The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-ty... more The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-type cation pumps. These couple ATP hydrolysis with cation transport, generating cation gradients across membranes. Ca2+-ATPase specifically maintains the low cytoplasmic calcium concentration of resting muscle by pumping calcium into the sarcoplasmic reticulum; subsequent release is used to initiate contraction. No high-resolution structure of a P-type pump has yet been determined, although a 14-A structure of Ca2+-ATPase, obtained by electron microscopy of frozen-hydrated, tubular crystals, showed a large cytoplasmic head connected to the transmembrane domain by a narrow stalk. We have now improved the resolution to 8A and can discern ten transmembrane alpha-helices, four of which continue into the stalk On the basis of constraints from transmembrane topology, site-directed mutagenesis and disulphide crosslinking, we have made tentative assignments for these alpha-helices within the amino-acid sequence. A distinct cavity leads to the putative calcium-binding site, providing a plausible path for calcium release to the lumen of the sarcoplasmic reticulum.
Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallogr... more Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state, and its final state structure has not been solved because of the low efficiencies of the S-state transitions in the crystals. Here we analyzed the structure of PSII in solution at 1.95 Ă… resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, which was reduced by reducing the electron dose. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.
Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs... more Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs) for capturing solar energy and conveying it to photosynthetic reaction centers. PBSs are compositionally and structurally diverse, and exceedingly complex, all of which pose a challenge for a comprehensive understanding of their function. To date, three detailed architectures of PBSs by cryo-electron microscopy (cryo-EM) have been described: a hemiellipsoidal type, a block-type from rhodophytes, and a cyanobacterial hemidiscoidaltype. Here, we report cryo-EM structures of a pentacylindrical allophycocyanin core and phycocyanin-containing rod of a thermophilic cyanobacterial hemidiscoidal PBS. The structures define the spatial arrangement of protein subunits and chromophores, crucial for deciphering the energy transfer mechanism. They reveal how the pentacylindrical core is formed, identify key interactions between linker proteins and the bilin chromophores, and indicate pathways for unidirectional energy transfer.
Electron crystallography has the potential to analyze crystals of membrane proteins and macromole... more Electron crystallography has the potential to analyze crystals of membrane proteins and macromolecular complexes too small or too thin for X-ray crystallography, as electrons are scattered 4 5 orders of magnitude more strongly than X-rays. Electron crystallography yields Coulomb potential maps, rather than electron density maps as X-rays do, providing information on charged states of amino-acids and metals. We present such Coulomb potential maps at 3.4 Ă… and 3.2 Ă… resolution, respectively, of Ca2+-ATPase and catalase obtained from crystals of just a few layers thick. These maps demonstrate that it is indeed possible to build atomic models from such crystals and charge information is included, often critical in understanding protein function [1]. We also report a suitable treatment of electron scattering factors of charged atoms for refinement of atomic models against cryoEM maps [2]. The ScatCurve package developed supports various models for parameterization of scattering curves, a...
Cryo-electron microscopy (cryo-EM) is widely used to determine high-resolution structures from im... more Cryo-electron microscopy (cryo-EM) is widely used to determine high-resolution structures from images of protein molecules embedded in thin ice and from electron diffraction patterns of small and thin crystals. These two techniques of imaging and diffraction offer complementary structure analysis of various-sized molecules: The former single particle analysis is applied mainly to larger-sized molecules that yield good image contrast in vitreous ice; Electron 3D crystallography (3D ED / MicroED) is applicable to undersized crystals of smaller molecules, which are hard to solve by single particle analysis and do not grow to a suitable size for X-ray diffraction even with a high-intensity synchrotron radiation beam. The former application has value in structural biology, while the importance of the latter is becoming recognized in synthetic chemistry, material sciences and related areas.
Acaryochloris marina is one of the cyanobacteria that can use far-red light to drive photochemica... more Acaryochloris marina is one of the cyanobacteria that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of the photosystem I reaction center of A. marina determined by cryo-electron microscopy at 2.5 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments different from other type I reaction centers. The paired chlorophyll, so-called special pair, of P740 is a dimer of chlorophyll d/d′ and the primary electron acceptor is pheophytin a, a metal-less chlorin different from the chlorophyll a common to all other type I reaction centers. Here we show the architecture of the photosystem I reaction center is composed of 11 subunits and identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.
