Mol. Cells 2020; 43(12): 1035-1045
Published online December 23, 2020
https://doi.org/10.14348/molcells.2020.0192
© The Korean Society for Molecular and Cellular Biology
Correspondence to : ksj@kribb.re.kr (SJK); bku@kribb.re.kr (BK)
The Drosophila genome contains four low molecular weightprotein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited our understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophosphatases, but CG31469 was also suggested to be a putative protein arginine phosphatase. Herein, we present the crystal structures of CG31469 and Primo-1, which are the first Drosophila LMW-PTP structures. Structural analysis showed that the two proteins adopt the typical LMW-PTP fold and have a canonically arranged P-loop. Intriguingly, while Primo-1 is presumed to be a canonical LMW-PTP, CG31469 is unique as it contains a threonine residue at the fifth position of the P-loop motif instead of highly conserved isoleucine and a characteristically narrow active site pocket, which should facilitate the accommodation of phosphoarginine. Subsequent biochemical analysis revealed that Primo-1 and CG31469 are enzymatically active on phosphotyrosine and phosphoarginine, respectively, refuting their classification as pseudophosphatases. Collectively, we provide structural and biochemical data on two Drosophila proteins: Primo-1, the canonical LMW-PTP protein, and CG31469, the first investigated eukaryotic protein arginine phosphatase. We named CG31469 as DARP, which stands for Drosophila ARginine Phosphatase.
Keywords crystal structure, DARP, low molecular weightprotein tyrosine phosphatase, Primo-1, protein arginine phosphatase
Protein tyrosine phosphatases (PTPs) are a family of enzymes that mediate the reversible dephosphorylation of phosphorylated tyrosine residues on substrate proteins, one of the most common and critical post-translational modifications (Tonks, 2006). They are closely associated with the regulation of various intracellular signaling and cellular processes by controlling protein-protein interaction, protein stability, and enzyme activity (Ardito et al., 2017; Hunter, 1995). The
LMW-PTPs are found in a broad spectrum of genera from bacteria to higher eukaryotes, including
The four
The DNA fragments coding for DARP and Primo-1 were amplified by polymerase chain reaction and subcloned into the pET21a plasmid, which were then used as templates for preparing mutant proteins: DARP(C7S), DARP(T11I), DARP(Y127F), DARP(T11I·Y127F), and Primo-1(C9S). The proteins were produced in the
The phosphatase activity of Primo-1 toward
Substrate specificity was measured at room temperature using 1 μM wild-type or C7S mutant recombinant DARP and four types of 600 μM phosphoamino acids in a 200 μl reaction mixture containing 0.2 mM 2-amino-6-mercapto-7-methylpurine riboside, 0.2 units purine nucleoside phosphorylase, 50 mM Tris-HCl, and 1 mM MgCl2. The EnzChek Phosphate Assay Kit was used for determining the phosphatase activity of DARP against the four phosphoamino acids by detecting the absorbance at 360 nm for 60 min using a SpectraMax plus 384 spectrophotometer. Kinetic parameters were calculated from initial velocity data, which were obtained at room temperature using 800 nM recombinant DARP and 0 to 5 mM phosphoarginine in a 200 μl reaction mixture containing 0.2 mM 2-amino-6-mercapto-7-methylpurine riboside, 0.2 units purine nucleoside phosphorylase, 50 mM Tris (pH 7.0), and 1 mM MgCl2. Specific activities of the wild-type and mutant DARP proteins toward phosphoarginine and phosphotyrosine were calculated from initial velocity data (0-10 min), which were determined at room temperature by using the EnzChek Phosphate Assay Kit using a 200 μl reaction mixture containing 500 μM protein and 800 nM phosphoarginine at pH 7.0 or 10 μM protein and 5 mM phosphotyrosine at pH 7.5. The nonenzymatic hydrolysis of substrates was also measured for baseline correction using a control sample. Substrate specificity and kinetic constant measurements were performed in triplicate, and specific activity measurements were performed in duplicate.
The atomic coordinates and the structure factors of DARP(C7S) and Primo-1 have been deposited in the Protein Data Bank under the accession codes 7BTG and 7CUY, respectively.
