New measures of anisotropy of cryo-EM maps.

We propose two new measures of resolution anisotropy for cryogenic electron microscopy maps: Fourier shell occupancy (FSO), and the Bingham test (BT). FSO varies from 1 to 0, with 1 representing perfect isotropy, and lower values indicating increasing anisotropy. The threshold FSO = 0.5 occurs at Fourier shell correlation resolution. BT is a hypothesis test that complements the FSO to ensure the existence of anisotropy. FSO and BT allow visualization of resolution anisotropy. We illustrate their use with different experimental cryogenic electron microscopy maps.

Emerging Themes in CryoEM─Single Particle Analysis Image Processing.

Cryo-electron microscopy (CryoEM) has become a vital technique in structural biology. It is an interdisciplinary field that takes advantage of advances in biochemistry, physics, and image processing, among other disciplines. Innovations in these three basic pillars have contributed to the boosting of CryoEM in the past decade. This work reviews the main contributions in image processing to the current reconstruction workflow of single particle analysis (SPA) by CryoEM. Our review emphasizes the time evolution of the algorithms across the different steps of the workflow differentiating between two groups of approaches: analytical methods and deep learning algorithms. We present an analysis of the current state of the art. Finally, we discuss the emerging problems and challenges still to be addressed in the evolution of CryoEM image processing methods in SPA.

Customized retarders based on waveplates.

Phase control is a critical parameter in polarization measurements. It is well known that a proper combination of wave plates allows to obtain achromatic phase shift, i.e., a constant retardation in certain spectral ranges. This paper is focused on a different, but more useful, goal, as it is to achieve customized variable retarders in broad spectral ranges. To do that, a merit function was used to measure the similarity between the overall phase shift of the wave plate combinations and the desired target. The control variables are the thicknesses and orientations of the wave plates. All possible combinations with four and five wave plates of quartz and MgF2 were analyzed, but our approach can be perfectly extended to deal with more wave plates. The result of an optimization process determines the thicknesses and orientations of the wave plates, which results in the closest retarder to the desired one. Numerical results show deviations below 10% between the target and the obtained retardation. These systems are of special interest in those fields and instruments in which polarization control plays a fundamental role.

Protective Strategies of Haberlea rhodopensis for Acquisition of Freezing Tolerance: Interaction between Dehydration and Low Temperature.

Resurrection plants are able to deal with complete dehydration of their leaves and then recover normal metabolic activity after rehydration. Only a few resurrection species are exposed to freezing temperatures in their natural environments, making them interesting models to study the key metabolic adjustments of freezing tolerances. Here, we investigate the effect of cold and freezing temperatures on physiological and biochemical changes in the leaves of Haberlea rhodopensis under natural and controlled environmental conditions. Our data shows that leaf water content affects its thermodynamical properties during vitrification under low temperatures. The changes in membrane lipid composition, accumulation of sugars, and synthesis of stress-induced proteins were significantly activated during the adaptation of H. rhodopensis to both cold and freezing temperatures. In particular, the freezing tolerance of H. rhodopensis relies on a sucrose/hexoses ratio in favor of hexoses during cold acclimation, while there is a shift in favor of sucrose upon exposure to freezing temperatures, especially evident when leaf desiccation is relevant. This pattern was paralleled by an elevated ratio of unsaturated/saturated fatty acids and significant quantitative and compositional changes in stress-induced proteins, namely dehydrins and early light-induced proteins (ELIPs). Taken together, our data indicate that common responses of H. rhodopensis plants to low temperature and desiccation involve the accumulation of sugars and upregulation of dehydrins/ELIP protein expression. Further studies on the molecular mechanisms underlying freezing tolerance (genes and genetic regulatory mechanisms) may help breeders to improve the resistance of crop plants.

Automatic local resolution-based sharpening of cryo-EM maps.

