Abstract The recent ‘resolution revolution’ in structural analyses of cryo-electron microscopy (cryo-EM) has drastically changed the research strategy for structural biology. In addition to X-ray crystallography and nuclear magnetic resonance spectroscopy, cryo-EM has achieved the structural analysis of biological molecules at near-atomic resolution, resulting in the Nobel Prize in Chemistry 2017. The effect of this revolution has spread within the biology and medical science fields affecting everything from basic research to pharmaceutical development by visualizing atomic structure. As we have used cryo-EM as well as X-ray crystallography since 2000 to elucidate the molecular mechanisms of the fundamental phenomena in the cell, here we review our research history and summarize our findings. In the first half of the review, we describe the structural mechanisms of microtubule-based motility of molecular motor kinesin by using a joint cryo-EM and X-ray crystallography method. In the latter half, we summarize our structural studies on transcriptional regulation by X-ray crystallography of in vitro reconstitution of a multi-protein complex. structural biology, X-ray crystallography, cryo-electron microscopy, microtubule, kinesin, mediator complex Introduction Structural biology is the study of the molecular structure of biological macromolecules, particularly proteins and nucleic acids, providing a comprehensive understanding of how molecular architecture performs the biological reactions that are central to life. To date we have made full use of X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for visualizing proteins, DNA or RNA, however, prior to the 1950s, we did not have any idea about how biomolecules such as proteins and nucleic acids were organized three-dimensionally. It was only in the 1950s when researchers began to expose protein crystals to X-ray beams; visualization occurred of the long chains of amino acids, showing how myoglobin coiled into specific three-dimensional configurations [1,2]. For this discovery, Max Peruz and John Kendrew won the 1962 Nobel Prize in Chemistry. In the early 1980s, NMR spectroscopy was implemented for structural determination of biomolecules, becoming another possibility for studying their atomic structure. NMR has a limit in target size, generally <50 kDa; however, it can analyze the dynamics of the targets and their structures, providing a great advantage in revealing how they move and interact with other molecules. X-ray crystallography and NMR provided us with a tremendous number of atomic models of biomolecules, which we now use in everything from basic research to drug discovery. However, both methods have fundamental difficulties; X-ray crystallography requires high-quality crystal suitable for structural analysis, and NMR has limited application for small proteins. If the target has a large size and is difficult to arrange in crystals, it is impossible to understand the atomic structure of the biomolecules. Richard Henderson  decided to abandon X-ray crystallography and this is where the story of the 2017 Nobel Prize in Chemistry begins. Henderson believed that EM, once previously thought to be only suitable for studying non-living materials, could provide images of biomolecules as detailed as those using X-ray crystallography. Because an electron wavelength is much shorter than that of light, the electron microscope theoretically could visualize structural details of the targets. However, the harsh electron beam, intense enough to obtain high-resolution images, incinerates biological material. Henderson and his colleagues overcame this problem by using a mathematical approach similar to that used in X-ray crystallography. At the same time as their trial, the electron microscope analyses gradually improved through improvements in lenses and the development of cryotechnology. Henderson gradually added more details to the model and finally achieved a visualized structure of bacteriorhodopsin at atomic resolution in 1990 . Jacques Dubochet also won the 2017 Nobel Prize in Chemistry for successfully avoiding the disruption of electron beams by ice crystals through the vitrification method, which by rapid cooling causes water to a glass instead of crystals [5,6]. Joachim Frank, who is the third winner of the 2017 Nobel Prize in Chemistry, contributed to developing cryo-EM analysis technology by refining the image processing method, now referred to as ‘single particle analysis’. He developed an image analysis method that allows a computer to identify different recurring patterns in the image and generate a sharper image [7–10]. The two-dimensional high-resolution images of biomolecules from different angles were assembled and integrated into high- resolution 3D images by computational analysis. All these technological developments have improved the resolution, Ångström by Ångström. In addition to these essential cryo-EM technologies, development in 2013 of a new type of direct electron detection device [11,12] and progress of computers and software further pushed the limit of biological molecular resolution up to an atomic resolution of 3 Ångström or better, in 2013 [13,14]. Nowadays, cryo-EM structural analyses can routinely provide images that show individual atoms as detailed as those generated using X-ray crystallography, indicating this technique has come to the forefront of structural analyses. Simultaneously, critical technical maturation of X-ray crystallography analysis has allowed crystallographers to compete for arranging more difficult targets, such as huge multi-protein or/and nucleic acid complexes in crystals. In this way, marked progress has been achieved this decade in this field. In this review, we will introduce our structural studies using the cryo-EM and X-ray crystallography during the last two decades and will discuss the future direction of structural biology. Structural study of the molecular motor on the filamentous structure Microtubule-based molecular motor kinesins move processively along microtubules by using energy derived from Adenosine triphosphate (ATP) hydrolysis [15,16]. Since 2000, we have used structural studies to solve how kinesins move along the microtubule. Molecular motor kinesin moves unidirectionally toward the plus-end of the microtubule and our research question has focused on how kinesin moves along the filamentous structure microtubule toward one specific end. In order to elucidate the molecular mechanisms of kinesin motility, the shape of a moving molecule should be analyzed. The kinesin motor domain, the minimal domain for the microtubule-based motility, is 4–5 nm in diameter and its molecular weight is around 40 kDa. Since its size is less than the diffraction limit of light, X-ray crystallography, as well as cryo-EM, can be used to assess the structure. However, proteins in these methods must be frozen to the temperature of liquid nitrogen so that the protein structure is visualized as a freeze-frame (not a movie). Thus, we need to look for methods that can illustrate the structure of moving molecules. Around 2000, a pioneering study on nanosecond time-resolved crystallography was reported for photolysis of the myoglobin–carbon monoxide complex . However, applicable molecules for time-resolved crystallography were very limited then and our study using kinesin motor also failed because of protein damage by the diffraction experiment at room temperature and a prolonged interval time of the serial X-ray beam shoot. However, time-resolved crystallography has entered a new era during the past decade. The development of X-ray free-electron laser has become more applicable for various proteins. Recent studies using the SPring-8 Angstrom Compact free-electron LAser (SACLA) have revealed the fundamental structural changes that occur during photo-induced reactions of several proteins [18,19]. However, we had to find another approach. The first one that we chose to reveal the structure of moving motor of kinesin was using the nucleotide analogs that stop kinesin movement at various time points during ATP hydrolysis. The second one was slowing down ATPase reactions by crystallizing proteins. Kinesin still retains an ATPase activity even in the crystallized protein, although the rate of ATPase reaction slows significantly. Thus, we could easily observe the kinesin structures in the various intermediate forms. Kinesin was crystallized in the presence of adenosine diphosphate (ADP). After the kinesin crystals in the ADP state grew, both ethylenediaminetetraacetic acid and apyrase were soaked into the crystals to release Mg and ADP from kinesin. Crystals were frozen every 24 h after setup. In this way, we finally solved four structures of the nucleotide analogs during ATP hydrolysis by using the nucleotide analogs  and five successive structures during ADP release by utilizing slow ATPase reaction in crystals (Fig. 1) . With a combination of these two methods, we obtained nine successive snapshots during the ATPase cycle, thus visualizing the kinesin motility at the atomic resolution. Fig. 1. View largeDownload slide Crystal structures of the motor domain of KIF1A kinesin in the successive nine states during the ATPase cycle. The accession codes of Protein Data Bank (PDB) are shown in parentheses. The first five structures (upper column) were solved by inducing slow Mg and ADP release in the crystal . The next four structures (lower column) were solved using the nucleotide analogs . Fig. 1. View largeDownload slide Crystal structures of the motor domain of KIF1A kinesin in the successive nine states during the ATPase cycle. The accession codes of Protein Data Bank (PDB) are shown in parentheses. The first five structures (upper column) were solved by inducing slow Mg and ADP release in the crystal . The next four structures (lower column) were solved using the nucleotide analogs . Hybrid method During the 2000s, before the resolution revolution in cryo-EM [22,23], the resolution limit of cryo-EM structural