Acta Crystallographica Section A Foundations and Advances, Dec 1, 2017
Electron crystallography has the potential to analyze crystals of membrane proteins and macromole... more Electron crystallography has the potential to analyze crystals of membrane proteins and macromolecular complexes too small or too thin for X-ray crystallography, as electrons are scattered 4-5 orders of magnitude more strongly than X-rays. Electron crystallography yields Coulomb potential maps, rather than electron density maps as X-rays do, providing information on charged states of amino-acids and metals. We present such Coulomb potential maps at 3.4 Ă… and 3.2 Ă… resolution, respectively, of Ca2+-ATPase and catalase obtained from crystals of just a few layers thick. These maps demonstrate that it is indeed possible to build atomic models from such crystals and charge information is included, often critical in understanding protein function [1]. We also report a suitable treatment of electron scattering factors of charged atoms for refinement of atomic models against cryo-EM maps [2]. The ScatCurve package developed supports various models for parameterization of scattering curves, and the parameter tables can be implemented in major refinement programs in structural biology. We changed partial charge values of charged amino acid in crystal structures in steps of 0.1 for refinement of the atomic models against electron diffraction data from three-dimensional crystals. By exploring a range of partial charges, we found the proper electrostatic setting to produce atomic models with improved statistics and to better reflect experimental data. Structure refinement for single particle analysis also benefits from the more accurate analysis, and our programs could find wide use for model refinement against cryo-EM maps.
Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis... more Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis, orchitis, meningitis, and encephalitis. MuV-related hearing loss is also a serious complication because it is usually irreversible. MuV outbreaks have been reported in many countries, even in high-vaccine-coverage areas. MuV has tropism toward glandular tissues and the central nervous system. To understand the unique MuV tropism, revealing the mechanism of receptor recognition by MuV is very important. Here, using a large-scale glycan array and X-ray crystallography, we show that MuV recognizes sialyl Lewis X and GM2 ganglioside as receptors, in addition to a previously reported MuV receptor, a trisaccharide containing an α2,3-linked sialic acid. The flexible recognition of these glycan receptors by MuV may explain the unique tropism and pathogenesis of MuV. Structures will also provide a template for the development of effective entry inhibitors targeting the receptor-binding site of ...
Sarcoplasmic reticulum Ca2+-ATPase is an integral membrane protein of Mr 110k and pumps Ca2+ agai... more Sarcoplasmic reticulum Ca2+-ATPase is an integral membrane protein of Mr 110k and pumps Ca2+ against a large concentration gradient during relaxation of muscle.
This paper reports a suitable treatment of electron scattering factors of charged atoms for refin... more This paper reports a suitable treatment of electron scattering factors of charged atoms for refinement of atomic models against cryo-electron microscopy (cryo-EM) maps. The ScatCurve package developed here supports various curve models for parameterization of scattering factors and the parameter tables can be implemented in major refinement programs in structural biology. Partial charge values of charged amino acids in crystal structures were changed in small steps for refinement of the atomic models against electron diffraction data from three-dimensional crystals. By exploring a range of partial charges, the authors found the electrostatic setting that produces atomic models with improved statistics and better reflects experimental data. Structure refinement for single-particle analysis also benefits from the more accurate analysis and the programs could find wide use for model refinement against cryo-EM maps.
Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-pr... more Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-protein coupled receptors (GPCRs), and the extracellular ligand binding domains (LBDs) of T1r1/T1r3 and T1r2/T1r3 heterodimers are responsible for binding of chemical substances eliciting umami or sweet taste. However, molecular analyses of T1r have been hampered due to the difficulties in recombinant expression and protein purification, and thus little is known about mechanisms for taste perception. Here we show the first molecular view of reception of a taste substance by a taste receptor, where the binding of the taste substance elicits a different conformational state of T1r2/T1r3 LBD heterodimer. Electron microscopy has showed a characteristic dimeric structure. Förster resonance energy transfer and X-ray solution scattering have revealed the transition of the dimerization manner of the ligand binding domains, from a widely spread to compactly organized state upon taste substance bind...
There are many huge macromolecular complexes in living organisms. They are often hard to crystall... more There are many huge macromolecular complexes in living organisms. They are often hard to crystallize because of their size, complexity and heterogeneity. Cryo-electron microscopy (cryo-EM) is a suitable method to analyze the structures of such biological macromolecules, because it can be applied to various forms of samples, e.g. two-dimensional crystal, helical assembly, spherical virus, dispersed particle, cell organelle and cell, although attainable resolution depends on the system. In this review, I introduce these techniques and examples of the structure analysis, and briefly review the perspective of cryo-EM.