MOTIVATION: Recent technological advances and computational developments have allowed the reconstruction of Cryo-Electron Microscopy (cryo-EM) maps at near-atomic resolution. On a typical workflow and once the cryo-EM map has been calculated, a sharpening process is usually performed to enhance map visualization, a step that has proven very important in the key task of structural modeling. However, sharpening approaches, in general, neglects the local quality of the map, which is clearly suboptimal. RESULTS: Here, a new method for local sharpening of cryo-EM density maps is proposed. The algorithm, named LocalDeblur, is based on a local resolution-guided Wiener restoration approach of the original map. The method is fully automatic and, from the user point of view, virtually parameter-free, without requiring either a starting model or introducing any additional structure factor correction or boosting. Results clearly show a significant impact on map interpretability, greatly helping modeling. In particular, this local sharpening approach is especially suitable for maps that present a broad resolution range, as is often the case for membrane proteins or macromolecules with high flexibility, all of them otherwise very suitable and interesting specimens for cryo-EM. To our knowledge, and leaving out the use of local filters, it represents the first application of local resolution in cryo-EM sharpening. AVAILABILITY AND IMPLEMENTATION: The source code (LocalDeblur) can be found at https://github.com/I2PC/xmipp and can be run using Scipion (http://scipion.cnb.csic.es) (release numbers greater than or equal 1.2.1). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Validation of electron microscopy initial models via small angle X-ray scattering curves.

MOTIVATION: Cryo electron microscopy (EM) is currently one of the main tools to reveal the structural information of biological macromolecules. The re-construction of three-dimensional (3D) maps is typically carried out following an iterative process that requires an initial estimation of the 3D map to be refined in subsequent steps. Therefore, its determination is key in the quality of the final results, and there are cases in which it is still an open issue in single particle analysis (SPA). Small angle X-ray scattering (SAXS) is a well-known technique applied to structural biology. It is useful from small nanostructures up to macromolecular ensembles for its ability to obtain low resolution information of the biological sample measuring its X-ray scattering curve. These curves, together with further analysis, are able to yield information on the sizes, shapes and structures of the analyzed particles. RESULTS: In this paper, we show how the low resolution structural information revealed by SAXS is very useful for the validation of EM initial 3D models in SPA, helping the following refinement process to obtain more accurate 3D structures. For this purpose, we approximate the initial map by pseudo-atoms and predict the SAXS curve expected for this pseudo-atomic structure. The match between the predicted and experimental SAXS curves is considered as a good sign of the correctness of the EM initial map. AVAILABILITY AND IMPLEMENTATION: The algorithm is freely available as part of the Scipion 1.2 software at http://scipion.i2pc.es/.

Thermostructural Behavior in a Series of Lanthanide-Containing Polyoxotungstate Hybrids with Copper(II) Complexes of the Tetraazamacrocycle Cyclam: A Single-Crystal-to-Single-Crystal Transformation Study.

A series of 14 isostructural [Cu(cyclam)]2[{Cu(cyclam)}4{(α-GeW11O39)Ln(H2O)(OAc)}2]·18H2O (1-Ln, where Ln = La-Lu; cyclam = 1,4,8,11-tetraazacyclotetradecane) polyoxometalate-based hybrids reported herein represent (i) the first example of a two-dimensional covalent hybrid lattice involving the [{(α-XW11O39)Ln(H2O)(OAc)}2] n- archetype and (ii) the first structural characterization of such a dimeric polyoxotungste for Ln = La and Pr as well as for the combination of X = Ge and Ln = Ce, Nd, Sm, or Lu. Compounds 1-Ln have been characterized by elemental analyses, infrared spectroscopy, and thermogravimetric analysis, and their thermostructural behavior has been monitored by powder and single-crystal X-ray diffraction. The title compounds undergo two single-crystal-to-single-crystal transformations triggered by thermal dehydration leading to the [{Cu(cyclam)}6{(α-GeW11O39)Ln(H2O)(OAc)}2]·4H2O intermediate (2-Ln, where Ln = Eu or Er) and [Cu(cyclam)]0.5[{Cu(cyclam)}5.5{(α-GeW11O39)Ln(OAc)}2] (3-Ln, where Ln = Ce or Eu) final anhydrous phases, the latter evidencing a coordinatively unsaturated derivative of the dimeric archetype for the first time. These transitions involve formation and disruption of Cu-OPOM bonds that result in different {Cu(cyclam)}2+ moieties grafting onto and being released from Keggin surfaces, which reduces the dimensionality of 1-Ln to one-dimensional covalent assemblies for 2-Ln and 3-Ln. While all 3-Ln phases rehydrate fully upon exposure to air for 24 h, the kinetics governing the crystal transitions back toward 1-Ln through 2-Ln depend on the nature of Ln. Under ambient moisture, the anhydrous structures fully revert back to the parent framework for Ln = La-Sm, while the samples containing Eu to Lu afford mixtures of 1-Ln and 2-Ln and require immersion in water for the structural reversion to reach completion. Single-crystal X-ray diffraction analyses of the rehydrated 1R-Ln samples (Ln = Ce, Eu, and Er) support these observations.

