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Masatsune Kainosho - One of the best experts on this subject based on the ideXlab platform.
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nano mole scale side chain Signal Assignment by 1h detected protein solid state nmr by ultra fast magic angle spinning and stereo array isotope labeling
PLOS ONE, 2015Co-Authors: Songlin Wang, Sudhakar Parthasarathy, Yusuke Nishiyama, Yuki Endo, Takahiro Nemoto, Kazuo Yamauchi, Tetsuo Asakura, Mitsuhiro Takeda, Tsutomu Terauchi, Masatsune KainoshoAbstract:We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain Signal Assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain Assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C Assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain Signal Assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.
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Alternative SAIL-Trp for robust aromatic Signal Assignment and determination of the χ_2 conformation by intra-residue NOEs
Journal of Biomolecular NMR, 2011Co-Authors: Yohei Miyanoiri, Mitsuhiro Takeda, Tsutomu Terauchi, Kosuke Okuma, Masatsune KainoshoAbstract:Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U–^13C,^15N]-proteins, the observation and Assignment of the ring Signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR Assignment, exclusively through the ^13C–^13C and ^13C–^1H spin coupling networks (Kainosho et al. in Nature 440:52–57, 2006 ). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3-^2H_2; δ1,ε3,η2-^13C_3; ε1-^15N]-indole ring ([^12C _γ, ^ 12 C_ε2] SAIL-Trp), which provides a more robust way to correlate the ^1H_β, ^1H_α, and ^1H_N to the ^1H_δ1 and ^1H_ε3 through the intra-residue NOEs. The Assignment of the ^1H_δ1/^13C_δ1 and ^1H_ε3/^13C_ε3 Signals can thus be transferred to the ^1H_ε1/^15N_ε1 and ^1H_η2/^13C_η2 Signals, as with the previous type of SAIL-Trp, which has an extra ^13C at the C_γ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was ^1H_β2 in this experiment, one can determine the side-chain conformation of the Trp residue including the χ_2 angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [^12C_γ,^12C_ε2] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.
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alternative sail trp for robust aromatic Signal Assignment and determination of the χ 2 conformation by intra residue noes
Journal of Biomolecular NMR, 2011Co-Authors: Yohei Miyanoiri, Mitsuhiro Takeda, Tsutomu Terauchi, Masatsune Kainosho, Kosuke OkumaAbstract:Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U–13C,15N]-proteins, the observation and Assignment of the ring Signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR Assignment, exclusively through the 13C–13C and 13C–1H spin coupling networks (Kainosho et al. in Nature 440:52–57, 2006). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3-2H2; δ1,e3,η2-13C3; e1-15N]-indole ring ([12C γ, 12 Ce2] SAIL-Trp), which provides a more robust way to correlate the 1Hβ, 1Hα, and 1HN to the 1Hδ1 and 1He3 through the intra-residue NOEs. The Assignment of the 1Hδ1/13Cδ1 and 1He3/13Ce3 Signals can thus be transferred to the 1He1/15Ne1 and 1Hη2/13Cη2 Signals, as with the previous type of SAIL-Trp, which has an extra 13C at the Cγ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was 1Hβ2 in this experiment, one can determine the side-chain conformation of the Trp residue including the χ2 angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [12Cγ,12Ce2] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.
Kiyoshi Fukuchi - One of the best experts on this subject based on the ideXlab platform.
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Crosstalk Reduction With Bidirectional Signal Assignment on Square Lattice Structure 16-Core Fiber Over WDM Transmission for Gradual Upgrade of SMF-Based Lines
Journal of Lightwave Technology, 2016Co-Authors: Manabu Arikawa, Emmanuel Le Taillandier De Gabory, Kiyoshi FukuchiAbstract:C-band and L-band wavelength division multiplexing (WDM) transmission performance was improved by using bidirectional Signal Assignment (BSA) to reduce crosstalk (XT) in square lattice structure 16-core fiber. Transmission of 16 WDM channels with 256-Gb/s polarization multiplexed-16 quadrature amplitude modulation Signals on a 50-GHz grid with a net spectral efficiency of 4 b/s/Hz over 55-km of 16-core fiber spans was achieved. Use of BSA to reduce XT extended the transmission distance to 880 km in both types of transmission. Furthermore, transmission was achieved over eight mixed spans consisting of 16-core fiber and conventional single-mode fiber (SMF), demonstrating the possibility of gradually upgrading standard SMF-based lines to multicore fiber-based lines. With BSA, six 16-core fiber spans can be acceptable if the Q penalty from XT is below 0.5 dB.
