Crystal structures of γ-glutamylmethylamide synthetase provide insight into bacterial metabolism of oceanic monomethylamine
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015952
ISSN1083-351X
AutoresNing Wang, Xiu‐Lan Chen, Chao Gao, Ming Peng, Peng Wang, Na Zhang, Fuchuan Li, Gui‐Peng Yang, Qing-Tao Shen, Shengying Li, Yin Chen, Yu‐Zhong Zhang, Chunyang Li,
Tópico(s)Methane Hydrates and Related Phenomena
ResumoMonomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. γ-Glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide, the first step in MMA metabolism in many marine bacteria. The gmaS gene occurs in ∼23% of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria. However, the catalytic mechanism of GmaS has not been studied because of the lack of structural information. Here, the GmaS from Rhodovulum sp. 12E13 (RhGmaS) was characterized, and the crystal structures of apo-RhGmaS and RhGmaS with different ligands in five states were solved. Based on structural and biochemical analyses, the catalytic mechanism of RhGmaS was explained. ATP is first bound in RhGmaS, leading to a conformational change of a flexible loop (Lys287-Ile305), which is essential for the subsequent binding of glutamate. During the catalysis of RhGmaS, the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate; Asp177 is responsible for the deprotonation of MMA, assisting the attack of MMA on γ-glutamyl phosphate to produce a tetrahedral intermediate; and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate glutamylmethylamide. Sequence analysis suggested that the catalytic mechanism of RhGmaS proposed in this study has universal significance in bacteria containing GmaS. Our results provide novel insights into MMA metabolism, contributing to a better understanding of MMA catabolism in global carbon and nitrogen cycles. Monomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. γ-Glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide, the first step in MMA metabolism in many marine bacteria. The gmaS gene occurs in ∼23% of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria. However, the catalytic mechanism of GmaS has not been studied because of the lack of structural information. Here, the GmaS from Rhodovulum sp. 12E13 (RhGmaS) was characterized, and the crystal structures of apo-RhGmaS and RhGmaS with different ligands in five states were solved. Based on structural and biochemical analyses, the catalytic mechanism of RhGmaS was explained. ATP is first bound in RhGmaS, leading to a conformational change of a flexible loop (Lys287-Ile305), which is essential for the subsequent binding of glutamate. During the catalysis of RhGmaS, the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate; Asp177 is responsible for the deprotonation of MMA, assisting the attack of MMA on γ-glutamyl phosphate to produce a tetrahedral intermediate; and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate glutamylmethylamide. Sequence analysis suggested that the catalytic mechanism of RhGmaS proposed in this study has universal significance in bacteria containing GmaS. Our results provide novel insights into MMA metabolism, contributing to a better understanding of MMA catabolism in global carbon and nitrogen cycles. Methylated amines, such as monomethylamine (MMA), dimethylamine, trimethylamine, and trimethylamine N-oxide, are widely distributed in marine environments and play important roles in marine carbon (C) and nitrogen (N) cycles (1Gibb S.W. Mantoura R.F.C. Liss P.S. Barlow R.G. Distributions and biogeochemistries of methylamines and ammonium in the Arabian Sea.Deep Sea Res. Part II. 1999; 46: 593-615Crossref Scopus (41) Google Scholar, 2Stein L.Y. Methylamine: a vital nitrogen (and carbon) source for marine microbes.Environ. Microbiol. 