Metal Nanoparticles Embedded in Cellulose Nanocrystal Based Films: Material Properties and Post-use Analysis.

The dispersion of nanoparticles having different size-, shape-, and composition-dependent properties is an exciting approach to design and synthesize multifunctional materials and devices. This work shows a detailed investigation of the preparation and properties of free-standing nanocomposite films based on cellulose nanocrystals (CNC) loaded with three different types of metal nanoparticles. CNC-based nanocomposites having zinc oxide (ZnO), titanium dioxide (TiO2), and silver oxide (Ag2O) have been obtained through evaporation-induced self-assembly (EISA) in acqueous solution. Morphological and optical characteristics, chemical properties, wettability, and antimicrobial assays of the produced films were conducted. Furthermore, disintegrability in composting condition of CNC based nanocomposites was here investigated for the first time. The morphological observations revealed the formation of a chiral nematic structure with uniformly distributed nanoparticles. The bionanocomposite films based on the metal nanoparticles had effective antimicrobial activity, killing both Escherichia coli RB ( E. coli RB) and Staphylococcus aureus 8325-4 ( S. aureus 8325-4). The simplicity method of film preparation, the large quantity of cellulose in the world, and the free-standing nature of the nanocomposite films offer highly advantageous characteristics that can for the new development of multifunctional materials.

Thermally-Triggered Crystal Dynamics and Permanent Porosity in the First Heptatungstate-Metalorganic Three-Dimensional Hybrid Framework.

The hybrid compound [{Cu(cyclam)}3 (W7 O24 )]⋅15.5 H2 O (1) (cyclam=1,4,8,11-tetraaza-cyclotetradecane) was synthesized by reacting the {Cu(cyclam)}2+ complex with a tungstate source in water at pH 8. Compound 1 exhibits an unprecedented three-dimensional covalent structure built of heptatungstate clusters linked through metalorganic complexes in a POMOF-like framework that displays water-filled channels. This dynamic architecture undergoes two sequential single-crystal-to-single-crystal transformations upon thermal evacuation of water molecules to result in the partially dehydrated [{Cu(cyclam)}3 (W7 O24 )]⋅12 H2 O (2) and anhydrous [Cu(cyclam)]0.5 [{Cu(cyclam)}2.5 (W7 O24 )] (3) crystalline phases. These transitions are associated with cluster rotations and modifications in the CuII coordination geometries, which reduce the dimensionality of the original lattice to layered systems but preserving the porous nature. Phase 3 reverts to 2 upon exposure to ambient moisture, whereas the transition between 1 and 2 proved to be irreversible. The permanent microporosity of 3 was confirmed by gas sorption measurements (N2 , CO2 ), which reveal a system of parallel channels made of wide cavities connected through narrow necks that limit the adsorption process. This observation is in good agreement with Grand Canonical Monte Carlo simulations.

A Robust Open Framework Formed by Decavanadate Clusters and Copper(II) Complexes of Macrocyclic Polyamines: Permanent Microporosity and Catalytic Oxidation of Cycloalkanes.