Among the four
Given that DARP and Primo-1 were previously classified as enzymatically inactive pseudophosphatases (Hatzihristidis et al., 2015), we investigated whether the two proteins have any structural feature in their catalytic pocket region that negatively affects their catalytic activity. We first analyzed the P-loop arrangement of the two proteins, which is a key factor restricting phosphatase activity in a number of PTP proteins such as PTPRU, DUSP6, DUSP9, and Tk-PTP (Fjeld et al., 2000; Hay et al., 2020; Hong et al., 2005; Yun et al., 2018; Zhou and Zhang, 1999). The catalytic motifs of DARP and Primo-1 are composed of C7IGNTCR13 and C9LGNICR15, respectively, and are located at the β1−α1 loop, which is called the phosphate-binding loop or simply P-loop (Fig. 1A). The P-loop motif contains the catalytic cysteine residue (substituted with serine in the DARP structure), which functions as a nucleophile, and the conserved arginine residue (Arg13 in DARP and Arg15 in Primo-1), which anchors the phosphate group of the substrate during dephosphorylation (Fig. 2A). The center of P-loop is occupied by a single phosphate ion in the DARP structure and by the sulfonic group of a HEPES molecule in the Primo-1 structure. Oxygen atoms of these molecules mediate hydrogen bonds with the protein main chain amides and electrostatic interactions with the guanidinium group of the conserved arginine residue, which not only stabilize their positioning in the catalytic pocket but also maintain the active loop conformation of the
In contrast to a previous report classifying DARP as a pseudophosphatase (Hatzihristidis et al., 2015), a study by Fuhrmann et al. (2013) identifying
Next, to analyze the accessibility of the catalytic pocket more precisely, we structurally compared the size of the catalytic pocket between eight LMW-PTPs, including DARP and Primo-1, that contain a bound negatively charged ion or a phosphotyrosine-mimetic molecule. Intriguingly, DARP has a characteristically narrow catalytic pocket compared to that of other LMW-PTPs, while the Primo-1 catalytic pocket is comparable to those of canonical enzymes (Fig. 3C). The distance between the Cγ atom of Trp43 and the Cζ atom of Tyr127 is 7.8 Å in DARP, which is shorter than the corresponding distances in human LMW-PTP (9.3 Å), Primo-1 (9.6 Å), and SP-PTP (9.8 Å). Consistently, structural alignments demonstrate that phosphoarginine, rather than phosphotyrosine, might fit into the narrow active pocket of DARP (Fig. 3D). Atomic-level structural analysis revealed several features that presumably contribute to the shape of the DARP catalytic pocket: the bulky side chain of Trp43, which is pushed toward the cleft by the aliphatic chain of Arg41; nonconserved electrostatic interactions between Arg41 and Glu92; and a hydrogen bond between Asn44 and Tyr127 that attracts this tyrosine residue toward the pocket (Fig. 3E). These features are unique to DARP, as they are absent in other LMW-PTP structures, including that of Primo-1, YwlE, and human LMW-PTP (Fig. 3E, Supplementary Fig. S1C). In contrast, the catalytic pocket composition of Primo-1 is highly homologous to that of conventional LMW-PTPs such as human LMW-PTP (Supplementary Fig. S1C). Collectively, these data strongly suggest that the accessibility of the catalytic pocket region, which is controlled by the presence of nonconserved threonine in the P-loop region (Figs. 3A and 3B) and the pocket size (Figs. 3C-3E), determines the substrate preference of DARP.
Our structural data implied that DARP and Primo-1 might be enzymatically active phosphatases rather than pseudophosphatases.
Next, we conducted
The enzymatic property of DARP was further analyzed by comparing the specific activities of wild-type and mutant proteins toward phosphoarginine and phosphotyrosine. Compared to the wild-type, substitution of Thr11 to isoleucine caused a remarkable decrease in the dephosphorylation of phosphoarginine and a noticeable increase in the dephosphorylation of phosphotyrosine (Fig. 5C). These results support the finding of the previous study reporting that the threonine residue at position 5 of the P-loop significantly contributes to the substrate specificity of DARP (Fuhrmann et al., 2013). We also mutated Tyr127 to phenylalanine to abrogate the hydrogen bond between Tyr127 and Asn44 (Fig. 3E), which, according to our crystal structure, is expected to widen the catalytic pocket of DARP and thereby improve phosphotyrosine accessibility (Figs. 3C-3E), without critically altering the active loop arrangement. Activity tests demonstrated that the Y127F mutant was slightly less active on phosphotyrosine than the wild-type enzyme. However, the dephosphorylation activity of the double mutant T11I·Y127F toward phosphotyrosine was noticeably higher than that of the T11I single mutant protein, suggesting that the pocket size of DARP (Fig. 3) is an additional factor controlling the access of phosphotyrosine into the catalytic site (Fig. 5C). Collectively, our biochemical analysis indicates that DARP is a catalytically active arginine phosphatase, and its substrate specificity depends on the amino acid composition of the catalytic pocket region.