2017; 19: 2117-2118Crossref PubMed Scopus (2) Google Scholar, 3Carpenter L.J. Archer S.D. Beale R. Ocean-atmosphere trace gas exchange.Chem. Soc. Rev. 2012; 41: 6473-6506Crossref PubMed Scopus (131) Google Scholar). In the ocean, the major source of methylated amines is likely the breakdown of quaternary amine osmolytes, such as betaine, choline, and carnitine (4Chen Y. Scanlan J. Song L. Crombie A. Rahman M.T. Schäfer H. Murrell J.C. Gamma-glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylocella silvestris.Appl. Environ. Microbiol. 2010; 76: 4530-4537Crossref PubMed Scopus (42) Google Scholar, 5Poste A.E. Grung M. Wright R.F. 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Ocean-atmosphere trace gas exchange.Chem. Soc. Rev. 2012; 41: 6473-6506Crossref PubMed Scopus (131) Google Scholar, 10Lidbury I. Mausz M.A. Scanlan D.J. Chen Y. Identification of dimethylamine monooxygenase in marine bacteria reveals a metabolic bottleneck in the methylated amine degradation pathway.ISME J. 2017; 11: 1592-1601Crossref PubMed Scopus (12) Google Scholar). There are two pathways identified from bacteria for aerobic MMA metabolism, a direct MMA-oxidation pathway and an indirect MMA-oxidation pathway (6Wischer D. Kumaresan D. Johnston A. El Khawand M. Stephenson J. Hillebrand-Voiculescu A.M. Chen Y. Colin Murrell J. Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave.ISME J. 2015; 9: 195-206Crossref PubMed Scopus (27) Google Scholar). The direct MMA-oxidation pathway is only found in methylotrophic bacteria, through which MMA is metabolized to formaldehyde and ammonium by a single enzyme (4Chen Y. Scanlan J. Song L. Crombie A. Rahman M.T. Schäfer H. Murrell J.C. Gamma-glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylocella silvestris.Appl. Environ. Microbiol. 2010; 76: 4530-4537Crossref PubMed Scopus (42) Google Scholar, 6Wischer D. Kumaresan D. Johnston A. El Khawand M. Stephenson J. Hillebrand-Voiculescu A.M. Chen Y. Colin Murrell J. Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave.ISME J. 2015; 9: 195-206Crossref PubMed Scopus (27) Google Scholar). This enzyme is an MMA oxidase in gram-positive bacteria such as Arthrobacter (11Zhang X. Fuller J.H. McIntire W.S. Cloning, sequencing, expression, and regulation of the structural gene for the copper/topa quinone-containing methylamine oxidase from Arthrobacter strain P1, a gram-positive facultative methylotroph.J. 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Murrell J.C. Gamma-glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylocella silvestris.Appl. Environ. Microbiol. 2010; 76: 4530-4537Crossref PubMed Scopus (42) Google Scholar, 8Latypova E. Yang S. Wang Y.S. Wang T. Chavkin T.A. Hackett M. Schäfer H. Kalyuzhnaya M.G. Genetics of the glutamate-mediated methylamine utilization pathway in the facultative methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5.Mol. Microbiol. 2010; 75: 426-439Crossref PubMed Scopus (61) Google Scholar). Because NMG is an essential intermediate of the indirect MMA-oxidation pathway, this pathway is also termed as the NMG pathway. Unlike the direct MMA-oxidation pathway which is only found in methylotrophic bacteria to date, the NMG pathway is adopted by both methylotrophic and nonmethylotrophic bacteria, in particular, by many bacteria of the marine Roseobacter clade (MRC) (14Lidbury I.D.E.A. Murrell J.C. Chen Y. Trimethylamine and trimethylamine N-oxide are supplementary energy sources for a marine heterotrophic bacterium: implications for marine carbon and nitrogen cycling.ISME J. 2015; 9: 760-769Crossref PubMed Scopus (45) Google Scholar). MRC bacteria are ubiquitous and numerically abundant in marine environments (15Selje N. Simon M. Brinkhoff T. A newly discovered Roseobacter cluster in temperate and polar oceans.Nature. 