Relative Orientation Of Domains Research Articles

Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst-A Structural and Functional Analysis.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Analysis of the inter-domain orientation of tandem RRM domains with diverse linkers: connecting experimental with AlphaFold2 predicted models.

The RNA recognition motif (RRM) is the most prevalent RNA binding domain in eukaryotes and is involved in most RNA metabolism processes. Single RRM domains have a limited RNA specificity and affinity and tend to be accompanied by other RNA binding domains, frequently additional RRMs that contribute to an avidity effect. Within multi-RRM proteins, the most common arrangement are tandem RRMs, with two domains connected by a variable linker. Despite their prevalence, little is known about the features that lead to specific arrangements, and especially the role of the connecting linker. In this work, we present a novel and robust way to investigate the relative domain orientation in multi-domain proteins using inter-domain vectors referenced to a stable secondary structure element. We apply this method to tandem RRM domains and cluster experimental tandem RRM structures according to their inter-domain and linker-domain contacts, and report how this correlates with their orientation. By extending our analysis to AlphaFold2 predicted structures, with particular attention to the inter-domain predicted aligned error, we identify new orientations not reported experimentally. Our analysis provides novel insights across a range of tandem RRM orientations that may help for the design of proteins with a specific RNA binding mode.

Highly Ordered Monolayers of μm-Sized Polystyrene Spheres Studied by Grazing-Incidence Small-Angle X-ray Scattering, Simulations, and Geometrical Calculations.

Unraveling the two-dimensional (2D) structural ordering of colloidal particles assembled at a flat surface is essential for understanding and optimizing their physical properties. So far, grazing-incidence small-angle X-ray scattering (GISAXS) has been widely used to determine crystallographic information on 2D self-assembled structures of nanosize objects. However, solving the structure of 2D lattices consisting of micrometer (μm)-sized objects still remains a challenge using scattering methods. Here, a model 2D SCALMS (supported catalytically active liquid metal solution) template is fabricated from μm-sized polystyrene (PS) spheres that form a monolayer on top of the flat solid support. GISAXS patterns of the sample were collected for rotation angles around its surface normal in steps of 3°. For every rotation angle, different Bragg-type interference maxima along the out-of-plane (qz) direction were observed. On the basis of simulations of GISXAS patterns of single domains of ordered particle arrangements using the distorted wave Born approximation (DWBA) and validation against a simple geometrical scattering model, the interference maxima could nicely be interpreted to originate from a monolayer of the μm-sized spherical particles which are arranged in domains of hexagonal 2D paracrystalline order. This novel GISAXS evaluation technique serves as a proof of principle for determining the μm-size periodicity of 2D crystalline domains and demonstrates its potential to spatially resolve the relative orientations of such domains with respect to a reference direction.

Putting AlphaFold models to work with phenix.process_predicted_model and ISOLDE.

AlphaFold has recently become an important tool in providing models for experimental structure determination by X-ray crystallography and cryo-EM. Large parts of the predicted models typically approach the accuracy of experimentally determined structures, although there are frequently local errors and errors in the relative orientations of domains. Importantly, residues in the model of a protein predicted by AlphaFold are tagged with a predicted local distance difference test score, informing users about which regions of the structure are predicted with less confidence. AlphaFold also produces a predicted aligned error matrix indicating its confidence in the relative positions of each pair of residues in the predicted model. The phenix.process_predicted_model tool downweights or removes low-confidence residues and can break a model into confidently predicted domains in preparation for molecular replacement or cryo-EM docking. These confidence metrics are further used in ISOLDE to weight torsion and atom-atom distance restraints, allowing the complete AlphaFold model to be interactively rearranged to match the docked fragments and reducing the need for the rebuilding of connecting regions.

Using Single-Molecule Optical Tweezers to Study the Conformational Cycle of the Hsp90 Molecular Chaperone.