In this study, we demonstrated that the two
Arginine, one of the twenty standard amino acids, contains a side chain composed of three-carbon aliphatic straight chain capped by a positively charged guanidinium group where a phosphate moiety can be covalently linked to. To date, phosphorylation and dephosphorylation of this amino acid, which are nonconventional compared to those of serine, threonine, and tyrosine, were mostly investigated in gram-positive bacteria; these reactions are involved in diverse bacterial physiological processes such as stress responses and protein degradation (Elsholz et al., 2012; Fuhrmann et al., 2016; Schmidt et al., 2014; Trentini et al., 2016). Contrastively, even though arginine phosphorylation was first reported in mouse proteins more than 30 years ago (Matthews, 1995; Wakim and Aswad, 1994), its precise biological functionality and associated proteins in eukaryotes remain to be elucidated. Therefore, the physiological role of DARP in
In summary, we determined the crystal structures of DARP and Primo-1, which are catalytically active phosphatases with an LMW-PTP fold. Structural and biochemical analyses revealed that DARP and Primo-1 dephosphorylate phosphoarginine and phosphotyrosine, respectively. The results of the present study will aid in finding unknown substrates for these proteins and signaling pathways in which the proteins are involved. This, in turn, might expand our understanding of post-translational phosphorylation and dephosphorylation controlling signaling pathways in
We thank the beamline 7A at the Pohang Accelerator Laboratory in Korea for assistance during the data collection. This work was supported by the National Research Foundation of Korea funded by the Ministry of Science and ICT (MSIT; 2019M3E5D6063955) and by the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program (to B.K.).
H.S.L. and Y.M. conceived and analyzed experiments and wrote the manuscript. H.C.S. provided expertise and feedback. S.J.K. provided supervision. B.K. conceived and analyzed experiments, wrote the manuscript, secured funding, and provided supervision.
The authors have no potential conflicts of interest to disclose.
Data collection and structure refinement statistics
DARP(C7S) | Primo-1 | |
---|---|---|
Data collection | ||
Space group | ||
Unit cell dimensions | ||
a, b, c (Å) | 40.2, 62.2, 71.1 | 45.9, 59.9, 60.9 |
α, β, γ (°) | 90, 90, 90 | 90, 110.2, 90 |
Wavelength (Å) | 0.9793 | 0.9793 |
Resolution (Å) | 50.0-2.2 (2.24-2.20) | 50.0-2.1 (2.14-2.10) |
7.2 (26.1) | 9.5 (25.2) | |
39.9 (6.2) | 23.8 (4.3) | |
Completeness (%) | 98.2 (96.1) | 97.4 (94.2) |
Redundancy | 5.3 | 4.7 |
Refinement | ||
Resolution (Å) | 50.0-2.2 | 50.0-2.1 |
No. of reflections | 9,483 | 18,040 |
20.3/ 25.4 | 18.0/ 24.0 | |
No. of atoms | ||
Protein | 1,247 | 2,396 |
Water and ion | 48 | 123 |
HEPES | - | 30 |
RMSD | ||
Bond length (Å) | 0.008 | 0.008 |
Bond angle (°) | 1.051 | 0.954 |
Ramachandran plot (%) | ||
Most favored region | 97.4 | 98.0 |
Additionally allowed region | 2.6 | 2.0 |
Average B-value (Å2) | ||
Protein | 38.7 | 29.4 |
Water and ion | 41.5 | 32.9 |
HEPES | - | 30.7 |
Mol. Cells 2020; 43(12): 1035-1045
Published online December 31, 2020 https://doi.org/10.14348/molcells.2020.0192
Copyright © The Korean Society for Molecular and Cellular Biology.