2004; 427: 445-448Crossref PubMed Scopus (211) Google Scholar, 16González J.M. Moran M.A. Numerical dominance of a group of marine bacteria in the alpha-subclass of the class Proteobacteria in coastal seawater.Appl. Environ. Microbiol. 1997; 63: 4237-4242Crossref PubMed Google Scholar) and are important participants in MMA metabolism (14Lidbury I.D.E.A. Murrell J.C. Chen Y. 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Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave.ISME J. 2015; 9: 195-206Crossref PubMed Scopus (27) Google Scholar, 18Chen Y. Comparative genomics of methylated amine utilization by marine Roseobacter clade bacteria and development of functional gene markers (tmm, gmaS).Environ. Microbiol. 2012; 14: 2308-2322Crossref PubMed Scopus (44) Google Scholar). The gmaS gene occurs in ∼23% of microbial genomes in the surface ocean (17Chen Y. Patel N.A. Crombie A. Scrivens J.H. Murrell J.C. Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 17791-17796Crossref PubMed Scopus (76) Google Scholar), suggesting that GmaS plays an important role in marine N and C cycles. However, despite that the biochemical characteristics of GmaS from several strains have been reported (19Kimura T. Sugahara I. Hanai K. Tonomura Y. Purification and characterization of gamma-glutamylmethylamide synthetase from Methylophaga sp. AA-30.Biosci. Biotechnol. Biochem. 1992; 56: 708-711Crossref PubMed Scopus (21) Google Scholar, 20Yamamoto S. Wakayama M. Tachiki T. Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production.Biosci. Biotechnol. Biochem. 2007; 71: 545-552Crossref PubMed Scopus (25) Google Scholar, 21Yamamoto S. Wakayama M. Tachiki T. Cloning and expression of Methylovorus mays No. 9 gene encoding gamma-glutamylmethylamide synthetase: an enzyme usable in theanine formation by coupling with the alcoholic fermentation system of baker's yeast.Biosci. Biotechnol. Biochem. 2008; 72: 101-109Crossref PubMed Scopus (27) Google Scholar, 22Yang S.Y. Han Y.H. Park Y.L. Park J.Y. No S.Y. Jeong D. Park S. Park H.Y. Kim W. Seo S.O. Yang Y.H. Production of L-Theanine Using Escherichia coli whole-cell overexpressing gamma-glutamylmethylamide synthetase with baker's yeast.J. Microbiol. Biotechnol. 2020; 30: 785-792Crossref PubMed Scopus (6) Google Scholar), little is known about the molecular mechanism of GmaS catalyzing the conversion of MMA to GMA. Sequence analysis indicates that GmaS is closely related to, but distinct from, the glutamine synthetase (GS) family, one of the oldest and most ubiquitously existing families of enzymes in biota (4Chen Y. Scanlan J. Song L. Crombie A. Rahman M.T. Schäfer H. Murrell J.C. Gamma-glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylocella silvestris.Appl. Environ. Microbiol. 2010; 76: 4530-4537Crossref PubMed Scopus (42) Google Scholar, 23Kumada Y. Benson D.R. Hillemann D. Hosted T.J. Rochefort D.A. Thompson C.J. Wohlleben W. Tateno Y. Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes.Proc. Natl. Acad. 