The heat shock protein 90 (Hsp90) family of chaperones are well-known, highly important components of the cellular systems which regulate protein homeostasis. Essential in eukaryotes, Hsp90s is also found in prokaryotes, including archaea. Hsp90 is a dimeric protein, with each monomer consisting of three separate structural domains, and undergoes large conformational changes as part of its functional cycle. This cycle is driven by interactions with nucleotides, cochaperone proteins, client proteins and allosteric effects enacted by these and by posttranslational modifications. All of these influence the rate and degree of the opening and closing of the dimer as well as the relative domain orientations and its overall rigidity. Optical tweezers, which can access many of these functionally important conformational changes, therefore provide a unique tool for the study of this large and complex molecular chaperone. Here, we provide protocols for the design and implementation of different Hsp90 constructs and optical tweezers experiments for addressing the many open questions about the function of this important molecular chaperone.

Advances in NMR Spectroscopy of Weakly Aligned Biomolecular Systems.

The measurement and application of residual dipolar couplings (RDCs) in solution NMR studies of biological macromolecules has become well established over the past quarter of a century. Numerous methods for generating the requisite anisotropic orientational molecular distribution have been demonstrated, each with its specific strengths and weaknesses. In parallel, an enormous number of pulse schemes have been introduced to measure the many different types of RDCs, ranging from the most widely measured backbone amide 15N-1H RDCs, to 1H-1H RDCs and couplings between low-γ nuclei. Applications of RDCs range from structure validation and refinement to the determination of relative domain orientations, the measurement of backbone and domain motions, and de novo structure determination. Nevertheless, it appears that the power of the RDC methodology remains underutilized. This review aims to highlight the practical aspects of sample preparation and RDC measurement while describing some of the most straightforward applications that take advantage of the exceptionally precise information contained in such data. Some emphasis will be placed on more recent developments that enable the accurate measurement of RDCs in larger systems, which is key to the ongoing shift in focus of biological NMR spectroscopy from structure determination toward gaining improved understanding of how molecular flexibility drives protein function.

HERMES - A Software Tool for the Prediction and Analysis of Magnetic-Field-Induced Residual Dipolar Couplings in Nucleic Acids.

Field-Induced Residual Dipolar Couplings (fiRDC) are a valuable source of long-range information on structure of nucleic acids (NA) in solution. A web application (HERMES) was developed for structure-based prediction and analysis of the (fiRDCs) in NA. fiRDC prediction is based on input 3D model structure(s) of NA and a built-in library of nucleobase-specific magnetic susceptibility tensors and reference geometries. HERMES allows three basic applications: (i) the prediction of fiRDCs for a given structural model of NAs, (ii) the validation of experimental or modeled NA structures using experimentally derived fiRDCs, and (iii) assessment of the oligomeric state of the NA fragment and/or the identification of a molecular NA model that is consistent with experimentally derived fiRDC data. Additionally, the program's built-in routine for rigid body modeling allows the evaluation of relative orientation of domains within NA that is in agreement with experimental fiRDCs.

Probing Relative Domain Mobility of Class-I Major Histocompability Complex Molecules through Residual Dipolar Couplings

The highly polymorphic proteins of class I major histocompatibility complex (MHC-I) are 45 kDa multi-domain heterodimers that play a key role in orchestrating the human adaptive immune response. Their function is to present endogenous and exogenous peptide ligands to cytotoxic T cells and Natural Killer cells. Despite a wealth of X-ray structural information on these proteins and their complexes, dynamic studies by NMR have been restricted to the study of local (chemical shift-based) changes occurring at different timescales, using relaxation experiments recorded for amide probes. Recent work from our group using methyl probes has suggested the presence of an allosteric network on the MHC structure, which is modulated in different alleles with functional consequences for the recognition of peptides and molecular chaperones. However, the precise structural mechanism underlying how conformational changes at the peptide-binding groove induced by the binding of different ligands affect the distal α3 domain remains unclear. Here we have obtained full NMR backbone assignments for a common human MHC-I molecule, HLA-A∗02:01, bound to two different high-affinity peptides. Using transverse relaxation optimized spectroscopy-based methods (ARTSY), we collected residual dipolar couplings (RDCs) for both systems, and utilized this data as restraints for structural refinement towards a rigorous analysis of alignment tensors for the different protein domains, relative to an overall molecular frame. Our results suggest that the local conformation of the peptide-binding groove may affect the global structure of the molecule via subtle changes in relative domain orientation and mobility. Our NMR-based mechanism has important implications for how MHC-I molecules of different peptide occupancies interact with their CD8 co-receptors and molecular chaperones.