Hye Seon Lee1,2 , Yeajin Mo1,2, Ho-Chul Shin1
, Seung Jun Kim1,*
, and Bonsu Ku1,*
1Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea,2These authors contributed equally to this work.
Correspondence to:ksj@kribb.re.kr (SJK); bku@kribb.re.kr (BK)
The Drosophila genome contains four low molecular weightprotein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited our understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophosphatases, but CG31469 was also suggested to be a putative protein arginine phosphatase. Herein, we present the crystal structures of CG31469 and Primo-1, which are the first Drosophila LMW-PTP structures. Structural analysis showed that the two proteins adopt the typical LMW-PTP fold and have a canonically arranged P-loop. Intriguingly, while Primo-1 is presumed to be a canonical LMW-PTP, CG31469 is unique as it contains a threonine residue at the fifth position of the P-loop motif instead of highly conserved isoleucine and a characteristically narrow active site pocket, which should facilitate the accommodation of phosphoarginine. Subsequent biochemical analysis revealed that Primo-1 and CG31469 are enzymatically active on phosphotyrosine and phosphoarginine, respectively, refuting their classification as pseudophosphatases. Collectively, we provide structural and biochemical data on two Drosophila proteins: Primo-1, the canonical LMW-PTP protein, and CG31469, the first investigated eukaryotic protein arginine phosphatase. We named CG31469 as DARP, which stands for Drosophila ARginine Phosphatase.
Keywords: crystal structure, DARP, low molecular weightprotein tyrosine phosphatase, Primo-1, protein arginine phosphatase
Protein tyrosine phosphatases (PTPs) are a family of enzymes that mediate the reversible dephosphorylation of phosphorylated tyrosine residues on substrate proteins, one of the most common and critical post-translational modifications (Tonks, 2006). They are closely associated with the regulation of various intracellular signaling and cellular processes by controlling protein-protein interaction, protein stability, and enzyme activity (Ardito et al., 2017; Hunter, 1995). The
LMW-PTPs are found in a broad spectrum of genera from bacteria to higher eukaryotes, including
The four
The DNA fragments coding for DARP and Primo-1 were amplified by polymerase chain reaction and subcloned into the pET21a plasmid, which were then used as templates for preparing mutant proteins: DARP(C7S), DARP(T11I), DARP(Y127F), DARP(T11I·Y127F), and Primo-1(C9S). The proteins were produced in the
The phosphatase activity of Primo-1 toward
Substrate specificity was measured at room temperature using 1 μM wild-type or C7S mutant recombinant DARP and four types of 600 μM phosphoamino acids in a 200 μl reaction mixture containing 0.2 mM 2-amino-6-mercapto-7-methylpurine riboside, 0.2 units purine nucleoside phosphorylase, 50 mM Tris-HCl, and 1 mM MgCl2. The EnzChek Phosphate Assay Kit was used for determining the phosphatase activity of DARP against the four phosphoamino acids by detecting the absorbance at 360 nm for 60 min using a SpectraMax plus 384 spectrophotometer. Kinetic parameters were calculated from initial velocity data, which were obtained at room temperature using 800 nM recombinant DARP and 0 to 5 mM phosphoarginine in a 200 μl reaction mixture containing 0.2 mM 2-amino-6-mercapto-7-methylpurine riboside, 0.2 units purine nucleoside phosphorylase, 50 mM Tris (pH 7.0), and 1 mM MgCl2. Specific activities of the wild-type and mutant DARP proteins toward phosphoarginine and phosphotyrosine were calculated from initial velocity data (0-10 min), which were determined at room temperature by using the EnzChek Phosphate Assay Kit using a 200 μl reaction mixture containing 500 μM protein and 800 nM phosphoarginine at pH 7.0 or 10 μM protein and 5 mM phosphotyrosine at pH 7.5. The nonenzymatic hydrolysis of substrates was also measured for baseline correction using a control sample. Substrate specificity and kinetic constant measurements were performed in triplicate, and specific activity measurements were performed in duplicate.
The atomic coordinates and the structure factors of DARP(C7S) and Primo-1 have been deposited in the Protein Data Bank under the accession codes 7BTG and 7CUY, respectively.
