Diffracted X-ray Tracking Research Articles

Time-resolved local dynamics measurement of supersaturated solution by diffracted X-ray tracking

Time-resolved local dynamics measurement of supersaturated solution by diffracted X-ray tracking

Characterization of group II chaperonins from an acidothermophilic archaeon Picrophilustorridus.

Chaperonins are a type of molecular chaperone that assist in the folding of proteins. Group II chaperonins play an important role in the proteostasis in the cytosol of archaea and eukarya. In this study, we expressed, purified, and characterized group II chaperonins from an acidothermophilic archaeon Picrophilus torridus. Two genes exist for group II chaperonins, and both of the gene products assemble to form double‐ring complexes similar to other archaeal group II chaperonins. One of the Picrophilus chaperonins, PtoCPNα, was able to refold denatured GFP at 50 °C. As expected, PtoCPNα exhibited an ATP‐dependent conformational change that is observed by the change in fluorescence and diffracted X‐ray tracking (DXT). In contrast, PtoCPNα lost its protein folding ability at moderate temperatures, becoming unable to interact with unfolded proteins. At lower temperatures, the release rate of the captured GFP from PtoCPNα was accelerated, and the affinity of denatured protein to PtoCPNα was weakened at the lower temperatures. Unexpectedly, in the DXT experiment, the fine motions were enhanced at the lower temperatures. Taken together, the results suggest that the fine tilting motions of the apical domain might correlate with the affinity of group II chaperonins for denatured proteins.

Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl

Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.

X-Ray Digital Aggregated Dynamics of Intrinsically Disordered Proteins

Parkinson's Disease (PD) and Alzheimer Disease (AD) is one of the neurodegenerative pathology. These diseases are occurred by anomalous aggregations of causative proteins.Alpha-synuclein (Syn) is PD's causative proteins and Tau is AD's ones. But these protein's aggregation process haven’t been clear. Additionally, these proteins are one of the famous intrinsically disorder proteins (IDP). Until now, it is difficult to understand aggregation process from static structural information of IDP. Therefore, in this study, we observed that characteristic of single molecule structural fluctuations which occurred anomalous aggregation. Furthermore, we observed dynamical digital aggregation process (from monomer to dimer or trimer). In order to understand single molecular dynamics which occurred aggregation, we measured single molecule structural fluctuations of Syn's wild type (WT) and mutants (E46K, A53T). And, we observed digital aggregations of Tau proteins for the purpose of detecting dynamical process. At these experiments, we used Diffracted X-ray Tracking (DXT). In order to measure movements of the specific binding sites of proteins, DXT monitor X-ray diffraction spots from labeled gold nanocrystal. DXT experiments used the energy of quasi-white x-rays (energy peak-width of 2%, 10-20 keV, BL40XU, SPring-8), for that reason DXT has high time-resolution (0.1ms/frame) and high super-precision (0.1nm scale). As a result from Syn's DXT data, The motion's histograms in WT has simple single Gaussian distribution. However, these in other Syn's mutants which occurred anomalous aggregation have two Gaussian distributions. From DXT results, we confirmed that mutant's structural fluctuation is more rigid than that of WT. In the other hand, Tau dimer is very more flexible than that of monomer and trimer. The motion histograms show that there are very different process between dimerization and trimerization in the digital aggregation observations of Tau proteins.

Catalyzing of Immune Peptide/MHC Class II Complex with DM Molecule Observed by Diffracted X-ray Tracking (DXT)

Major Histocompatibility complex (MHC) Class II Molecule has important role on activation of the immune system. Antigen presenting cells engulf extracellular molecules by endocytosis, then the molecules are decomposed into small peptides. MHC class II molecule binds a one of small peptides, then be transported onto the cell surface and present the peptide to T cell receptor to activate the immune system. While MHC class II molecule binds a peptide, it is said that DM molecule which is homolog of MHC class II molecule catalyzes and controls the peptide binding process of MHC class II molecule. But, the detailed information about the control has not been revealed obviously yet.In order to elucidate the mechanism of DM molecule's catalyzation, we performed Diffracted X-ray Tracking (DXT) to observe motions of peptides bound onto MHC class II molecule with or without DM molecule effects. In DXT, we immobilize proteins on the prepared surface and attach a single gold nanocrystal on a single protein on the surface by using thiol-specific binding. Then, by irradiating pulse x-rays from synchrotron sources, x-rays are diffracted as time-resolvable diffraction spots by gold nanocrystals on proteins. By tracking diffracted spots, we obtain two-dimensional motions of single gold nanocrystal which is related to single molecule's ones.We observed motions of two types of peptides whose length are different from each other (14 residues and 9 residues) to observe DM molecule's dependency on a peptide length. Furthermore, we also observed other two peptides whose sequences are slightly modified in order to change a location of labeled nanocrystals. In the poster session, we are going to discuss results from these experiments and MD simulations we are performing.