VH -VL interdomain dynamics observed by computer simulations and NMR.

The relative orientation of the two variable domains, VH and VL, influences the shape of the antigen binding site, that is, the paratope, and is essential to understand antigen specificity. ABangle characterizes the VH‐VL orientation by using five angles and a distance and compares it to other known structures. Molecular dynamics simulations of antibody variable domains (Fvs) reveal fluctuations in the relative domain orientations. The observed dynamics between these domains are confirmed by NMR experiments on a single‐chain variable fragment antibody (scFv) in complex with IL‐1β and an antigen‐binding fragment (Fab). The variability of these relative domain orientations can be interpreted as a structural feature of antibodies, which increases the antibody repertoire significantly and can enlarge the number of possible binding partners substantially. The movements of the VH and VL domains are well sampled with molecular dynamics simulations and are in agreement with the NMR ensemble. Fast Fourier transformation of the ABangle metrics allows to assign timescales of 0.1‐10 GHz to the fastest collective interdomain movements. The results clearly show the necessity of dynamics to understand and characterize the favorable orientations of the VH and VL domains implying a considerable binding interface flexibility and reveal in all antibody fragments (Fab, scFv, and Fv) very similar VH‐VL interdomain variations comparable to the distributions observed for known X‐ray structures of antibodies.Significance StatementAntibodies have become key players as therapeutic agents. The binding ability of antibodies is determined by the antigen‐binding fragment (Fab), in particular the variable fragment region (Fv). Antigen‐binding is mediated by the complementarity‐determining regions consisting of six loops, each three of the heavy and light chain variable domain VH and VL. The relative orientation of the VH and VL domains influences the shape of the antigen‐binding site and is a major objective in antibody design. In agreement with NMR experiments and molecular dynamics simulations, we show a considerable binding site flexibility in the low nanosecond timescale. Thus we suggest that this flexibility and its implications for binding and specificity should be considered when designing and optimizing therapeutic antibodies.

IFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains.

Recent years have witnessed increasing efforts to engineer artificial biological functions through recombination of modular-organized toolboxes of protein scaffolds and parts. A critical, yet frequently neglected aspect concerns the identity of peptide linkers or spacers connecting individual domains which remain poorly understood and challenging to assemble. Addressing these limitations, iFlinkC comprises a highly scalable DNA assembly process that facilitates the combinatorial recombination of functional domains with linkers of varying length and flexibility, thereby overcoming challenges with high GC-content and the repeat nature of linker elements. The capacity of iFLinkC is demonstrated in the construction of synthetic protease switches featuring PDZ-FN3-based affinity clamps and single-chain FKBP12-FRB receptors as allosteric inputs. Library screening experiments demonstrate that linker space is highly plastic as the induction of allosterically regulated protease switches can vary from >150-fold switch-ON to >13-fold switch-OFF solely depending on the identity of the connecting linkers and relative orientation of functional domains. In addition, Pro-rich linkers yield the most potent switches contradicting the conventional use of flexible Gly-Ser linkers. Given the ease and efficiency how functional domains can be readily recombined with any type of linker, iFLinkC is anticipated to be widely applicable to the assembly of any type of fusion protein.