analyses was around 10 to 20 Å. However, cryo-EM structural analysis works well for huge protein complexes like microtubules. Thus, we combined cryo-EM structural analysis with X-ray crystallography (Hybrid Method). Cryo-EM analysis revealed the medium resolution structure of the kinesin–microtubule complex, while X-ray crystallography solved the atomic resolution structure of relatively small homogenous proteins, such as the kinesin motor domain. Thus, we could visualize the crystal structures of the kinesin motor domain around 40 kDa and analyze the medium resolution cryo-EM structures of a kinesin–microtubule complex. Finally, the crystal structures of kinesin were docked into the cryo-EM structures of a kinesin–microtubule complex in silico, providing the pseudo-atomic structures of the kinesin–microtubule complex (Fig. 2). Fig. 2. View largeDownload slide A hybrid method combining X-ray crystallography and cryo-EM. Atomic structures of the motor domain of KIF1A kinesin were solved by X-ray crystallography. Cryo-EM separately solved the medium resolution structures of the kinesin–microtubule complex. Resulting crystal structures of kinesin were docked into the cryo-EM map of the kinesin–microtubule complex in silico, achieving the pseudo-atomic structures of the kinesin–microtubule complex. Fig. 2. View largeDownload slide A hybrid method combining X-ray crystallography and cryo-EM. Atomic structures of the motor domain of KIF1A kinesin were solved by X-ray crystallography. Cryo-EM separately solved the medium resolution structures of the kinesin–microtubule complex. Resulting crystal structures of kinesin were docked into the cryo-EM map of the kinesin–microtubule complex in silico, achieving the pseudo-atomic structures of the kinesin–microtubule complex. Structural mechanisms of kinesin motility along the microtubule We visualized almost all of the intermediate structures of this ATPase reaction cycle for the monomeric kinesin-3 family KIF1A by using the combination of X-ray crystallography and cryo-EM. We first figured out the cryo-EM structure of the KIF1A–microtubule complex in the ATP and ADP state [24,25], and also visualized the crystal structures of KIF1A at the nine successive states during the ATP hydrolysis [20,21]. We then docked the crystal structures into the cryo-EM structure to obtain the pseudo-atomic model of the KIF1A–microtubule complex to elucidate molecular mechanisms of KIF1A kinesin motility, as described below . Kinesin motor is one of the Walker-type NTPases and has a common ancestry with G-proteins or small GTPases . ATP/GTP hydrolysis generates conformational changes at the interface for the effector molecule, sometimes leading to attachment/detachment on/from the effector . G-protein uses this for switching signals on and off. The effector molecule of the motile kinesins such as KIF1A is the protein polymer microtubule, so kinesin uses the attachment/detachment on/from the microtubule to produce unidirectional movement along the microtubule. The unique non-motile kinesin KIF2C/MCAK also uses the attachment/detachment cycle on/from the effector . In this case, however, the effector of KIF2C is not the polymerized microtubule but the depolymerized tubulins. This difference results in acquiring the distinct function of KIF2C from other motile kinesins. Instead of unidirectional movement along microtubules, it depolymerizes microtubules from both ends to lead several fundamental phenomena including the chromosome segregation. Kinesin in the ATP state is strongly bound to the microtubule (Fig. 3). During the ATP binding into the pocket, small conformational changes around ATP occur, further inducing the large conformational change of switch II at the interface for the microtubule. The resulting interface represents a zig-zag shape with the protrusion of the switch II helices, fitting very well to the zig-zag surface of the microtubule (Fig. 3a). Thus, kinesin in the ATP state can bind strongly to the microtubule. The energy from the ATP hydrolysis induces the active detachment of kinesin from the microtubule (Fig. 3). The γ-phosphate from the kinesin ATP pocket leads to the conformational change of switch II. The kinesin interface becomes flat; thus, kinesin detaches from the microtubule (Fig. 3a). Fig. 3. View largeDownload slide The molecular mechanism of kinesin motility along the microtubule. (a) Schematic model of kinesin motility, as seen from the right side of the microtubule protofilament. Dotted lines show the binding surfaces of kinesin and the microtubule. Alternative attachment and detachment of kinesin to/from the microtubule occur during the ATPase cycle. (b) Snapshots of kinesin movement during the ATPase cycles are illustrated. Please see the full movie of kinesin motility at our website, 'http://www.med.kobe-u.ac.jp/anato1/Anat1_home.html'. Fig. 3. View largeDownload slide The molecular mechanism of kinesin motility along the microtubule. (a) Schematic model of kinesin motility, as seen from the right side