The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-ty... more The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-type cation pumps. These couple ATP hydrolysis with cation transport, generating cation gradients across membranes. Ca2+-ATPase specifically maintains the low cytoplasmic calcium concentration of resting muscle by pumping calcium into the sarcoplasmic reticulum; subsequent release is used to initiate contraction. No high-resolution structure of a P-type pump has yet been determined, although a 14-A structure of Ca2+-ATPase, obtained by electron microscopy of frozen-hydrated, tubular crystals, showed a large cytoplasmic head connected to the transmembrane domain by a narrow stalk. We have now improved the resolution to 8A and can discern ten transmembrane alpha-helices, four of which continue into the stalk On the basis of constraints from transmembrane topology, site-directed mutagenesis and disulphide crosslinking, we have made tentative assignments for these alpha-helices within the amino-acid sequence. A distinct cavity leads to the putative calcium-binding site, providing a plausible path for calcium release to the lumen of the sarcoplasmic reticulum.
Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallogr... more Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state, and its final state structure has not been solved because of the low efficiencies of the S-state transitions in the crystals. Here we analyzed the structure of PSII in solution at 1.95 Ă… resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, which was reduced by reducing the electron dose. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.
Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs... more Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs) for capturing solar energy and conveying it to photosynthetic reaction centers. PBSs are compositionally and structurally diverse, and exceedingly complex, all of which pose a challenge for a comprehensive understanding of their function. To date, three detailed architectures of PBSs by cryo-electron microscopy (cryo-EM) have been described: a hemiellipsoidal type, a block-type from rhodophytes, and a cyanobacterial hemidiscoidaltype. Here, we report cryo-EM structures of a pentacylindrical allophycocyanin core and phycocyanin-containing rod of a thermophilic cyanobacterial hemidiscoidal PBS. The structures define the spatial arrangement of protein subunits and chromophores, crucial for deciphering the energy transfer mechanism. They reveal how the pentacylindrical core is formed, identify key interactions between linker proteins and the bilin chromophores, and indicate pathways for unidirectional energy transfer.
Electron crystallography has the potential to analyze crystals of membrane proteins and macromole... more Electron crystallography has the potential to analyze crystals of membrane proteins and macromolecular complexes too small or too thin for X-ray crystallography, as electrons are scattered 4 5 orders of magnitude more strongly than X-rays. Electron crystallography yields Coulomb potential maps, rather than electron density maps as X-rays do, providing information on charged states of amino-acids and metals. We present such Coulomb potential maps at 3.4 Ă… and 3.2 Ă… resolution, respectively, of Ca2+-ATPase and catalase obtained from crystals of just a few layers thick. These maps demonstrate that it is indeed possible to build atomic models from such crystals and charge information is included, often critical in understanding protein function [1]. We also report a suitable treatment of electron scattering factors of charged atoms for refinement of atomic models against cryoEM maps [2]. The ScatCurve package developed supports various models for parameterization of scattering curves, a...
Cryo-electron microscopy (cryo-EM) is widely used to determine high-resolution structures from im... more Cryo-electron microscopy (cryo-EM) is widely used to determine high-resolution structures from images of protein molecules embedded in thin ice and from electron diffraction patterns of small and thin crystals. These two techniques of imaging and diffraction offer complementary structure analysis of various-sized molecules: The former single particle analysis is applied mainly to larger-sized molecules that yield good image contrast in vitreous ice; Electron 3D crystallography (3D ED / MicroED) is applicable to undersized crystals of smaller molecules, which are hard to solve by single particle analysis and do not grow to a suitable size for X-ray diffraction even with a high-intensity synchrotron radiation beam. The former application has value in structural biology, while the importance of the latter is becoming recognized in synthetic chemistry, material sciences and related areas.
Acaryochloris marina is one of the cyanobacteria that can use far-red light to drive photochemica... more Acaryochloris marina is one of the cyanobacteria that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of the photosystem I reaction center of A. marina determined by cryo-electron microscopy at 2.5 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments different from other type I reaction centers. The paired chlorophyll, so-called special pair, of P740 is a dimer of chlorophyll d/d′ and the primary electron acceptor is pheophytin a, a metal-less chlorin different from the chlorophyll a common to all other type I reaction centers. Here we show the architecture of the photosystem I reaction center is composed of 11 subunits and identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.
