1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

A structure of substrate-bound Synaptojanin1 provides new insights in its mechanism and the effect of disease mutations

  1. Jone Paesmans
  2. Ella Martin
  3. Babette Deckers
  4. Marjolijn Berghmans
  5. Ritika Sethi
  6. Yannick Loeys
  7. Els Pardon
  8. Jan Steyaert
  9. Patrik Verstreken
  10. Christian Galicia  Is a corresponding author
  11. Wim Versées  Is a corresponding author
  1. VIB-VUB Center for Structural Biology, Belgium
  2. Structural Biology Brussels, Vrije Universiteit Brussel, Belgium
  3. VIB-KU Leuven Center for Brain and Disease Research, Belgium
  4. KU Leuven, Department of Neurosciences, Leuven Brain Institute, Belgium
https://doi.org/10.7554/eLife.64922
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Cite this article
  1. Jone Paesmans
  2. Ella Martin
  3. Babette Deckers
  4. Marjolijn Berghmans
  5. Ritika Sethi
  6. Yannick Loeys
  7. Els Pardon
  8. Jan Steyaert
  9. Patrik Verstreken
  10. Christian Galicia
  11. Wim Versées
(2020)
A structure of substrate-bound Synaptojanin1 provides new insights in its mechanism and the effect of disease mutations
eLife 9:e64922.
https://doi.org/10.7554/eLife.64922
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13 figures, 5 tables and 2 additional files

Figures

Figure 1 with 1 supplement
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Domain organization of the inositol polyphosphate 5-phosphatase family (5PPases).

The domain organization of the ten human 5PPases, subdivided in four groups (type I–IV), is shown schematically. The different splice forms for Synaptojanin 1 (Synj1) and 2 (Synj2) are also shown. The domain boundaries of the 5PPase domain of Synj1 145 kDa used in this study and the disease mutations under study are indicated. 5PPase = 5-phosphatase domain; PH = Pleckstrin homology domain; ASH = ASPM, SPD-2, Hydin domain; Rho-GAP = Rho GTPase-activating protein domain; CB = clathrin-binding domain; PRD = proline-rich domain; SKICH = SKIP carboxyl homology domain; SAC1 = suppressor of actin 1-like domain; RRM = RNA recognition motif; SH2 = Src homology two domain; SAM = sterile alpha motif.

Figure 1—figure supplement 1
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Sequence alignment of the 5PPase domains of ten human 5-phosphatases and Schizosaccaromyces pombe Synaptojanin (SPSynj).

The sequence alignment of the 5PPase domain of all human 5PPases and SPSynj was obtained via Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Secondary structure assignment was performed using ESPript (http://espript.ibcp.fr/ESPript/ESPript/). Residues important for substrate or metal-ion interactions are indicated as described in the figure legend. The loop containing the P4-interacting-motif (P4IM) and the lipid chain 1 (LC1R) and 2 (LC2R) recognition motifs are indicated by magenta, green-blue, and cyan lines, respectively. Completely conserved residues are indicated by a red background and residues that show similarity across the 5PPases are written in red. Similar sequence motifs are indicated with a blue square. α-helices and 310-helices are displayed as helices and indicated by α or η respectively. β-strands are displayed as arrows and strict β-turns as TT.

Figure 2 with 3 supplements
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Structure of the Nb15-Synj1528–873 complex in presence or absence of the substrate diC8-PI(3,4,5)P3.

(A) Apo-structure of the Nb15-Synj1528–873 complex. Synj1528–873 (chain E) is represented in different shades of magenta, while the Nb (chain F) is represented in grey with indication of the different CDR regions. The Mg2+-ion is shown as a salmon sphere. (B) The Nb15-Synj1528–873 complex bound to diC8-PI(3,4,5)P3. Synj1528–873 (chain A) is represented in different shades of green, while the Nb (chain B) is represented similar as in (A). The Mg2+-ion is shown as an orange sphere and diC8-PI(3,4,5)P3 is shown as yellow sticks. (C) Zoom-in on the active site region of Synj1528–873 with bound diC8-PI(3,4,5)P3 (yellow sticks), Mg2+ (orange sphere) and the nucleophilic water molecule (red sphere) shown with their corresponding 2FO-FC-map contoured at 1σ. Residues D730 and N732, which play a role in the activation of the nucleophilic water, are shown as green sticks. (D) Superposition of the apo (magenta) and diC8-PI(3,4,5)P3-bound (green) Synj1528–873 structure.

Figure 2—figure supplement 1
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Content of the asymmetric unit (AU) of the apo and the diC8-PI(3,4,5)P3-bound Nb15-Synj1528–873 complex.