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Crosstalk reduction using bidirectional Signal Assignment over square lattice structure 16-core fiber for gradual upgrade of SSMF-based lines
2015 European Conference on Optical Communication (ECOC), 2015Co-Authors: Manabu Arikawa, Emmanuel Le Taillandier De Gabory, Kiyoshi FukuchiAbstract:We demonstrate crosstalk reduction using bidirectional Signal Assignment on WDM transmission of 256 Gb/s PM-16QAM over square lattice structure 16-core fiber. Mixed SSMF and MCF transmission with 6 spans of MCF is possible with Q crosstalk penalty below 0.5 dB.
Hideo Akutsu - One of the best experts on this subject based on the ideXlab platform.
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spectral fitting for Signal Assignment and structural analysis of uniformly 13c labeled solid proteins by simulated annealing based on chemical shifts and spin dynamics
Journal of Biomolecular NMR, 2007Co-Authors: Yoh Matsuki, Hideo Akutsu, Toshimichi FujiwaraAbstract:We describe an approach for the Signal Assignment and structural analysis with a suite of two-dimensional 13C–13C magic-angle-spinning solid-state NMR spectra of uniformly 13C-labeled peptides and proteins. We directly fit the calculated spectra to experimental ones by simulated annealing in restrained molecular dynamics program CNS as a function of atomic coordinates. The spectra are calculated from the conformation dependent chemical shift obtained with SHIFTX and the cross-peak intensities computed for recoupled dipolar interactions. This method was applied to a membrane-bound 14-residue peptide, mastoparan-X. The obtained C′, Cα and Cβ chemical shifts agreed with those reported previously at the precisions of 0.2, 0.7 and 0.4 ppm, respectively. This spectral fitting program also provides backbone dihedral angles with a precision of about 50° from the spectra even with resonance overlaps. The restraints on the angles were improved by applying protein database program TALOS to the obtained chemical shifts. The peptide structure provided by these restraints was consistent with the reported structure at the backbone RMSD of about 1 A.
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Signal Assignment and secondary structure analysis of a uniformly 13c 15n labeled membrane protein h atp synthase subunit c by magic angle spinning solid state nmr
Journal of Biomolecular NMR, 2006Co-Authors: Masatoshi Kobayashi, Yoh Matsuki, Toshimichi Fujiwara, Ikuko Yumen, Hideo AkutsuAbstract:Signal Assignment and secondary structural analysis of uniformly [13C, 15N] labeled H+-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the 13C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue 13C–13C and 13C–15N chemical shift correlations, 78% of \({\rm C}^{\upalpha}\), 72% of \({\rm C}^{\upbeta}\), 62% of C′ and 61% of NH Signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific Assignment. On top of this, the 13C–13C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and Signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by \(\upalpha\)-helices with a loop structure in the middle (at sequence 41–45) as in the case of the solution structure in spite of additional extended conformations at 76–79 at the C-terminus.
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Signal Assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H +-ATP synthase subunit c, by magic-angle spinning solid-state NMR.
Journal of Biomolecular NMR, 2006Co-Authors: Masatoshi Kobayashi, Yoh Matsuki, Toshimichi Fujiwara, Ikuko Yumen, Hideo AkutsuAbstract:Signal Assignment and secondary structural analysis of uniformly [13C, 15N] labeled H+-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the 13C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue 13C–13C and 13C–15N chemical shift correlations, 78% of \({\rm C}^{\upalpha}\), 72% of \({\rm C}^{\upbeta}\), 62% of C′ and 61% of NH Signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific Assignment. On top of this, the 13C–13C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and Signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by \(\upalpha\)-helices with a loop structure in the middle (at sequence 41–45) as in the case of the solution structure in spite of additional extended conformations at 76–79 at the C-terminus.
Toshimichi Fujiwara - One of the best experts on this subject based on the ideXlab platform.
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noise peak filtering in multi dimensional nmr spectra using convolutional neural networks
Bioinformatics, 2018Co-Authors: Naohiro Kobayashi, Yoshikazu Hattori, Takashi Nagata, Shoko Shinya, Peter Guntert, Chojiro Kojima, Toshimichi FujiwaraAbstract:Motivation: Multi-dimensional NMR spectra are generally used for NMR Signal Assignment and structure analysis. There are several programs that can achieve highly automated NMR Signal Assignments and structure analysis. On the other hand, NMR spectra tend to have a large number of noise peaks even for data acquired with good sample and machine conditions, and it is still difficult to eliminate these noise peaks. Results: We have developed a method to eliminate noise peaks using convolutional neural networks, implemented in the program package Filt_Robot. The filtering accuracy of Filt_Robot was around 90-95% when applied to 2D and 3D NMR spectra, and the numbers of resulting non-noise peaks were close to those in corresponding manually prepared peaks lists. The filtering can strongly enhance automated NMR spectra analysis. Availability and implementation: The full package of the program, documents and example data are available from http://bmrbdep.pdbj.org/en/nmr_tool_box/Filt_Robot.html. Supplementary information: Supplementary data are available at Bioinformatics online.