Acta Crystallographica Section A Foundations and Advances, Dec 1, 2017
Electron crystallography has the potential to analyze crystals of membrane proteins and macromole... more Electron crystallography has the potential to analyze crystals of membrane proteins and macromolecular complexes too small or too thin for X-ray crystallography, as electrons are scattered 4-5 orders of magnitude more strongly than X-rays. Electron crystallography yields Coulomb potential maps, rather than electron density maps as X-rays do, providing information on charged states of amino-acids and metals. We present such Coulomb potential maps at 3.4 Ă… and 3.2 Ă… resolution, respectively, of Ca2+-ATPase and catalase obtained from crystals of just a few layers thick. These maps demonstrate that it is indeed possible to build atomic models from such crystals and charge information is included, often critical in understanding protein function [1]. We also report a suitable treatment of electron scattering factors of charged atoms for refinement of atomic models against cryo-EM maps [2]. The ScatCurve package developed supports various models for parameterization of scattering curves, and the parameter tables can be implemented in major refinement programs in structural biology. We changed partial charge values of charged amino acid in crystal structures in steps of 0.1 for refinement of the atomic models against electron diffraction data from three-dimensional crystals. By exploring a range of partial charges, we found the proper electrostatic setting to produce atomic models with improved statistics and to better reflect experimental data. Structure refinement for single particle analysis also benefits from the more accurate analysis, and our programs could find wide use for model refinement against cryo-EM maps.
Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis... more Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis, orchitis, meningitis, and encephalitis. MuV-related hearing loss is also a serious complication because it is usually irreversible. MuV outbreaks have been reported in many countries, even in high-vaccine-coverage areas. MuV has tropism toward glandular tissues and the central nervous system. To understand the unique MuV tropism, revealing the mechanism of receptor recognition by MuV is very important. Here, using a large-scale glycan array and X-ray crystallography, we show that MuV recognizes sialyl Lewis X and GM2 ganglioside as receptors, in addition to a previously reported MuV receptor, a trisaccharide containing an α2,3-linked sialic acid. The flexible recognition of these glycan receptors by MuV may explain the unique tropism and pathogenesis of MuV. Structures will also provide a template for the development of effective entry inhibitors targeting the receptor-binding site of ...
Sarcoplasmic reticulum Ca2+-ATPase is an integral membrane protein of Mr 110k and pumps Ca2+ agai... more Sarcoplasmic reticulum Ca2+-ATPase is an integral membrane protein of Mr 110k and pumps Ca2+ against a large concentration gradient during relaxation of muscle.
This paper reports a suitable treatment of electron scattering factors of charged atoms for refin... more This paper reports a suitable treatment of electron scattering factors of charged atoms for refinement of atomic models against cryo-electron microscopy (cryo-EM) maps. The ScatCurve package developed here supports various curve models for parameterization of scattering factors and the parameter tables can be implemented in major refinement programs in structural biology. Partial charge values of charged amino acids in crystal structures were changed in small steps for refinement of the atomic models against electron diffraction data from three-dimensional crystals. By exploring a range of partial charges, the authors found the electrostatic setting that produces atomic models with improved statistics and better reflects experimental data. Structure refinement for single-particle analysis also benefits from the more accurate analysis and the programs could find wide use for model refinement against cryo-EM maps.
Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-pr... more Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-protein coupled receptors (GPCRs), and the extracellular ligand binding domains (LBDs) of T1r1/T1r3 and T1r2/T1r3 heterodimers are responsible for binding of chemical substances eliciting umami or sweet taste. However, molecular analyses of T1r have been hampered due to the difficulties in recombinant expression and protein purification, and thus little is known about mechanisms for taste perception. Here we show the first molecular view of reception of a taste substance by a taste receptor, where the binding of the taste substance elicits a different conformational state of T1r2/T1r3 LBD heterodimer. Electron microscopy has showed a characteristic dimeric structure. Förster resonance energy transfer and X-ray solution scattering have revealed the transition of the dimerization manner of the ligand binding domains, from a widely spread to compactly organized state upon taste substance bind...
There are many huge macromolecular complexes in living organisms. They are often hard to crystall... more There are many huge macromolecular complexes in living organisms. They are often hard to crystallize because of their size, complexity and heterogeneity. Cryo-electron microscopy (cryo-EM) is a suitable method to analyze the structures of such biological macromolecules, because it can be applied to various forms of samples, e.g. two-dimensional crystal, helical assembly, spherical virus, dispersed particle, cell organelle and cell, although attainable resolution depends on the system. In this review, I introduce these techniques and examples of the structure analysis, and briefly review the perspective of cryo-EM.
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Papers by Koji YONEKURA