The asymmetric unit (AU) of both structures contains three Nb15-Synj1528–873 complexes. (A) AU of the apo-structure. (B) Superposition of the three Synj1528–873 chains from the apo-structure. (C) AU of the diC8-PI(3,4,5)P3-bound structure. (D) Superposition of the three Synj1528–873 chains from the diC8-PI(3,4,5)P3-bound structure. Mg2+-ions are represented as orange, yellow, and salmon spheres for chain A, C, and E, respectively. diC8-PI(3,4,5)P3, free phosphates, and glycerol are shown as sticks in yellow, orange, and purple, respectively.

Figure 2—figure supplement 2
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Close-up view on the active site of different Synj1528–873-chains of the apo- and diC8-PI(3,4,5)P3-bound Nb15-Synj1528–873 structure.

The close-up views of the different Synj1528–873 chains of the apo-structure are show on the left, while the same chains of the diC8-PI(3,4,5)P3-bound structure are shown on the right. The Mg2+-ions are shown as orange, yellow or salmon spheres for chain A (green), chain C (cyan) of chain E (magenta), respectively. The Mg2+-interacting residues, N543 and E591, are shown as sticks in every chain. In chain A of the diC8-PI(3,4,5)P3-bound Synj1528–873 structure (top right panel) residues D730 and N732, which play a direct role in activation of the nucleophilic water (red sphere), are also shown as sticks. Free orthophosphates and the substrate diC8-PI(3,4,5)P3 are shown as orange and yellow sticks, respectively. The omit map, contoured at 3 σ, is shown as a grey mesh around the phosphates, Mg2+-ions, diC8-PI(3,4,5)P3 and the nucleophilic water.

Figure 2—figure supplement 3
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Superposition of the catalytic (5PPase) domain of human Synj1528–873 on the available structures of the other human 5-phosphatases and SPSynj.

Superposition of Synj1528–873 (green, PDB 7A17) on the 5PPase domain of (A) INPP5B (magenta, PDB 4CML), (B) SHIP2 (blue, PDB 4A9C), (C) OCRL (salmon, PDB 4CMN), (D) INPP5E (grey, PDB 2XSW) and (E) SPSynj (cyan, PDB 1I9Z). The Mg2+-ion and the diC8-PI(3,4,5)P3 substrate of the Synj1528–873 structure are shown as an orange sphere and yellow sticks, respectively. (F) Zoom-in on the active site of the superposed 5PPases described in (A to E). For clarity, only the Mg2+-ion and the diC8-PI(3,4,5)P3 substrate of the Synj1528–873 structure are shown as an orange sphere and yellow sticks, respectively.

Figure 3
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Enzyme-substrate interactions in the Synj1528–873-diC8-PI(3,4,5)P3 complex.

(A) Zoom-in on the active site where Synj1528–873 forms interactions with different groups of the diC8-PI(3,4,5)P3 substrate (yellow sticks). The residues coloured in gold are forming interactions with the 1 P group or inositol ring of the PIP (gold dashes), residues coloured in magenta form interactions with the 4 P group (magenta dashes), and residues shown in green are interacting with the 5 P group (green dashes). Residues important for activation of the nucleophilic water (red sphere) are shown in cyan, while two residues shown in salmon are interacting with the Mg2+-ion (orange sphere). (B) Schematic representation of the interactions between Synj1528–873 and diC8-PI(3,4,5)P3. The same colour-code as in (A) was used, except for the substrate that is shown in black. Distances (in Å) between interacting atoms are indicated.

Figure 4 with 4 supplements
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Kinetic analysis of the contribution of different substrate groups to the Synj1528–873 5-phosphatase activity.

(A) Michaelis-Menten curves obtained for Synj1528–873 with different substrates: diC8-PI(3,4,5)P3, diC8-PI(4,5)P2, diC8-PI(3,5)P2, diC8-PI(5)P, IP3, and diC8-PI(4,5)P2 in the absence of Mg2+. The turnover number (kcat), the Michaelis-Menten constant (KM) and specificity constant (kcat/KM) are given for every measurement, together with the standard error. Each datapoint is the average of three independent measurements with the error bars representing the standard deviation. (B) Schematic overview of the contribution of the different groups of the diC8-PI(3,4,5)P3-substrate to the Synj1528–873 mechanism, with the acyl chains coloured in gold, the 3 P group in blue, the 4 P group in magenta, the 5 P group in green and the Mg2+-ion in orange. The ΔΔG value shows the contribution of the acyl chains, 3 P, 4 P, and Mg2+-ion to catalysis (kcat), binding (KM) and overall catalytic efficiency (kcat/KM). The more positive the ΔΔG value, the larger the contribution of the respective group to either catalysis, binding or overall catalytic efficiency.

