Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

Anomalous diffraction of matter waves with minimal quantum metasurfaces

In the last few years, there is a huge upsurge in the number of closed deals regarding quantum technologies for materials, computing, communication and instrumentation. Such a trend has inevitably affected the research funding market; thus, large state initiatives are taken that are directly expected to drive the formulation of novel research concepts and the development of quantum device prototypes from sensors and circuitry to quantum memory and repeaters. A fundamental operation behind all these applications is the effective steering of electrons, constituting matter waves, along specific directions and with certain magnitudes, due to development of various reflective and refractive orders. The objective of this study is to optimize the simplest structure that supports such anomalous diffraction, namely a quantum metasurface comprising cylindrical rods embedded in suitable crystalline matter. Several highly-performing designs from these minimal setups are proven to work exceptionally as multiport components, employable to a variety of quantum engineering implementations.

Cation distribution in Cu2ZnSnSe4, Cu2FeSnS4 and Cu2ZnSiSe4 by multiple-edge anomalous diffraction

Multiple-Edge Anomalous Diffraction (MEAD) has been applied to various quaternary sulfosalts belonging to the adamantine compound family in order to validate the distribution of copper, zinc and iron cations in the structure. Semiconductors from this group of materials are promising candidates for photovoltaic applications. Their properties strongly depend on point defects, in particular related to cation order–disorder. However, Cu+, Zn2+ and Fe2+ have very similar scattering factors and are all but indistinguishable in usual X-ray diffraction experiments. Anomalous diffraction utilizes the dependency of the atomic scattering factors f′ and f′′ of the energy of the radiation, especially close to the element-specific absorption edges. In the MEAD technique, individual Bragg peaks are tracked over an absorption edge. The intensity changes depending on the structure factor can be highly characteristic for Miller indices selected for a specific structural problem, but require very exact measurements. Beamline KMC-2 at synchrotron BESSY II, Berlin, has been recently upgraded for this technique. Anomalous X-ray powder diffraction and XAFS compliment the data. Application of this technique confirmed established cation distribution in Cu2ZnSnSe4 (CZTSe) and Cu2FeSnS4 (CFTS). In contrast to the literature, cation distribution in Cu2ZnSiSe4 (CZSiSe) is shown to adopt a highly ordered wurtz-kesterite structure type.

IPCAS: a direct-method-based pipeline from phasing to model building and refinement for macromolecular structure determination

A new version (2.0) of the pipeline IPCAS (Iterative Protein Crystal structure Automatic Solution) has been released, in which the program OASIS performs direct-method single-wavelength anomalous diffraction/single isomorphous replacement phasing and direct-method-aided partial-structure extension. IPCAS incorporates the widely used packages CCP4 and PHENIX for locating heavy atoms, density modification, molecular replacement, model building and refinement. Important extensions to the previous version of IPCAS include a resolution screening method for non-crystallographic symmetry searching, an alternate model-building protocol for avoiding premature convergence and direct-method image processing for electron microscopy maps, including single-particle cryo-EM maps. Moreover, a new graphical user interface is provided for controlling and real-time monitoring of the whole dual-space iterative process, which works as a plugin to CCP4i. Applications of the new IPCAS to difficult cases have yielded promising results, including `direct-method phasing and fragment extension' from weak anomalous diffraction signal data and `direct-method-aided partial-structure extension' from low-homology models.

De novo Phasing Xenons Observed in the Frog Ependymin-Related Protein

Pressurizing Xe or Kr noble gas into the protein crystal for de novo phasing has been one method of choice when the introduction of other heavy-atom compounds fails. One reason is because, unlike other heavy-atom compounds, their immobilized sites are mostly hydrophobic cavities. Previously, the structure of frog ependymin-related protein (EPDR) has been determined using a single wavelength anomalous diffraction (SAD) on a Xe-pressurized crystal. Since no report on the four Xe binding sites has been made, these sites are analyzed in this study. Of the four Xe atoms, three are found along the hydrophobic interfaces created by the two crystallographic symmetry mates of EPDR. One final Xe atom occupies a Ca2+-binding site of the native protein entirely stabilized by the polar atoms of the surrounding EDPR residues. We believe that this atypical Xe location is very unique and merits further study.

Bayesian machine learning improves single-wavelength anomalous diffraction phasing

Single-wavelength X-ray anomalous diffraction (SAD) is a frequently employed technique to solve the phase problem in X-ray crystallography. The precision and accuracy of recovered anomalous differences are crucial for determining the correct phases. Continuous rotation (CR) and inverse-beam geometry (IBG) anomalous data collection methods have been performed on tetragonal lysozyme and monoclinic survivin crystals and analysis carried out of how correlated the pairs of Friedel's reflections are after scaling. A multivariate Bayesian model for estimating anomalous differences was tested, which takes into account the correlation between pairs of intensity observations and incorporates the a priori knowledge about the positivity of intensity. The CR and IBG data collection methods resulted in positive correlation between I(+) and I(−) observations, indicating that the anomalous difference dominates between these observations, rather than different levels of radiation damage. An alternative pairing method based on near simultaneously observed Bijvoet's pairs displayed lower correlation and it was unsuccessful for recovering useful anomalous differences when using the multivariate Bayesian model. In contrast, multivariate Bayesian treatment of Friedel's pairs improved the initial phasing of the two tested crystal systems and the two data collection methods.