of the microtubule protofilament. Dotted lines show the binding surfaces of kinesin and the microtubule. Alternative attachment and detachment of kinesin to/from the microtubule occur during the ATPase cycle. (b) Snapshots of kinesin movement during the ATPase cycles are illustrated. Please see the full movie of kinesin motility at our website, 'http://www.med.kobe-u.ac.jp/anato1/Anat1_home.html'. Kinesin in the ADP state weakly interacts with the microtubule (Fig. 3). Although most kinesin in the monomeric state is fully detached from the microtubule, KIF1A kinesin diffuses along the microtubule without detachment [29,30]. This is because KIF1A has highly basic residues in the long flexible loop L12 (K-loop) that ionically interacts with the C-terminal tail of tubulins (E-hook) rich in acidic residues (Fig. 3) . These flexible ionic interactions keep KIF1A hooked on the microtubule during the weak-binding ADP state. Furthermore, the basic residues align along the long axis of the kinesin interface in the ADP state, while acidic residues align along the center ridge along the microtubule (Fig. 3b). These linear ionic contacts also support the one-dimensional diffusion of kinesin along the microtubule. The latter property is conserved among all the plus-end-directed kinesin motors. In fact, conventional kinesin kinesin-1 (KIF5) diffuses along the microtubule, when kinesin-1 forms a dimer to keep attached to the microtubule . Finally, kinesin goes back to the strong-binding state to the microtubules during the release of ADP (Fig. 3). In other words, the microtubule serves as a nucleotide exchange factor to accelerate the ADP release from kinesin. During this step, kinesin binds to the microtubule with a stochastic unidirectional movement toward the microtubule plus-end . The source of the directionality could not be fully explained structurally, but several structural features contributing to the directional movement of kinesin have been reported . As described above, kinesin in the ADP state has a flat, smooth interface having a cluster of the basic residues along the center line. Thus, the interface in the ADP state appears as a forward-backward symmetry. However, ADP release induces the conformational change of switch II, breaking the forward-backward symmetry both for the shape and the alignment of ionic residues. The new interface represents the zig-zag shape with a forward-backward asymmetry. Further, the minus-end side of the basic residue clusters changes to the acidic residues. Therefore, kinesin fluctuates between the forward-backward symmetrical and asymmetrical potentials, so that kinesin stochastically moves forward during the transition from the symmetrical potential at the ADP state to the asymmetrical potential at the nucleotide-free state. Thus, kinesin uses the Brownian ratchet mechanism to move forward along the asymmetrical polymer microtubule. In vitro reconstitution of a multi-protein complex for structural study We now move to review a different topic, the structural analysis of the globular, multi-protein complex using the in vitro reconstitution system. We have implicated in the study of the Mediator complex, one of the essential players in the regulation of RNA polymerase II transcription [33–36]. However, its low abundance, large size (~1 MDa in yeast and ~1.2 MDa in human), and the complexity (25 subunits in yeast and more than 30 subunits in human) have hampered the progress of in vitro studies of Mediator. In order to decipher the mechanisms of Mediator, structural information is required. Most of the structural studies on Mediator have been carried out using X-ray crystallography and EM. A pioneering study of a low-resolution negative stain projection of Mediator was revealed by the Kornberg group . They showed the stable multi-subunit assembly of Mediator and direct binding of Mediator to the RNA polymerase II by EM analysis, opening the door for the structural study of Mediator. Further structural study of a 3D EM-reconstruction of a Negative-stained Mediator revealed that it is composed of three distinguishable modules: the head, middle and tail . However, high-resolution structural information was expected to reveal precise complex assembly between each subunit, and the physiological function of Mediator. The major challenges for the high-resolution structural study of Mediator are its complexity, heterogeneity, large size and low abundance in the native source. In particular, its large size (>1 MDa with over 20 subunits) prevents the recombinant expression of Mediator complex requisite for X-ray crystallography. At that time, X-ray crystallography was the only technology that was able to obtain atomic resolution structures of large multi-protein complexes. The purification of the Mediator complex from Saccharomyces cerevisiae cells for EM study requires 100–1000 g cells . To overcome these difficulties, we decided not to target the whole Mediator complex, but only the Mediator head module, which consists of seven