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spectral fitting for Signal Assignment and structural analysis of uniformly 13c labeled solid proteins by simulated annealing based on chemical shifts and spin dynamics
Journal of Biomolecular NMR, 2007Co-Authors: Yoh Matsuki, Hideo Akutsu, Toshimichi FujiwaraAbstract:We describe an approach for the Signal Assignment and structural analysis with a suite of two-dimensional 13C–13C magic-angle-spinning solid-state NMR spectra of uniformly 13C-labeled peptides and proteins. We directly fit the calculated spectra to experimental ones by simulated annealing in restrained molecular dynamics program CNS as a function of atomic coordinates. The spectra are calculated from the conformation dependent chemical shift obtained with SHIFTX and the cross-peak intensities computed for recoupled dipolar interactions. This method was applied to a membrane-bound 14-residue peptide, mastoparan-X. The obtained C′, Cα and Cβ chemical shifts agreed with those reported previously at the precisions of 0.2, 0.7 and 0.4 ppm, respectively. This spectral fitting program also provides backbone dihedral angles with a precision of about 50° from the spectra even with resonance overlaps. The restraints on the angles were improved by applying protein database program TALOS to the obtained chemical shifts. The peptide structure provided by these restraints was consistent with the reported structure at the backbone RMSD of about 1 A.
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Signal Assignment and secondary structure analysis of a uniformly 13c 15n labeled membrane protein h atp synthase subunit c by magic angle spinning solid state nmr
Journal of Biomolecular NMR, 2006Co-Authors: Masatoshi Kobayashi, Yoh Matsuki, Toshimichi Fujiwara, Ikuko Yumen, Hideo AkutsuAbstract:Signal Assignment and secondary structural analysis of uniformly [13C, 15N] labeled H+-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the 13C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue 13C–13C and 13C–15N chemical shift correlations, 78% of \({\rm C}^{\upalpha}\), 72% of \({\rm C}^{\upbeta}\), 62% of C′ and 61% of NH Signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific Assignment. On top of this, the 13C–13C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and Signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by \(\upalpha\)-helices with a loop structure in the middle (at sequence 41–45) as in the case of the solution structure in spite of additional extended conformations at 76–79 at the C-terminus.
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Signal Assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H +-ATP synthase subunit c, by magic-angle spinning solid-state NMR.
Journal of Biomolecular NMR, 2006Co-Authors: Masatoshi Kobayashi, Yoh Matsuki, Toshimichi Fujiwara, Ikuko Yumen, Hideo AkutsuAbstract:Signal Assignment and secondary structural analysis of uniformly [13C, 15N] labeled H+-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the 13C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue 13C–13C and 13C–15N chemical shift correlations, 78% of \({\rm C}^{\upalpha}\), 72% of \({\rm C}^{\upbeta}\), 62% of C′ and 61% of NH Signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific Assignment. On top of this, the 13C–13C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and Signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by \(\upalpha\)-helices with a loop structure in the middle (at sequence 41–45) as in the case of the solution structure in spite of additional extended conformations at 76–79 at the C-terminus.
Manabu Arikawa - One of the best experts on this subject based on the ideXlab platform.
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Crosstalk Reduction With Bidirectional Signal Assignment on Square Lattice Structure 16-Core Fiber Over WDM Transmission for Gradual Upgrade of SMF-Based Lines
Journal of Lightwave Technology, 2016Co-Authors: Manabu Arikawa, Emmanuel Le Taillandier De Gabory, Kiyoshi FukuchiAbstract:C-band and L-band wavelength division multiplexing (WDM) transmission performance was improved by using bidirectional Signal Assignment (BSA) to reduce crosstalk (XT) in square lattice structure 16-core fiber. Transmission of 16 WDM channels with 256-Gb/s polarization multiplexed-16 quadrature amplitude modulation Signals on a 50-GHz grid with a net spectral efficiency of 4 b/s/Hz over 55-km of 16-core fiber spans was achieved. Use of BSA to reduce XT extended the transmission distance to 880 km in both types of transmission. Furthermore, transmission was achieved over eight mixed spans consisting of 16-core fiber and conventional single-mode fiber (SMF), demonstrating the possibility of gradually upgrading standard SMF-based lines to multicore fiber-based lines. With BSA, six 16-core fiber spans can be acceptable if the Q penalty from XT is below 0.5 dB.
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Crosstalk reduction using bidirectional Signal Assignment over square lattice structure 16-core fiber for gradual upgrade of SSMF-based lines
2015 European Conference on Optical Communication (ECOC), 2015Co-Authors: Manabu Arikawa, Emmanuel Le Taillandier De Gabory, Kiyoshi FukuchiAbstract:We demonstrate crosstalk reduction using bidirectional Signal Assignment on WDM transmission of 256 Gb/s PM-16QAM over square lattice structure 16-core fiber. Mixed SSMF and MCF transmission with 6 spans of MCF is possible with Q crosstalk penalty below 0.5 dB.