Among the four
Given that DARP and Primo-1 were previously classified as enzymatically inactive pseudophosphatases (Hatzihristidis et al., 2015), we investigated whether the two proteins have any structural feature in their catalytic pocket region that negatively affects their catalytic activity. We first analyzed the P-loop arrangement of the two proteins, which is a key factor restricting phosphatase activity in a number of PTP proteins such as PTPRU, DUSP6, DUSP9, and Tk-PTP (Fjeld et al., 2000; Hay et al., 2020; Hong et al., 2005; Yun et al., 2018; Zhou and Zhang, 1999). The catalytic motifs of DARP and Primo-1 are composed of C7IGNTCR13 and C9LGNICR15, respectively, and are located at the β1−α1 loop, which is called the phosphate-binding loop or simply P-loop (Fig. 1A). The P-loop motif contains the catalytic cysteine residue (substituted with serine in the DARP structure), which functions as a nucleophile, and the conserved arginine residue (Arg13 in DARP and Arg15 in Primo-1), which anchors the phosphate group of the substrate during dephosphorylation (Fig. 2A). The center of P-loop is occupied by a single phosphate ion in the DARP structure and by the sulfonic group of a HEPES molecule in the Primo-1 structure. Oxygen atoms of these molecules mediate hydrogen bonds with the protein main chain amides and electrostatic interactions with the guanidinium group of the conserved arginine residue, which not only stabilize their positioning in the catalytic pocket but also maintain the active loop conformation of the
In contrast to a previous report classifying DARP as a pseudophosphatase (Hatzihristidis et al., 2015), a study by Fuhrmann et al. (2013) identifying
Next, to analyze the accessibility of the catalytic pocket more precisely, we structurally compared the size of the catalytic pocket between eight LMW-PTPs, including DARP and Primo-1, that contain a bound negatively charged ion or a phosphotyrosine-mimetic molecule. Intriguingly, DARP has a characteristically narrow catalytic pocket compared to that of other LMW-PTPs, while the Primo-1 catalytic pocket is comparable to those of canonical enzymes (Fig. 3C). The distance between the Cγ atom of Trp43 and the Cζ atom of Tyr127 is 7.8 Å in DARP, which is shorter than the corresponding distances in human LMW-PTP (9.3 Å), Primo-1 (9.6 Å), and SP-PTP (9.8 Å). Consistently, structural alignments demonstrate that phosphoarginine, rather than phosphotyrosine, might fit into the narrow active pocket of DARP (Fig. 3D). Atomic-level structural analysis revealed several features that presumably contribute to the shape of the DARP catalytic pocket: the bulky side chain of Trp43, which is pushed toward the cleft by the aliphatic chain of Arg41; nonconserved electrostatic interactions between Arg41 and Glu92; and a hydrogen bond between Asn44 and Tyr127 that attracts this tyrosine residue toward the pocket (Fig. 3E). These features are unique to DARP, as they are absent in other LMW-PTP structures, including that of Primo-1, YwlE, and human LMW-PTP (Fig. 3E, Supplementary Fig. S1C). In contrast, the catalytic pocket composition of Primo-1 is highly homologous to that of conventional LMW-PTPs such as human LMW-PTP (Supplementary Fig. S1C). Collectively, these data strongly suggest that the accessibility of the catalytic pocket region, which is controlled by the presence of nonconserved threonine in the P-loop region (Figs. 3A and 3B) and the pocket size (Figs. 3C-3E), determines the substrate preference of DARP.
Our structural data implied that DARP and Primo-1 might be enzymatically active phosphatases rather than pseudophosphatases.
