ectodomain (amino acids 1-348) of human death receptor 6 (DR6) encompassing the CRD region was phased using the anomalous signal from S atoms. In order to explore the feasibility of S-SAD phasing at longer wavelengths (beyond 2.5 Å), a comparative study was performed on data collected at wavelengths of 2.0 and 2.7 Å. In spite of sub-optimal experimental conditions, the 2.7 Å wavelength used for data collection showed potential for S-SAD phasing. The results showed that the R(ano)/R(p.i.m.) ratio is a good indicator for monitoring the anomalous data quality when the anomalous signal is relatively strong, while d''/sig(d'') calculated by SHELXC is a more sensitive and stable indicator applicable for grading a wider range of anomalous data qualities. The use of the `parameter-space screening method' for S-SAD phasing resulted in solutions for data sets that failed during manual attempts. SAXS measurements on the ectodomain suggested that a dimer defines the minimal physical unit of an unliganded DR6 molecule in solution.

UV LED lighting for automated crystal centring.

A direct outcome of the exponential growth of macromolecular crystallography is the continuously increasing demand for synchrotron beam time, both from academic and industrial users. As more and more projects entail screening a profusion of sample crystals, fully automated procedures at every level of the experiments are being implemented at all synchrotron facilities. One of the major obstacles to achieving such automation lies in the sample recognition and centring in the X-ray beam. The capacity of UV light to specifically react with aromatic residues present in proteins or with DNA base pairs is at the basis of UV-assisted crystal centring. Although very efficient, a well known side effect of illuminating biological samples with strong UV sources is the damage induced on the irradiated samples. In the present study the effectiveness of a softer UV light for crystal centring by taking advantage of low-power light-emitting diode (LED) sources has been investigated. The use of UV LEDs represents a low-cost solution for crystal centring with high specificity.

Crystallization of small proteins assisted by green fluorescent protein.

The generation of crystal lattice contacts by proteinaceous tags fused to target proteins is an attractive approach to aid in the crystallization of otherwise intractable proteins. Here, the use of green fluorescent protein (GFP) fusions for this purpose is demonstrated, using ubiquitin and the ubiquitin-binding motif (UBM) of Y-family polymerase ι as examples. The structure of the GFP-ubiquitin fusion protein revealed that the crystal lattice was formed by GFP moieties. Ubiquitin was accommodated in the lattice through interactions with the peripheral loops of GFP. However, in the GFP-UBM fusion crystal UBM formed extensive interactions with GFP and these interactions, together with UBM dimerization, mediated the crystal packing. Interestingly, the tyrosine residues that are involved in mediating crystal contacts in both GFP-ubiquitin and GFP-UBM crystals are arranged in a belt on the surface of the ß-barrel structure of GFP. Therefore, it is likely that GFP can assist in the crystallization of small proteins and of protein domains in general.

[Advancement of synchrotron radiation protein crystallography in the targeted protein research program: beamline developments at the photon factory].

The Targeted Protein Research Program (TPRP) started in 2007 as a sequel of the Protein 3000 Project which lasted from 2002 to 2007. In the new project, four cores, Protein Production, Structure Analysis, Control of Protein Functions with Compounds, and Informatics, have been established as focus of methodology developments critical for functional and structural studies by the target protein research teams. Within the "Analysis Core" synchrotron radiation plays a pivotal role providing X-ray beams for structural analyses of the target proteins. The two large Japanese synchrotron radiation facilities, SPring-8 and Photon Factory (PF), along with three protein crystallography groups from Hokkaido, Kyoto and Osaka Universities have teamed up to develop two complementary micro-beam beamlines, one on each synchrotron site, and associated technologies for cutting edge structural biology research. At the PF, there are 5 operational beamlines which are equipped with state-of-the-art instrumentation for high-throughput protein crystallography experiments. Within the TPRP framework, the PF is developing a micro-focus beamline optimized for a lower energy single anomalous diffraction (SAD) experiment. This will be particularly useful for structure determination of difficult protein targets for which heavy atom derivatives or selenomethionine substitution does not work and other standard phasing methods fail to give structure solutions. This will augment the capabilities of the PF structural biology beamlines with similar look-and-feel experimental environments.