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In this study, we report the structure of a GmaS enzyme and its molecular mechanism for the conversion of MMA to GMA. The gmaS gene from strain Rhodovulum sp. 12E13 (RhgmaS) was expressed, and the recombinant RhGmaS was purified and characterized. Six crystal structures of RhGmaS in different states were solved. Based on structural and mutational assays, we proposed the molecular mechanism of RhGmaS for the conversion of MMA to GMA. The results provide novel insights into MMA metabolism, leading to a better understanding of MMA catabolism in global C and N cycles. Full-length RhgmaS of R. sp. 12E13 contains 1293 nucleotides and encodes a 430-amino-acid polypeptide, which shows 61% identity to the functional GmaS from a type strain of MRC, Ruegeria pomeroyi DSS-3 (18Chen Y. Comparative genomics of methylated amine utilization by marine Roseobacter clade bacteria and development of functional gene markers (tmm, gmaS).Environ. Microbiol. 2012; 14: 2308-2322Crossref PubMed Scopus (44) Google Scholar, 32Chen Y. McAleer K.L. Murrell J.C. Monomethylamine as a nitrogen source for a nonmethylotrophic bacterium, Agrobacterium tumefaciens.Appl. Environ. Microbiol. 2010; 76: 4102-4104Crossref PubMed Scopus (26) Google Scholar). We chemically synthesized RhgmaS, expressed it in Escherichia coli BL21 (DE3), and characterized the recombinant RhGmaS. The recombinant RhGmaS was active to catalyze the ligation of MMA and glutamate to produce GMA, with ATP and Mg2+ as cofactors. The optimal pH for RhGmaS enzymatic activity was ∼8.0 (Fig. 1A), similar to that of Methylovorus mays No.9 GmaS (7.5–8.0) (21Yamamoto S. Wakayama M. Tachiki T. Cloning and expression of Methylovorus mays No. 9 gene encoding gamma-glutamylmethylamide synthetase: an enzyme usable in theanine formation by coupling with the alcoholic fermentation system of baker's yeast.Biosci. Biotechnol. Biochem. 2008; 72: 101-109Crossref PubMed Scopus (27) Google Scholar). The optimal temperature of RhGmaS was 60 °C (Fig. 1B), which is higher than that of Methylophaga sp. AA-30 GmaS (40 °C) (19Kimura T. Sugahara I. Hanai K. Tonomura Y. Purification and characterization of gamma-glutamylmethylamide synthetase from Methylophaga sp. AA-30.Biosci. Biotechnol. Biochem. 1992; 56: 708-711Crossref PubMed Scopus (21) Google Scholar) and of M. mays No.9 GmaS (50 °C) (22Yang S.Y. Han Y.H. Park Y.L. Park J.Y. No S.Y. Jeong D. Park S. Park H.Y. Kim W. Seo S.O. Yang Y.H. Production of L-Theanine Using Escherichia coli whole-cell overexpressing gamma-glutamylmethylamide synthetase with baker's yeast.J. Microbiol. Biotechnol. 2020; 30: 785-792Crossref PubMed Scopus (6) Google Scholar). We noticed that the optimal temperatures of these GmaS enzymes are much higher than those of typical marine surface water column. Then, we purified two other GmaS homologs from MRC strains R. pomeroyi DSS-3 and Dinoroseobacter shibae DFL12. These two GmaS homologs also presented a high optimal temperature of 60 °C (Fig. 1, C–D), indicating that the relatively high optimal temperature is a common trait of GmaS proteins. Even so, RhGmaS still maintains a specific activity of ∼0.71 μmol min−1 mg−1 at 20 °C, suggesting that RhGmaS could work properly under the physiological temperature. The Km of RhGmaS for glutamate was 67.18 mM (Fig. 1E), and that for ATP was 0.42 mM (Fig. 1F). RhGmaS exhibited a Km value of 26.94 μM for MMA (Table 1), which is threefold lower than that of M. sp. AA-30 GmaS (89 μM) (19Kimura T. Sugahara I. Hanai K. Tonomura Y. Purification and characterization of gamma-glutamylmethylamide synthetase from Methylophaga sp. AA-30.Biosci. Biotechnol. Biochem. 1992; 56: 708-711Crossref PubMed Scopus (21) Google Scholar) and sixfold lower than that of M. mays No.9 GmaS (180 μM) (20Yamamoto S. Wakayama M. Tachiki T. Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production.Biosci. Biotechnol. Biochem. 2007; 71: 545-552Crossref PubMed Scopus (25) Google Scholar). The micromolar level of Km values of GmaS enzymes for MMA indicates that GmaS enzymes possess high affinities to MMA.Table 1Kinetic parameters for recombinant RhGmaS with different substratesaThe experiments were performed at pH 8.0, 30 °C. The data shown in the table are from triplicate experiments (means ± SDs).SubstrateKm (μM)Vmax (μM min−1)kcat (s−1)MMA26.94 ± 1.733.58 ± 0.114.18 ± 0.13Ethylamine58.10 ± 4.493.51 ± 0.114.09 ± 0.13Hydroxylamine211.03 ± 9.873.56 ± 0.054.15 ± 0.06Propylamine1.79 × 103 ± 0.12 × 1033.63 ± 0.084.23 ± 0.09NH4Cl8.72 × 103 ± 0.97 × 1033.65 ± 0.174.26 ± 0.20DMA16.07 × 103 ± 1.15 × 1032.20 ± 0.092.57 ± 0.10TMA22.91 × 103 ± 1.50 × 1032.13 ± 0.062.48 ± 0.07DMA, dimethylamine; MMA, monomethylamine; TMA, trimethylamine.a The experiments were performed at pH 8.0, 30 °C. The data shown in the table are from triplicate experiments (means ± SDs). Open table in a new tab DMA, dimethylamine; MMA, monomethylamine; TMA, trimethylamine. Amine donors, such as hydroxylamine and ethylamine, can replace ammonia as substrate for some GS enzymes (20Yamamoto S. Wakayama M. Tachiki T. Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production.Biosci. Biotechnol. Biochem. 2007; 71: 545-552Crossref PubMed Scopus (25) Google Scholar, 24Kamini Sharma R. Punekar N.S. Phale P.S. Carbaryl as a carbon and nitrogen source: an inducible methylamine metabolic pathway at the biochemical and molecular levels in Pseudomonas sp. strain C5pp.Appl. Environ. Microbiol. 2018; 84e01866-18Crossref PubMed Scopus (6) Google Scholar, 30Unno H. Uchida T. Sugawara H. Kurisu G. Sugiyama T. Yamaya T. Sakakibara H. Hase T. Kusunoki M. Atomic structure of plant glutamine synthetase: a key enzyme for plant productivity.J. Biol. Chem. 2006; 281: 29287-29296Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). To determine whether RhGmaS can catalyze different ammonia analogs, we analyzed the substrate specificity of RhGmaS (Table 1). In addition to MMA, RhGmaS can accept ethylamine, hydroxylamine, propylamine, ammonium chloride, dimethylamine, or trimethylamine as a substrate, indicating that this enzyme has a relatively broad substrate specificity. The Km of RhGmaS for MMA was the lowest among the tested ammonia analogs, while the Km for NH4Cl is much higher (Table 1), suggesting that MMA is likely the natural substrate of RhGmaS. This result is consistent with the previous reports that GmaS prefers MMA as its substrate rather than ammonia (4Chen Y. Scanlan J. Song L. Crombie A. Rahman M.T. Schäfer H. Murrell J.C. Gamma-glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylocella silvestris.Appl. Environ. Microbiol. 2010; 76: 4530-4537Crossref PubMed Scopus (42) Google Scholar, 19Kimura T. Sugahara I. Hanai K. Tonomura Y. Purification and characterization of gamma-glutamylmethylamide synthetase from Methylophaga sp. AA-30.Biosci. Biotechnol. Biochem. 1992; 56: 708-711Crossref PubMed Scopus (21) Google Scholar, 20Yamamoto S. Wakayama M. Tachiki T. Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production.Biosci. Biotechnol. Biochem. 2007; 71: 545-552Crossref PubMed Scopus (25) Google Scholar, 24Kamini Sharma R. Punekar N.S. Phale P.S. Carbaryl as a carbon and nitrogen source: an inducible methylamine metabolic pathway at the biochemical and molecular levels in Pseudomonas sp. strain C5pp.Appl. Environ. Microbiol. 