The first decavanadate-based microporous hybrid, namely, [Cu(cyclam)][{Cu(cyclam)}2(V10O28)]·10H2O (1, cyclam = 1,4,8,11-tetraazacyclotetradecane) was prepared by reaction of (VO3)(-) anions and {Cu(cyclam)}(2+) complexes in NaCl (aq) at pH 4.6-4.7 and characterized by elemental analyses, thermogravimetry, and X-ray diffraction (powder, single-crystal) techniques. Compound 1 exhibits a POMOF-like supramolecular open-framework built of covalent decavanadate/metalorganic layers with square-like voids, the stacking of which is aided by interlamellar cementing complexes and generates water-filled channels with approximate cross sections of 10.4 × 8.8 Å(2). The framework is robust enough to remain virtually unaltered upon thermal evacuation of all water molecules of hydration, as demonstrated through single-crystal X-ray diffraction studies on the anhydrous phase 1a. This permanent microporosity renders interesting functionality to 1, such as selective adsorption of CO2 over N2 and remarkable activity as heterogeneous catalyst toward the H2O2-based oxidation of the highly-stable, tricyclic alkane adamantane.

Temperature dependence of birefringence in ethanol-filled suspended core fiber.

The temperature dependence of the birefringence in a suspended core fiber (SCF) has been experimentally analyzed by using a polarimetric setup. The used configuration consists of two linear polarizers and the SCF as birefringent medium. A theoretical study based on Jones matrices has been carried out to analyze the experimental observed behavior. For this, a polarimetric all-fiber configuration was used whose sensitivity depends on the wavelength variation with temperature. Results obtained show that it is strongly affected when the air holes of SCF are partially filled with ethanol.

Design of superachromatic quarter-wave retarders in a broad spectral range.

A superachromatic quarter-wave retarder using an arbitrary number of waveplates in a broadband spectral range has been proposed. Their design is based on the optimization of a merit function, the achromatism degree (AcD), which represents a global behavior metric for the retardation. By means of this technique, the thickness and azimuth of each waveplate is determined. The achromatism degree is a measure of the distance between the overall retardation and a target retardation weighted by the spectrum of the incident light. We report on a particular case where all waveplates are made of quartz. As application examples, the design of a quarter-wave retarder using two, three, and four waveplates in the spectral ranges of 500-700 nm and 400-1000 nm was studied. The numerical results show that for these ranges, the best designs obtained present a maximum difference of 0.013° and 0.010° with respect to the target retardation, respectively. In addition, an analysis of their achromatic stability is presented. These results can be applied in the aerospace industry, spectroscopic ellipsometry, and spectrogoniometry, among others.

Circularly polarized light with high degree of circularity and low azimuthal error sensitivity.

The generation of circularly polarized light with a high circularity degree and low azimuthal error sensitivity was analyzed using a system composed by two waveplates. It is shown how the high circularity degree is achieved using a combination of a half- (λ/2) and a quarter- (λ/4) waveplate λ/2+λ/4 configuration. However, the lowest azimuthal sensitivity under small variations in the azimuths of the waveplates is obtained by employing a λ/4+λ/2 configuration. Analytical calculus particularized for quartz and MgF2 waveplates is presented.

Evidence for the absence of enzymatic reactions in the glassy state. A case study of xanthophyll cycle pigments in the desiccation-tolerant moss Syntrichia ruralis.

Desiccation-tolerant plants are able to withstand dehydration and resume normal metabolic functions upon rehydration. These plants can be dehydrated until their cytoplasm enters a 'glassy state' in which molecular mobility is severely reduced. In desiccation-tolerant seeds, longevity can be enhanced by drying and lowering storage temperature. In these conditions, they still deteriorate slowly, but it is not known if deteriorative processes include enzyme activity. The storage stability of photosynthetic organisms is less studied, and no reports are available on the glassy state in photosynthetic tissues. Here, the desiccation-tolerant moss Syntrichia ruralis was dehydrated at either 75% or <5% relative humidity, resulting in slow (SD) or rapid desiccation (RD), respectively, and different residual water content of the desiccated tissues. The molecular mobility within dry mosses was assessed through dynamic mechanical thermal analysis, showing that at room temperature only rapidly desiccated samples entered the glassy state, whereas slowly desiccated samples were in a 'rubbery' state. Violaxanthin cycle activity, accumulation of plastoglobules, and reorganization of thylakoids were observed upon SD, but not upon RD. Violaxanthin cycle activity critically depends on the activity of violaxanthin de-epoxidase (VDE). Hence, it is proposed that enzymatic activity occurred in the rubbery state (after SD), and that in the glassy state (after RD) no VDE activity was possible. Furthermore, evidence is provided that zeaxanthin has some role in recovery apparently independent of its role in non-photochemical quenching of chlorophyll fluorescence.