proteins  by introducing three recombinant multi-protein complex expression technologies: sequence and ligation independent cloning (SLIC) based multi-fragment cloning technology for efficient construction , baculovirus-insect cell-based multi-protein expression system MultiBac for simple recombinant multi-protein expression, and titer estimation for quality control (TEQC)  for recombinant multi-protein expression optimization by baculovirus expression vector system (BEVS) (Fig. 4a). Fig. 4. View largeDownload slide Mediator head module reconstitution by SLIC, MultiBac, and TEQC. (a) Schematic diagram of the Mediator head module reconstitution. Target genes were cloned into MultiBac plasmids by SLIC. Multiple plasmids containing target genes were assembled into 1 plasmid through in vitro assembly by Cre-lox reaction. The plasmid harboring all target genes were integrated into MultiBac bacmid through Tn7 reaction in E.coli, transfected into insect cells, and virus was amplified. The virus titer was estimated and multi-protein complex expression was optimized by TEQC. (b) Expressed multi-protein complex was purified, crystallized, and structure was determined by X-ray crystallography. The Mediator head module is composed of seven different subunits with three domains named neck, movable jaw, and fixed jaw. Seven subunits are shown in different colors. Slice view from the perpendicular side of the neck is shown in the dashed-square figure (PDB ID: 3RJ1 and 4GWP) [39,57]. Fig. 4. View largeDownload slide Mediator head module reconstitution by SLIC, MultiBac, and TEQC. (a) Schematic diagram of the Mediator head module reconstitution. Target genes were cloned into MultiBac plasmids by SLIC. Multiple plasmids containing target genes were assembled into 1 plasmid through in vitro assembly by Cre-lox reaction. The plasmid harboring all target genes were integrated into MultiBac bacmid through Tn7 reaction in E.coli, transfected into insect cells, and virus was amplified. The virus titer was estimated and multi-protein complex expression was optimized by TEQC. (b) Expressed multi-protein complex was purified, crystallized, and structure was determined by X-ray crystallography. The Mediator head module is composed of seven different subunits with three domains named neck, movable jaw, and fixed jaw. Seven subunits are shown in different colors. Slice view from the perpendicular side of the neck is shown in the dashed-square figure (PDB ID: 3RJ1 and 4GWP) [39,57]. Reduce complexity by targeting stable multi-subunit entity of Mediator for structural study Hence, we focused not on the whole Mediator composed of more than 20 subunits, but to a Mediator head module composed of seven subunits, for the structural study of the Mediator. Mediator head module is an essential module of the Mediator, which dysfunction shows a cessation of the all mRNA synthesis in S. cerevisiae . Biochemical reconstitution study showed that the individual expression of each head module subunits produces lesser amounts of proteins. However, simultaneous expressions of all seven subunits yielded a much larger amount of proteins, which stably assembled in vitro , indicating the importance of multi-gene expressions for multi-subunit complex preparation. The evidence suggests the Mediator head module is a stable entity suitable for X-ray crystallography analysis. MultiBac with LIC-based cloning for simplifying molecular biology manipulation of multi-subunit expression We first streamlined molecular biology manipulation for the recombinant multi-protein complex production by introducing SLIC, which is a ligation independent cloning (LIC)  based multi-gene cloning method, and MultiBac, for simplifying the cloning step. SLIC could assemble multiple DNA fragments into one plasmid by recombination through the complementary sequence at the end of each DNA fragments  (Fig. 4a: SLIC). SLIC drastically simplified the multi-complex gene cloning containing two or three open reading frames of the target genes. Once multi-gene plasmids are generated by SLIC, these cassettes are further assembled to each other by MultiBac, a BEVS based technology, to generate a different combination for multi-protein expression containing many genes  (Fig. 4a: MultiBac). MultiBac could assemble gene cassettes into one huge gene cassette containing all genes of the target multi-protein complex by the Cre-lox recombination. The assembled gene cassette is integrated into a MultiBac bacmid, which lacks some proteolytic genes to alleviate proteolytic degradation through Tn7 reaction; the virus is produced by following the virus production protocol . These advances of the MultiBac enabled us to deal with multi-genes as a single gene [39,44], guarantees the presence of the all genes at cloning stage. In addition to the simple procedure, MultiBac produces a higher quality and quantity of the multi-protein complex than the previous method . Optimization of the multi-protein complex expression by TEQC Consistent production of the recombinant