Next, we conducted
The enzymatic property of DARP was further analyzed by comparing the specific activities of wild-type and mutant proteins toward phosphoarginine and phosphotyrosine. Compared to the wild-type, substitution of Thr11 to isoleucine caused a remarkable decrease in the dephosphorylation of phosphoarginine and a noticeable increase in the dephosphorylation of phosphotyrosine (Fig. 5C). These results support the finding of the previous study reporting that the threonine residue at position 5 of the P-loop significantly contributes to the substrate specificity of DARP (Fuhrmann et al., 2013). We also mutated Tyr127 to phenylalanine to abrogate the hydrogen bond between Tyr127 and Asn44 (Fig. 3E), which, according to our crystal structure, is expected to widen the catalytic pocket of DARP and thereby improve phosphotyrosine accessibility (Figs. 3C-3E), without critically altering the active loop arrangement. Activity tests demonstrated that the Y127F mutant was slightly less active on phosphotyrosine than the wild-type enzyme. However, the dephosphorylation activity of the double mutant T11I·Y127F toward phosphotyrosine was noticeably higher than that of the T11I single mutant protein, suggesting that the pocket size of DARP (Fig. 3) is an additional factor controlling the access of phosphotyrosine into the catalytic site (Fig. 5C). Collectively, our biochemical analysis indicates that DARP is a catalytically active arginine phosphatase, and its substrate specificity depends on the amino acid composition of the catalytic pocket region.
In this study, we demonstrated that the two
Arginine, one of the twenty standard amino acids, contains a side chain composed of three-carbon aliphatic straight chain capped by a positively charged guanidinium group where a phosphate moiety can be covalently linked to. To date, phosphorylation and dephosphorylation of this amino acid, which are nonconventional compared to those of serine, threonine, and tyrosine, were mostly investigated in gram-positive bacteria; these reactions are involved in diverse bacterial physiological processes such as stress responses and protein degradation (Elsholz et al., 2012; Fuhrmann et al., 2016; Schmidt et al., 2014; Trentini et al., 2016). Contrastively, even though arginine phosphorylation was first reported in mouse proteins more than 30 years ago (Matthews, 1995; Wakim and Aswad, 1994), its precise biological functionality and associated proteins in eukaryotes remain to be elucidated. Therefore, the physiological role of DARP in
In summary, we determined the crystal structures of DARP and Primo-1, which are catalytically active phosphatases with an LMW-PTP fold. Structural and biochemical analyses revealed that DARP and Primo-1 dephosphorylate phosphoarginine and phosphotyrosine, respectively. The results of the present study will aid in finding unknown substrates for these proteins and signaling pathways in which the proteins are involved. This, in turn, might expand our understanding of post-translational phosphorylation and dephosphorylation controlling signaling pathways in
We thank the beamline 7A at the Pohang Accelerator Laboratory in Korea for assistance during the data collection. This work was supported by the National Research Foundation of Korea funded by the Ministry of Science and ICT (MSIT; 2019M3E5D6063955) and by the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program (to B.K.).
H.S.L. and Y.M. conceived and analyzed experiments and wrote the manuscript. H.C.S. provided expertise and feedback. S.J.K. provided supervision. B.K. conceived and analyzed experiments, wrote the manuscript, secured funding, and provided supervision.
The authors have no potential conflicts of interest to disclose.
. Data collection and structure refinement statistics.
DARP(C7S) | Primo-1 | |
---|---|---|
Data collection | ||
Space group | ||
Unit cell dimensions | ||
a, b, c (Å) | 40.2, 62.2, 71.1 | 45.9, 59.9, 60.9 |
α, β, γ (°) | 90, 90, 90 | 90, 110.2, 90 |
Wavelength (Å) | 0.9793 | 0.9793 |
Resolution (Å) | 50.0-2.2 (2.24-2.20) | 50.0-2.1 (2.14-2.10) |
7.2 (26.1) | 9.5 (25.2) | |
39.9 (6.2) | 23.8 (4.3) | |
Completeness (%) | 98.2 (96.1) | 97.4 (94.2) |
Redundancy | 5.3 | 4.7 |
Refinement | ||
Resolution (Å) | 50.0-2.2 | 50.0-2.1 |
No. of reflections | 9,483 | 18,040 |
20.3/ 25.4 | 18.0/ 24.0 | |
No. of atoms | ||
Protein | 1,247 | 2,396 |
Water and ion | 48 | 123 |
HEPES | - | 30 |
RMSD | ||
Bond length (Å) | 0.008 | 0.008 |
Bond angle (°) | 1.051 | 0.954 |
Ramachandran plot (%) | ||
Most favored region | 97.4 | 98.0 |
Additionally allowed region | 2.6 | 2.0 |
Average B-value (Å2) | ||
Protein | 38.7 | 29.4 |
Water and ion | 41.5 | 32.9 |
HEPES | - | 30.7 |
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