Realtime Single Molecular Motion Analysis of Nicotinic Acetylcholine Receptor Alpha 7 by Diffracted X-Ray Tracking Method

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel. Binding of ligands (e.g. acetylcholine, nicotine) to nAChR induces ion flux through the channel formed by pentameric subunits, which leads to the changes of cell excitability, and then triggers the following signaling cascades in the peripheral and the central nervous system. In the previous study we applied Diffracted X-ray Tracking (DXT) method to acetylcholine binding protein (AChBP) and Torpedo nAChR. DXT is a method to track the X-ray diffraction spots from the gold nanocrystal labeled on an individual single protein in real time and real space. We observed molecular fluctuations of the proteins even without ligand, and enhancement of tilting and twisting motions with ligand [Sekiguchi, et al. (2014) Sci. Rep. 4: 6384].Here, to acquire more precise information on molecular motions of nAChR in the three sub-states (close, open, and desensitization) and their sate-to-state transitions, we applied the DXT for the neuronal nAChR alpha 7. The alpha 7 receptor in the central nervous system plays essential roles in the cholinergic neuro-signal transduction, and it is one of the drug targets for therapeutics of Alzheimer diseases and cognitive impairment associated with schizophrenia (CIAS). Alpha 7 receptor was recombinantly prepared from the cDNA expressed in Xenopus oocytes. To orderly configure the receptors defined tags were introduced at the defined positions; e.g. “Met tag” for labeling with gold nanocrystal in the N-terminal, and “His tag” for absorption on mica substrate in the second cytoplasmic loop of the receptor. Characteristic molecular motion(s) during the three states of the nAChR alpha 7 were revealed by the DXT analysis. Possible transition modeling will be discussed.

X-ray Observation of Novel Nucleation Factor in Protein Supersaturated Solution

Recently, Protein solution and the micro scale structure are receiving a lot of attention in diverse protein study. Protein crystal observation In particular, protein aggregation process is a typical micro scale phenomenon of protein solution. Moreover, it is a one of the unclear mechanism of protein characteristics.In this study, we approached to observe the time resolved micro scale dynamics of protein solution by Diffracted X-ray Tracking (DXT) as known as single molecule measurement method. This method is able to direct observation of local solution dynamics for Target solution by detecting angular rotational displacement of a coexisted and dispersed single gold nanocrystal (approximately 100 nm) with pico-meter scale positional accuracy and micro-second time resolution. For target sample, we choose a crystal precursor metastable state of lysozyme solution.From detailed DXT analysis, we observed as crystal precursor state solution of lysozyme are containing slow and fast dynamics and the fast diffusion corresponded relaxation process of metastable state of nano scale clusters which is a important factor of maintenance of metastable supersaturated condition, driving force of morphology determination of final product from crystallization and nucleation process from supersaturation. From this study, DXT measurement results and detailed analysis process for a protein solution such as crystal precursor metastable state of lysozyme solution are concerned that this technique is a powerful tool for observing nano-scale protein structures and its local dynamics in bulk solution. For next work, we will planning to tackle research in order to observe morphology control factor discovery inhibitation techniques of supersaturated condition, and approaching for elucidation of protein abnormal aggregation process.

Photoresist Micro-Chamber for the Diffracted X-ray Tracking Method Recording Single-Molecule Conformational Changes

The diffracted X-ray tracking method has been established for capturing conformational changes of single KcsA potassium channels, which records position of X-ray diffraction spot from a gold nanocrystal attached to the channel. The conventional observation chamber setup could trace a diffraction spot; however relatively high background noise resulting from X-ray scattering degraded a spatial resolution. This paper report a novel low-noise microfabricated observation chamber consisting of SiN membrane window supported by Si and embedded microchannel in negative photoresist. The measured noise exhibited lower level compared to the conventional chamber, which almost reached the detection limit of the employed imaging system.

Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks.

Supersaturation of a solution system is a metastable state containing more solute than can be normally solubilized. Moreover, this condition is thermodynamically important for a system undergoing a phase transition. This state plays critical roles in deposition morphology in inorganic, organic, polymer and protein solution systems. In particular, microscopic solution states under supersaturated conditions have recently received much attention. In this report, we observed the dynamic motion of individual ion-network domains (INDs) in a supersaturated sodium acetate trihydrate solution (6.4 M) by using microsecond time-resolved and high accuracy (picometre scale) X-ray observations (diffracted X-ray tracking; DXT). We found that there are femto-Newton (fN) anisotropic force fields in INDs that correspond to an Angstrom-scale relaxation process (continuous expansion and compression) of the INDs at 25 μs time scale. The observed anisotropic force-field (femto-Newton) from DXT can lead to new explanations of how material crystallization is triggered. This discovery could also influence the interpretation of supercooling, bio-polymer and protein aggregation processes, and supersaturated systems of many other materials.

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age.

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the 'frozen' crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane.

X‐Ray Single Molecule Tracking Reveal Motions of Peptides in the Binding Groove of MHC II Regulated by DM

Class II MHC can take loosely bound peptides conformations, which generated in the recycling endosomes or cell surface, that are recognized by autoimmune T cells. DM converts loosely bound complexes to tightly bound complexes; crystal structure advocated that DM bound form of MHC II generate novel threshold for peptide binding by creating a hydrogen bond inside the binding groove, that form loosely bound intermediate peptides complex. We used soluble I‐Ak and two peptides that differ in their length as a model system to detect such floating peptides. Fluorescein quenching study provided remarkable difference; fluorescein on the peptides gave about twice a strength signal with DM than without DM, suggests peptides floating half way from MHC II. Diffracted X‐ray tracking (DXT) that monitors real‐time motion of individual peptides/MHC in solution at micro‐second level, revealed that motions of peptides inside binding groove divided into two groups without DM. Addition of DM shifted the slow velocity peak to the fast one. Thus, we successfully detected the DM mediated loosely bound peptides intermediate by two distinct methods. We suggest that measuring the motion of peptide at micro‐second level presents new parameters for both peptide/MHC complex and DM function.

Alpha Synuclein's Anomalous Structural Fluctuations in X-ray Single Molecule Observations

The structures of alpha-synuclein (α-syn) in solutions resemble that of a random coil because α-syn is one of the intrinsically disordered protein (IDP). Alpha-syn is of great interest to Parkinson's researchers because it is a major constituent of Lewy bodies. Here, we observed characteristic of structural fluctuation of wild type (WT), phosphorylated mimic (S129E) and familial mutants (A53T, E46K) by using Diffracted X-ray Tracking (DXT) as x-ray single molecule detection systems. DXT is a method of measuring internal motions of the proteins by using trajectories of labeled gold nanocrystal. In order to measure movements of the specific binding sites of proteins, DXT monitors of X-ray diffraction spots from gold nanocrystal that were labeled in the active sites of the proteins. We measured structural fluctuations of individualα-syn molecules with both high time-resolution (36ms/frame and 0.1ms/frame) and high precision (0.1nm scale). DXT experiments used the energy of quasi-white x-rays (energy peak-width of 2%, 10-20 keV, BL40XU, SPring-8). As a result, we discovered that WT's fluctuation is lower than those of other mutants (S129E, A53T and E46K) from observed dynamical motion's histograms. Furthermore, we found that modes of motions in E46K and A53T have closer to that of S129E than that of WT. From motion's histograms in rotatory direction for the chain axis of α-syn's amino acid, we confirmed that motion's histograms in WT has simple single Gaussian distribution. However, those in other mutants have complex ones. From our DXT results, it is very clear that there is specific different motions between WT of α-syn and other mutants. In addition to, we found that there is different number of X-ray diffraction spots from gold nanocrystal between WT of α-syn and other mutants.