Structure of Urocanate Hydratase from the protozoan Trypanosoma cruzi

The enzyme Urocanate Hydratase (UH) participates in the catabolic pathway of L-histidine. Trypanosoma cruzi Urocanate Hydratase (TcUH) is identified as a therapeutic molecular target in the WHO/TDR Targets Database. We report the 3D structure determination and number of features of TcUH, and compared it to other few available bacterial UH structures. Each monomer presents two domains and one NAD+ molecule. Superpositions revealed differences in the relative orientation of domains within monomers, such that TcUH monomer A resembles Urocanate Hydratase from Geobacillus kaustophilus (GkUH) (open conformation), while monomer C resembles Urocanate Hydratase from Pseudomonas putida (PpUH) and Urocanate Hydratase from Bacillus subtilis (BsUH) (closed conformations). We use the structure of TcUH to make considerations about 3 non-deleterious and 2 deleterious mutations found in human UHs: non-deleterious mutations could be accommodated without large displacements or interaction interruptions, whereas deleterious mutations in one case might disrupt an α-helix (as previously suggested) and in the other case, besides disrupting the enzyme interaction with the substrate, might interfere with interdomain movement.

Understanding allosteric interactions in hMLKL protein that modulate necroptosis and its inhibition

Mixed Lineage Kinase domain-Like (MLKL), a key player in necroptosis, is a multi-domain protein with an N-terminal 4 helical bundle (4HB) and a pseudokinase domain (PsK) connected by brace helices. Phosphorylation of PsK domain of MLKL is a key step towards oligomerization of 4HB domain that causes cell death. Necrosulfonamide (NSA) binds to the 4HB domain of MLKL to inhibit necroptosis. To understand the molecular details of MLKL function and it’s inhibition, we have performed a molecular dynamic study on hMLKL protein in apo, phosphorylated and NSA-bound states for a total 3 μs simulation time. Our simulations show increased inter-domain flexibility, increased rigidification of the activation loop and increased alpha helical content in the brace helix region revealing a form of monomeric hMLKL necessary for oligomerization upon phosphorylation as compared to apo state. NSA binding disrupts this activated form and causes two main effects on hMLKL conformation: (1) locking of the relative orientation of 4HB and PsK domains by the formation of several new interactions and (2) prevention of key 4HB residues to participate in cross-linking for oligomer formation. This new understanding of the effect of hMLKL conformations on phosphorylation and NSA binding suggest new avenues for designing effective allosteric inhibitors of hMLKL.

Structural Aspects of Glycosylated Protein–Glycan Interactions by NMR

Characterization of glycosylated proteins and their response to interaction with glycans is challenging for any method, because of the heterogeneity of glycosylation and the extensive conformational sampling by most glycans. For NMR there are extra challenges because expression in mammalian cell cultures, which can preserve near‐native glycosylation, requires expensive substrates to achieve uniform isotopic labeling with 15N or 13C, and mammalian cells do not tolerate high levels of deuteration (something required to improve spectral resolution). We have developed a sparse‐labeling approach that preserves high resolution without deuteration, proves to be economical in most applications, and returns enough structural data to define geometries of multiple domain proteins and ligand protein complexes. The approach is based on labeling proteins with a single type of amino acid isotopically labeled in methyl groups with 13C (13C‐valine or 13C alanine). NMR signals from methyl groups are special in terms of the sensitivity and resolution that they provide. The addition of paramagnetic tags to specific sites in a protein provides long range distance and angular information on the location of labeled methyl groups. In combination with a newly developed approach to assign methyl resonances to specific sites in a protein, this information proves adequate to define relative domain orientations and facilitate the identification of ligand binding sites. Application to terminal domains from a cell surface signaling molecule, ROBO1, is used to illustrate the approach. It also provides an example of how binding of a heparan sulfate oligomer alters the relative orientation of domains.Support or Funding InformationThis work was supported by grants from the National Institutes of Health, P41GM103390 and R01GM033225.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantitative comparison of ABC membrane protein type I exporter structures in a standardized way