multi-protein complex is the key to structural study, which requires high-quality protein. However, optimization for maximizing the yield of the multi-protein complex is difficult; key parameters for expression of these complexes by single virus system such as MultiBac have not yet been studied. We investigated the multi-protein expression and developed modern technology ‘Titer estimation for quality control (TEQC)’ to establish consistency and maximize recombinant multi-protein expression in single virus harboring multi-genes . TEQC uses frozen virus prepared by TIPS method  for keeping the virus titer to be steady, and optimized expression by initial virus infectivity/Multiplicity of Infection (MOI) in 24 h, using the behavior of infected cells showing growth arrest in 24 h. We applied this TEQC technology to the Mediator head module MultiBac virus and established consistent and optimization of the multi-protein expression (Fig. 4a: TEQC). The obtained Mediator head module proteins were purified by His-tag affinity purification followed by size exclusion chromatography. Purified homogeneous recombinant Mediator head module was crystallized, and the architecture of S. cerevisiae Mediator head module by X-ray crystallography was successfully solved . Structure of the Mediator head module The structure of Mediator head module revealed its complex subunit assembly (Fig. 4b). The Mediator head module is composed of three distinct domains named the neck, fixed jaw and movable jaw, which are connected by a flexible joint. Consistent with the previous biochemical study, Med17, the largest subunit of the head module, played a key scaffold role in assembling six head module subunits. The most intriguing feature of the head module assembly is the 3D-architecture of the neck domain. The neck domain is formed by the integrated assembly of the 10 helix bundles composed of five subunits (Fig. 4b). The structure revealed how the multi-protein complex Mediator head module could establish stable and flexible assembly. Determining Mediator head module structure enabled us to map the known dysfunctional mutations of Mediator by docking the high-resolution structure into a low-resolution map. Conclusion and future directions The recent ‘resolution revolution’ in structural biology of cryo-EM reconstruction using single particle analysis has also influenced the structural studies of filamentous structures such as the microtubule and actin [22,23]. By handling the filamentous structure as the sequence of the single particles, several cryo-EM structures of filaments have been reported at near-atomic resolution [47–51]. We have also solved the kinesin–microtubule complex at a near 5-Å resolution, revealing unique functions of kinesin-8 and kinesin-14 [52,53]. Currently, cryo-EM technology can identify small protein structures whose molecular weight is <100 kDa, previously believed to be difficult to treat with cryo-EM . By decreasing the minimum size limitation of proteins applicable to cryo-EM, almost all molecules will be potential targets for cryo-EM structural analysis. Considering that the cryo-EM structures are determined in more physiological conditions than those solved by X-ray crystallography, the cryo-EM structural analysis provides a much greater hope for application in structure-based drug design. We do not mention here but the cryo-EM tomography (cryo-ET) technique, which can determine macromolecular structures in vitro or in the cell, and also a promising method for elucidating heterogeneous structures at a nanometer resolution [55,56]. Thus, cryo-EM will make great contributions not only to structure-based drug design by solving atomic resolution structures, but also to elucidate molecular mechanisms of pathophysiological functions in various cells by solving physiological structures at a nanometer resolution. Acknowledgements We are grateful to Professor Nobutaka Hirokawa for critical discussion. Funding This work was supported by JSPS KAKENHI (No.15K08168 to R.N.), Japan Agency for Medical Research and Development (JP17gm0810013 to R.N.), Takeda Science Foundation to E.N. and R.N., and Mochida Memorial Foundation for Medical and Pharmaceutical Research to R.N. References 1 Kendrew J C et al. . ( 1958 ) A three-dimensional model of the myoglobin molecule obtained by x-ray analysis . Nature 181 : 662 – 666 . Google Scholar CrossRef Search ADS PubMed 2 Perutz M F , and Weisz O ( 1947 ) Crystal structure of human carboxyhaemoglobin . Nature 160 : 786 . Google Scholar CrossRef Search ADS PubMed 3 Henderson R , and Unwin P N ( 1975 ) Three-dimensional model of purple membrane obtained by electron microscopy . Nature 257 : 28 – 32 . Google Scholar CrossRef Search ADS PubMed 4 Henderson R et al. . ( 1990 ) An atomic model for the structure of bacteriorhodopsin . Biochem. Soc. Trans. 18 : 844 . 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Microscopy – Oxford University Press
Published: May 16, 2018
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