Microsecond X-Ray Dynamics Observation of Nano Supersaturated Protein's Network

Today, comprehension of supersaturated phenomena in protein solution is one of the most important issue. For example, elucidation of amyloid fibril formation process which is known as a main causative agent causing Alzheimer disease[1] or bio-mineralization process of CaCO[2]. In a recent study, molecular dynamics simulation (MD) for crystallization process of CaCO3 was conducted by A.F. Wallace et al[3]. They simulated that the supersaturated solution has a local metastable state as a microscopic ion cluster (we called nano supersaturated network). On the other hand, until now, an experimental detection system for the nano supersaturated network has not been established so far.In our study, we utilized Diffracted X-ray Tracking (DXT) as a high time resolution (25µs) and high positional accuracy (-pm) detection system for the time resolved dynamical interactions between a single gold nanocrystal(-80nm) and localized prenucleation clusters as a nano supersaturated network at SPring-8(BL40XU). For the target samples, we choose two samples that are the sodium acetate trihydrate and lysozyme supersaturated solution as an inorganic and protein solution system, respectively. From DXT result, we succeeded in observing the localized high and low speed motions of gold nanocrystals interacted with nano supersaturated protein's networks. At this presentation, I explain the DXT method and obtained physical parameters of nano supersaturated networks in inorganic and protein supersaturated solution system from DXT analysis. In finally, I present a comprehensive model of nano supersaturated networks.

Single Molecule Motion Map of GLIC by Diffracted X-Ray Tracking

GLIC is a proton-gated bacterial ion channel from Gloeobacter violaceus and is a member of pentameric ligand-gated ion channels. Recently structural information of GLIC in open (acidic pH) and closed state (neutral pH) has become available (1,2), and the gating mechanism is discussed based on the structural information. However the dynamic information of each state in physical point of view is not available. Here we used the diffracted X-ray tracking (DXT) method (3) to detect the motion of the extracellular or transmembrane domain of GLIC. DXT has been considered as a powerful technique in biological science for detecting atomic-scale dynamic motion of the target protein at the single molecular level at several tens of microseconds time resolution. The dynamics of a single protein can be monitored through trajectory of a Laue spot from a nanocrystal which was attached to the target protein immobilized on the substrate surface (4,5).DXT observation was performed at BL40XU in SPring-8 Japan and showed that tilting motion of the transmembrane domain of GLIC and both tilting and twisting motions of the extracellular domain of GLIC were enhanced in open state (acidic pH). The detailed information on dynamic will be discussed.[1] N. Bocquet et al., Nature 457:111 (2009)[2] L. Sauguet et al., PNAS 111:966 (2014)[3] Y.C. Sasaki et al., Phys. Rev. E 62:3843 (2000)[4] H. Sekiguchi et al, PLOS ONE 8:e64176 (2013)[5] H. Sekiguchi et al, Scientific Reports 4:6384 (2014)

3D Dynamical Observations of Single Molecule Motions by X-Rays, Electron and Neutron

We have proposed that single molecule techniques using shorten wavelengths, for example, X-rays, electrons, and neutron [1]. Especially, Diffracted X-Ray Tracking (DXT) using normal synchrotron orbital radiation (SR) source (not XFEL) has been developed for obtaining the information of the 3D internal motions of single proteins with both high time-resolution (micro-seconds) and high precision (nm/1000) [2, 3]. DXT can be monitored through trajectories of the Laue diffraction spots from the nanocrystal which was labeled on the individual proteins. This concept can apply to utilize by using both electrons and neutron. Instead of the Laue diffraction using white X-ray, the Electron Back-Scattered Diffraction Pattern was adopted to monitor the 3D orientations of the nanocrystals linked to the single protein molecules[4]. We called Diffracted Electron Tracking (DET). Additionally, we call Diffracted Neutron Tracking (DNT) for new single molecule measuring method in which the long time observation from the non-destructivity of a neutron is possible.DXT, DET and DNT are assigned to labeling techniques through the nanocrystals. The size effect between intramolecular motions of individual proteins and the labeled nanocrystals becomes very important. We succeeded in the analysis of the quantitive size effects. As a result, we pointed out a possibility that determinations of the intramolecular motions without labeled nanocrystals are carried out quantitatively. Additionally, by progressing of the automatic DXT analysis corresponding to huge diffraction information, we obtained the time-resolved dynamical information that statistical reliability is sufficiently high.[1] Y. C. Sasaki, pp209-234 FUNDAMENTALS OF PICOSCIENCE, CRC Press (2013).[2] H. Sekiguchi et al, PLOS ONE 8:e64176 (2013)[3] H. Sekiguchi et al, Scientific Reports 4:6384 (2014)[4] N. Ogawa et al, Scientific Reports 3:2201 (2013)