Figure 4—source data 1

Steady-state enzyme kinetics data of Synj1528–873 wild-type in combination with different substrates.

https://cdn.elifesciences.org/articles/64922/elife-64922-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
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The 5-phosphatase activity of Synj1528–873 is affected by Nb15 and acidic pH.

(A) Michaelis-Menten curves obtained by using the Malachite green assay with 2.5 nM of human Synj1528–873 and various concentrations of diC8-PI(3,4,5)P3 in absence or presence of 100 nM Nb15. (B) Michaelis-Menten curves obtained by using the Malachite green assay with 2.5 nM of human Synj1528–873 and various concentration of diC8-PI(3,4,5)P3 in assay buffer containing 25 mM HEPES at pH 7.5 or in assay buffer containing 25 mM sodium citrate at pH 5.5. The turnover number (kcat), the Michaelis-Menten constant (KM), and specificity constant (kcat/KM) are given for the different measurements, together with the standard error. Each datapoint is the average of three independent measurements with the error bars representing the standard deviation.

Figure 4—figure supplement 1—source data 1

Steady-state enzyme kinetics data of Synj1528–873 wild-type using diC8-PI(3,4,5)P3 as substrate at pH 5.5 and in presence of an excess of Nb15 (comparable to the crystallization conditions).

https://cdn.elifesciences.org/articles/64922/elife-64922-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
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Steady-state enzyme kinetics of the Synj1528–873 Y793C mutant in combination with different substrates.

Michaelis-Menten curves obtained for Synj1528–873 Y793C with different substrates: diC8-PI(3,4,5)P3, diC8-PI(4,5)P2, diC8-PI(3,5)P2, diC8-PI(5)P, and IP3. The turnover number (kcat), the Michaelis-Menten constant (KM) and specificity constant (kcat/KM) are given for the different measurements, together with the standard error. Each datapoint is the average of three independent measurements with the error bars representing the standard deviation.

Figure 4—figure supplement 2—source data 1

Steady-state enzyme kinetics data of the Synj1528–873 Y793C mutant in combination with different substrates.

https://cdn.elifesciences.org/articles/64922/elife-64922-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
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Steady-state enzyme kinetics of the Synj1528–873 R800C mutant in combination with different substrates.

(A) Michaelis-Menten curves obtained for Synj1528–873 R800C with different substrates: diC8-PI(3,4,5)P3, diC8-PI(4,5)P2, diC8-PI(3,5)P2, diC8-PI(5)P, and IP3. The turnover number (kcat), the Michaelis-Menten constant (KM) and specificity constant (kcat/KM) are given for the different measurements, together with the standard error. Each datapoint is the average of three independent measurements with the error bars representing the standard deviation. (B) Thermodynamic squares with corresponding ΔΔG values calculated from kcat/KM. Each ΔΔG value corresponds to the contribution of either the substituted R800 side chain or the deleted 4 P group according to ΔΔG = -R.T.ln[(kcat/KM)2 / (kcat/KM)1]. ΔΔΔG shows the difference between ΔΔG1 and ΔΔG2 or ΔΔG3 and ΔΔG4, indicating that R800 performs its role in the Synj1528–873 mechanism through the 4 P group.

Figure 4—figure supplement 3—source data 1

Steady-state enzyme kinetics data of the Synj1528–873 R800C mutant in combination with different substrates.

https://cdn.elifesciences.org/articles/64922/elife-64922-fig4-figsupp3-data1-v2.xlsx
Figure 4—figure supplement 4
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The (GST-tagged) Synj1528–873 Y849C mutant shows no 5-phosphatase activity.

Time traces monitoring the activity (change in OD620nm) of 1 µM GST-Synj1528–873 Y849C using either 120 µM diC8-PI(3,4,5)P3, diC8-PI(4,5)P2 or IP3 are shown. After measuring 1 hr, the OD620nm in function of time curve was flat, indicating that no activity could be measured. These curves were compared to the corresponding curves of the wild-type Synj1528–873 at 100- to 1000-fold lower enzyme concentration (1–10 nM).

Figure 5
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Localization of the Y793C, R800C, and Y849C disease mutations in the Synj1528–873 structure.