Optimal achromatic wave retarders using two birefringent wave plates.

Two plates of different birefringence material can be combined to obtain an achromatic wave retarder. In this work, we achieve a correction for the overall retardation of the system that extends the relation to any azimuth. Current techniques for the design of achromatic wave retarders do not present a parameter that characterizes its achromatism on a range of wavelengths. Thus, an achromatic degree has been introduced, in order to determine the optimal achromatic design composed with retarder plates for a spectrum of incident light. In particular, we have optimized a quarter retarder using two wave plates for the visible spectrum. Our technique has been compared to previous results, showing significant improvement.

Optimal achromatic wave retarders using two birefringent wave plates: reply.

This reply attempts to cast some more light on the achromatic systems composed by wave plates, in particular to the calculus of the overall retardation and the use of the Jones matrix equivalence theorem. An equivalent expression for the overall retardation of the system in terms of the trace is also given.

Scipion3: A workflow engine for cryo-electron microscopy image processing and structural biology.

Image-processing pipelines require the design of complex workflows combining many different steps that bring the raw acquired data to a final result with biological meaning. In the image-processing domain of cryo-electron microscopy single-particle analysis (cryo-EM SPA), hundreds of steps must be performed to obtain the three-dimensional structure of a biological macromolecule by integrating data spread over thousands of micrographs containing millions of copies of allegedly the same macromolecule. The execution of such complicated workflows demands a specific tool to keep track of all these steps performed. Additionally, due to the extremely low signal-to-noise ratio (SNR), the estimation of any image parameter is heavily affected by noise resulting in a significant fraction of incorrect estimates. Although low SNR and processing millions of images by hundreds of sequential steps requiring substantial computational resources are specific to cryo-EM, these characteristics may be shared by other biological imaging domains. Here, we present Scipion, a Python generic open-source workflow engine specifically adapted for image processing. Its main characteristics are: (a) interoperability, (b) smart object model, (c) gluing operations, (d) comparison operations, (e) wide set of domain-specific operations, (f) execution in streaming, (g) smooth integration in high-performance computing environments, (h) execution with and without graphical capabilities, (i) flexible visualization, (j) user authentication and private access to private data, (k) scripting capabilities, (l) high performance, (m) traceability, (n) reproducibility, (o) self-reporting, (p) reusability, (q) extensibility, (r) software updates, and (s) non-restrictive software licensing.

Reversible First-Order Single Crystal to Single Crystal Thermal Phase Transition in [(CH3)3CNH3]4[V4O12].

The well-known compound tetrakis(tert-butylammonium)-cyclo-tetrametavanadate (V), [(CH3)3CNH3]4[V4O12] (1h_RT), which crystallizes in the tetragonal I4/m space group, undergoes an irreversible solid state transformation upon heating, constituting one of the few examples in which the initial and the final stages are structurally characterized by sc-XRD. Now, we observed the ability of the same compound to undergo an additional single-crystal-to-single-crystal (SCSC) transformation upon thermal stimuli, but this time at low temperatures (153 K). Compound 1h_RT contains a discrete unprotonated [V4O12]4- tetrahedral anion in which V and O bridging atoms are coplanar. In both phases, these tetrameric anions are linked through tert-butylammonium cations in an extensive network of hydrogen bonds, but at low temperatures, this phase loses its characteristic O-V-O coplanarity, with the resulting rearrangement of the crystal packing and hydrogen-bond network which provide its reversibility at low temperatures. Again, the initial and final stages have been characterized structurally by sc-XRD.

Measuring local-directional resolution and local anisotropy in cryo-EM maps.

The introduction of local resolution has enormously helped the understanding of cryo-EM maps. Still, for any given pixel it is a global, aggregated value, that makes impossible the individual analysis of the contribution of the different projection directions. We introduce MonoDir, a fully automatic, parameter-free method that, starting only from the final cryo-EM map, decomposes local resolution into the different projection directions, providing a detailed level of analysis of the final map. Many applications of directional local resolution are possible, and we concentrate here on map quality and validation.