放射光X線をプローブとする多量体タンパク質の機能的分子内運動・1分子解析

Diffracted X-ray Tracking (DXT) is one of single molecule techniques for investigating intra-molecule dynamics of functional proteins. In DXT, a nanocrystal is immobilized on a target protein and the trajectory of its diffracted spot is analyzed as the motion of the protein. We advanced the time resolution of this method and refined the analysis procedures in the last 5 years, and the method has been useful especially for cooperative motion analysis in multimeric proteins. In this article, we review the characteristic features and recent progress of DXT.

Dynamical X-Ray Single Molecule Observations of the Molecular Recognition Process in Major Histocompatibility Complex Molecule

Major histocompatibility complex (MHC) class II molecule has an important role in activities of the immune system. MHC binds peptides from exogenous antigens which is engulfed by endocytosis processes of antigen presenting cells and present it to T cell receptor, then stimulate and activate the immune system. In the peptide-loading process of MHC, it is said that DM which is a homolog of MHC has some activities on that process. The experiment of chromatography using peptide labeled with fluorescein on MHC with DM suggests that DM has a relationship between its activity and the length of peptides. Short peptides tend to be dissociated from MHC, on the other hand, long ones do not.In this research, we used Diffracted X-ray Tracking (DXT) to observe the dynamics of the peptide bound to MHC's groove with DM as a single molecule. DXT is the method of detecting the detailed motions of the targets for measurement by labeling a gold nanocrystal. The labeled nanocrystals diffract the incident x-rays and we obtained the motions of the target by tracking x-ray diffraction spots. We did experiments at SPring-8 (BL40XU, JAPAN).From the DXT experiments of observing the motions of peptides in MHC's groove with DM, we obtained the time-resolved (−100 microsecond) motion histograms of peptides. The motions of peptides are obviously different in that of being catalyzed by DM. Additionally, the peptide's motions with DM became more complicated than those without DM. We are going to discuss the results and consideration of the experiments.

Anomalous Stiffness Changes of Tau Protein in X-ray Single Molecule Observations

Alzheimer Disease (AD) is one of the most famous neurodegenerative disorders, and the neurofibrillary tangles serve as a hallmark of AD. They are composed of paired helical filaments of hyperphosphorylated Tau proteins, which are the microtubule-associated protein [1]. In order to understand the functions of a protein, we must determine its structures. But Tau protein's structure hasn’t been identified because it is famous for being one of the intrinsically disordered proteins (IDP), which lack stable tertiary structures or secondary structures [2].Here we observed Tau protein's fluctuations with the Diffracted X-ray tracking (DXT).This method is able to monitor the tilting and twisting motions of single protein molecules with nanometer resolution.In this method, gold nanocrystals are attached on target protein molecules to probe their intramolecular motions, and these proteins are irradiated with synchrotron X-ray to get time displacements of Laue diffraction patterns from a gold nanocrystal. As a synchrotron X-ray beamline, we used KEK NW-14A and SPring-8 BL40XU in Japan.In our experiment, in order to reveal the relation between an aggregation process and hyperphosphorylated Tau proteins, we phosphorylated wild-type and mutated recombinant Tau proteins with GSK-3β. Threonine and serine sites of these mutated Tau proteins were converted into Alanine in order not to be phosphorylated. And we found tau protein molecules were fluctuating between 0.3-1 nm in aqueous solution when a shutter speed was 36 ms/frame. More importantly, their fluctuations decreased after phosphorylated by GSK-3β. Finally, we specified the phosphorylation sites that affect structural fluctuations of Tau proteins.[1] A. Cavallini et al., J. Biol. Chem. 288, 23331-23347, (2013).[2] V. Uversky et al., Annu. Rev. Biophys. 37, 215-246, (2008).

Single-Molecule Motions of MHC Class II Rely on Bound Peptides

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.

Real time ligand-induced motion mappings of AChBP and nAChR using X-ray single molecule tracking.

We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 μs time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or α-bungarotoxin (αBtx), with views from two rotational axes. Our findings indicate that specific motion patterns represented as biaxial angular motion maps are associated with channel function in real time and on an atomic scale.