2018; 84e01866-18Crossref PubMed Scopus (6) Google Scholar). To investigate whether the N-terminal His-tag would affect the kinetic properties of the recombinant RhGmaS, we used thrombin to cut off the His-tag and measured the kinetic parameters of RhGmaS without His-tag (Fig. S1), which were similar to RhGmaS with His-tag. This result indicates that the presence of the His-tag has little effect on the kinetic properties of the enzyme. The RhGmaS proteins used in this study all contained the His-tag, unless otherwise noted. To gain insight into the catalytic mechanism of RhGmaS, the crystal structure of apo-RhGmaS was solved (Table 2). There are three monomers arranged as a trimer in an asymmetric unit (Fig. 2A), with each monomer composed of 15 α-helices and 13 β-strands. However, gel filtration analysis demonstrated that RhGmaS is a dodecamer in solution (Fig. 2B), which is consistent with the result of electron microscopic analysis (Fig. 2C). The negative staining electron micrograph clearly showed that RhGmaS consists of two hexameric rings, with each ring containing six monomers (Fig. 2C). Thus, RhGmaS should function as a dodecamer in the solution. During structural refinement, we could not find the electron densities of the N-terminal His-tag, suggesting that this tag is flexible. The electron microscopic analysis showed that RhGmaS without His-tag still maintains a dodecamer containing two hexameric rings in the solution (Fig. S2). These data suggest that the presence of the His-tag has little effect on the structural properties of RhGmaS. The overall structure of RhGmaS is similar to the structure of a GS enzyme from Bacillus subtilis (protein data bank [PDB] code: 4LNI), with an RMSD of 0.86 Å between these two structures. The B. subtilis GS also functions as a dodecamer (33Murray D.S. Chinnam N. Tonthat N.K. Whitfill T. Wray L.V. Fisher S.H. Schumacher M.A. Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism.J. Biol. Chem. 2013; 288: 35801-35811Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar).Table 2Crystallographic data collection and refinement of RhGmaSParametersApo-RhGmaSRhGmaS–AMPPCPRhGmaS–AMPPNP–MetSoxRhGmaS–ADP–MetSox-PRhGmaS–ADP-C1RhGmaS–ADP-C2Diffraction data Space groupI222I222I222I222I222I222 Unit cell a, b, c (Å)115.9, 179.0, 192.2110.9, 176.7, 191.8115.6, 174.5, 190.1116.6, 174.7, 190.6114.7, 176.0, 190.5113.4, 177.6, 190.5 α, β, γ (°)90.0, 90.0, 90.090.0, 90.0, 90.090.0, 90.0, 90.090.0, 90.0, 90.090.0, 90.0, 90.090.0, 90.0, 90.0 Resolution range (Å)50.0–2.8 (2.90–2.80) aNumbers in parentheses refer to data in the highest resolution shell.50.0–1.96 (1.99–1.96)50.0–2.3 (2.34–2.30)50.0–2.1 (2.18–2.10)50.0–2.3 (2.34–2.30)50.0–2.3 (2.38–2.30) Redundancy11.3 (10.7)4.6 (2.9)6.4 (6.0)4.0 (3.0)11.5 (10.5)13.4 (12.6) Completeness (%)98.6 (99.1)98.1 (87.7)100.0 (100.0)97.5 (94.5)99.5 (100.0)100.0 (100.0) RmergebRmerge=∑hkl∑i|I(hkl)i - |/∑hkl∑iI(hkl)i, where I is the observed intensity, and I(hkl)i represents the observed intensity of each unique reflection.0.1 (0.6)0.1 (0.4)0.1 (0.4)0.1 (0.5)0.2 (0.4)0.1 (0.5) I/σI54.0 (14.7)14.2 (1.8)18.6 (2.9)21.8 (3.3)34.9 (6.4)22.6 (5.4)Refinement statistics Rwork/Rfree0.20/0.250.17/0.200.16/0.200.16/0.200.17/0.220.18/0.21 RMSD from ideal geometry Bond lengths (Å)0.0090.0070.0070.0070.0070.007 Bond angles (°)1.21.11.11.11.11.1 Ramachandran plot (%) Favored93.697.297.197.695.796.3 Allowed6.22.82.92.34.13.7 Outliers0.2000.10.20 Overall B-factors (Å2)57.118.937.224.335.727.1a Numbers in parentheses refer to data in the highest resolution shell.b Rmerge
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