(A) Overall structure of Synj1528–873 (green) with the Y793, R800, and Y849 residues represented as blue, magenta, and purple sticks, respectively. The Y793 residue is present in a loop close to the active site, while R800 is present in the active site. The Y849 residue, on the other hand, is buried in the core of the 5PPase domain. The Mg2+-ion is represented as an orange sphere and the substrate, diC8-PI(3,4,5)P3, as yellow sticks. (B) Close-up view on Y793 and R800 and their surrounding residues. Y793 forms a hydrogen bond with Y786 and with the main chain of P782 (grey dashes) to potentially stabilize the conformation of the loop. R800 forms multiple hydrogen bonds with the 4 P group of diC8-PI(3,4,5)P3 (grey dashes). (C) Close-up view on Y849 and its surrounding residues. Y849 is buried in the hydrophobic core, where it forms a hydrogen bond with E775 and with the main chain NH of V808 (grey dashes).

Figure 6
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Proposed catalytic mechanism(s) of the 5-phosphatase reaction of Synj1.

The nucleophilic water is activated by proton transfer to the catalytic base D730, allowing attack on the scissile phosphate (P5), and resulting in a phosphorane transition state with excess negative charge that is stabilized by several surrounding residues. Two routes for leaving group activation are envisioned. In route 1 (upper pathway) a Mg2+-activated water molecule acts as general acid by donating a proton to the leaving hydroxylate. Route 2 (lower pathway) corresponds to a mechanism of substrate-assisted catalysis, where the adjacent 4 P group acts as general acid, potentially assisted by transfer of a proton from K798.

Appendix 1—figure 1
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Reciprocal lattices (axes a*, b*, c*) colour coded by mean I/σ(I) as given by STARANISO, showing the anisotropic diffraction of the crystal of (A) the apo Synj1528-873 and (B) the diC8-PI(3,4,5)P3-bound Synj1528-873.
Author response image 1
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Electron density in the active sites of protein molecules corresponding to chain E (top), C (middle) and A (bottom) of the Synj1528‐873 ‐ diC8‐PI(3,4,5)P3 structure.

The panels on the left show the Fo‐Fc and 2Fo‐Fc map contoured at 3σ and 1σ respectively, while the panels on the right show the omit map contoured at 2.5σ.

Author response image 2
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Electron density map around residues Y794 and K795 of S. pombe Synj (SPSynj).

2Fo‐Fc map at 1σ and Fo‐Fc map at 3σ around residues Y794 and K795 of SPSynj with (A) a trans‐peptide bond and (B) a cis‐peptide bond.

Author response image 3
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Electron density around diC8‐PI(3,4,5)P3 in the Synj1528‐873 structure (PDB 7A17) and around inositol‐(1,4)bisphosphate in the SPSynj structure (1I9Z).

The panels on the left show the Fo‐Fc and 2Fo‐Fc map contoured at 3σ and 1σ respectively, while the panels on the right show the omit map contoured at 3σ.

Author response image 4
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The 5‐phosphatase activity of Synj1528‐873 is affected by Nb15 and acidic pH.

(A) Michaelis‐Menten curves obtained by using the Malachite green assay with 2.5 nM of human Synj1528‐873 and various concentrations of diC8‐PI(3,4,5)P3 in absence or presence of 100 nM Nb15. (B) Michaelis‐Menten curves obtained by using the Malachite green assay with 2.5 nM of human Synj1528‐873 and various concentration of diC8‐PI(3,4,5)P3 in assay buffer containing 25 mM HEPES at pH 7.5 or in assay buffer containing 25 mM sodium citrate at pH 5.5.

Author response image 5
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Omit map contoured at 3σ around the diC8‐PI(3,4,5)P3 substrate of a structure (A) processed with STARANISO to 2.73 Å, (B) processed without STARANISSO to 2.73 Å and (C) processed without STARANISO to 3.0 Å.

Panel (D) Refined 2Fo‐Fc map contoured at 1σ around the diC8‐PI(3,4,5)P3 substrate of a structure processed with STARANISO to 2.73 Å.

Author response image 6
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2Fo‐Fc map contoured at 1 σ around (A) residues 670‐678 and (B) residues 723‐730 of chain A of the Synj1528‐873diC8‐PI(3,4,5)P3 structure.