SummaryAn increasing number of ABC membrane protein structures are determined by cryo-electron microscopy and X-ray crystallography, consequently identifying differences between their conformations has become an arising issue. Therefore, we propose to define standardized measures for ABC Type I exporter structure characterization. We set conformational vectors, conftors, which describe the relative orientation of domains and can highlight structural differences. In addition, continuum electrostatics calculations were performed to characterize the energetics of membrane insertion illuminating functionally crucial regions. In summary, the proposed metrics contribute to deeper understanding of ABC membrane proteins' structural features, structure validation, and analysis of movements observed in a molecular dynamics trajectory. Moreover, the concept of standardized metrics can be applied not only to ABC membrane protein structures (http://conftors.hegelab.org).

Reconstruction of 3D structures of MET antibodies from electron microscopy 2D class averages.

Dynamics of three MET antibody constructs (IgG1, IgG2, and IgG4) and the IgG4-MET antigen complex was investigated by creating their atomic models with an integrative experimental and computational approach. In particular, we used two-dimensional (2D) Electron Microscopy (EM) images, image class averaging, homology modeling, Rapidly exploring Random Tree (RRT) structure sampling, and fitting of models to images, to find the relative orientations of antibody domains that are consistent with the EM images. We revealed that the conformational preferences of the constructs depend on the extent of the hinge flexibility. We also quantified how the MET antigen impacts on the conformational dynamics of IgG4. These observations allow to create testable hypothesis to investigate MET biology. Our protocol may also help describe structural diversity of other antigen systems at approximately 5 Å precision, as quantified by Root-Mean-Square Deviation (RMSD) among good-scoring models.

Computational Investigation into the Solution Structure of Monomeric Lassa Virus Nucleoprotein and Insights into its RNA Binding Mechanism

Lassa is a dangerous Arenavirus, which is enveloped and has a single-stranded (ss)RNA genome. The nucleoprotein (NP) is the most abundant protein in the virion and functions to encapsidate the viral genome to form a ribonucleoprotein complex (RNP). Studies of the full-length NP show it forms a circular trimer and does not bind to RNA. However, a structure of just the N-terminal domain bound to RNA has also been determined, which indicates structural reorganization is required to bind ssRNA. Therefor it is believed that NP must undergo a large conformational change from the observed trimeric configuration in order to bind and polymerize on ssRNA to form the RNP.Using molecular dynamics and metadynamics simulations we have investigated the influence of RNA on the global motions of the N-terminal domain of Lassa NP, and have identified that RNA-binding reduces the free energy barrier to transition to the RNA bound state. We have gone on to investigate the conformational dynamics of the full-length NP in apo and RNA bound states when free in solution as a monomer. During multi-microsecond MD simulations performed on the Anton supercomputer, we observed significant conformational changes in the NP. In both apo and RNA bound simulations, we observe changes in interdomain contacts and relative domain orientation, as well formation of helical secondary structure in an unresolved domain linker loop. Most notably in the RNA bound simulation, the domain orientation is much more dramatic, and provides a putative template to understand the higher-order structural organization of the RNP. Kinetic network analysis and Brownian dynamics simulations have been employed to further analyze the structural transition pathways and to evaluate mechanisms of transition from the RNA-free trimer to the RNP.

A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a β-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.

DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography

DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain–domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept.

Conformational control of DNA target cleavage by CRISPR-Cas9.

Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by CRISPR (clustered regularly interspaced short palindromic repeats)1,2. Together with single-guide RNAs (sgRNA)3, Cas9 also functions as a powerful genome engineering tool in plants and animals4–6, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications7,8. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage9–11, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer (FRET) experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial PAM recognition12 and RNA–DNA base-pairing3 that serves as a final specificity checkpoint before DNA double-strand break formation.