Tables

Table 1
Data collection and refinement statistics.
Synj1528–873 - ApoSynj1528–873 - diC8-PI(3,4,5)P3
PDB code7A0V7A17
Data collection
SynchrotronDiamondSoleil
Beamlinei03Px2a
Wavelength (Å)0.980.98
Resolution range (Å)*87.06–2.30
(2.43–2.30)		87.39–2.73
(3.02–2.73)
Space groupC121C121
Unit cell dimensions (Å)a = 168.87a = 169.32
b = 108.79b = 109.21
c = 100.97c = 100.90
Unit cell angles (°)α = 90.00α = 90.00
β = 120.72β = 120.62
γ = 90.00γ = 90.00
Spherical completeness (%)*77.1 (26.4)76.2 (22.8)
Ellipsoidal completeness (%)*92.3 (95.0)91.5 (57.3)
Unique reflections5378932149
Mean (I)/SD(I)*11.2 (1.4)5.3 (1.4)
CC(1/2)*0.997 (0.512)0.964 (0.474)
Multiplicity*7.0 (5.7)3.5 (3.6)
Rmeas (%)*14.5 (125.1)28.2 (123.7)
Refinement
Resolution range (Å)86.81–2.3086.83–2.73
Rwork (%)19.6419.88
Rfree (%)†25.2325.74
Model content
Molecules per AU66
Protein atoms per AU1057410624
Ligand atoms per AU3985
Metal atoms per AU32
Water molecules per AU35971
Wilson B factors (Å2)38.0843.28
Average B factors (Å2)
Protein atoms47.3544.88
Ligand atoms65.6267.11
Metal atoms60.0842.62
Water molecules42.6822.93
Rmsd bonds (Å)0.0020.006
Rmsd angles (°)0.4561.15
Ramachandran plot (%) (favored, outliers)95.79, 0.2397.00, 0.46

  1. * Values in parentheses are for the high-resolution shell.

    † Rfree is based on a subset of 5% of reflections omitted during refinement.

  2. AU, asymmetric unit.

Table 2
Steady-state kinetic parameters of Synj1528–873 and the Synj1528–873 Y793C, R800C and Y849C mutants in combination with different substrates.
Synj1528–873Synj1528–873 Y793CSynj1528–873 R800CSynj1528–873 Y849C
diC8-PI(3,4,5)P3kcat (s−1)30.6 ± 2.47.6 ± 0.921.8 ± 4.2NMA
KM (µM)18 ± 529 ± 10155 ± 49NMA
kcat/KM
(•103 (M•s)−1)		1692 ± 474259 ± 97141 ± 52NMA
diC8-PI(4,5)P2kcat (s−1)85.3 ± 7.032.0 ± 3.26.4 ± 1.2NMA
KM (µM)39 ± 8117 ± 25161 ± 52NMA
kcat/KM
(•103 (M•s)−1)		2171 ± 482274 ± 6440 ± 15NMA
IP3kcat (s−1)16.4 ± 1.11.5 ± 0.20.055 ± 0.007NMA
KM (µM)96 ± 20864 ± 171289 ± 78NMA
kcat/KM
(•103 (M•s)−1)		171 ± 381.7 ± 0.40.19 ± 0.06NMA
diC8-PI(3,5)P2kcat (s−1)0.09 ± 0.010.012 ± 0.001NDND
KM (µM)133 ± 35101 ± 25NDND
kcat/KM
(•103 (M•s)−1)		0.62 ± 0.190.12 ± 0.030.29 ± 0.01ND
diC8-PI(5)Pkcat (s−1)NDNDNDND
KM (µM)NDNDNDND
kcat/KM
(•103 (M•s)−1)		1.45 ± 0.070.14 ± 0.011.2 ± 0.1ND
diC8-PI(4,5)P2 without Mg2+kcat (s−1)0.06 ± 0.01NDNDND
KM (µM)289 ± 85NDNDND
kcat/KM
(•103 (M•s)−1)		0.22 ± 0.08NDNDND

  1. ND, not determined.

    NMA, no measurable activity.

Key resources table
Reagent type (species)
or resource		DesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)SYNJ1NCBIGene ID: 8867
Strain, strain
background
(Escherichia coli)		BL21(DE3) pLysSWeiner et al., 1994Genotype: F-hsdSB
(rB-mB-) gal dcm
(DE3) pLysS (CmR)		Chemically (CaCl2) competent
Strain, strain
background
(E. coli)		WK6 (Su-)Zell and Fritz, 1987
(PMID:3038536)		Genotype: Δ(lac-proAB)
galE strA/F’
[lacIq lacZΔM15 proA+B+]		Chemically (CaCl2) competent
Strain, strain
background (M13
helper phage)		Kanamycin-resistant
VCSM13		Stratagene200251
Recombinant
DNA reagent		pET28a
(plasmid)		Novagen69864
Recombinant
DNA reagent		pGEX-4T1
(plasmid)		GE HealthcareGE28-9545-49
Recombinant
DNA reagent		pMESy4 (plasmid)Pardon et al., 2014
(DOI: 10.1038/nprot.2014.039)		GenBank KF415192
Chemical
compound, drug		IP6
(D-myo-inositol
1,2,3,4,5,6-hexakis
phosphate)		Merck Millipore407125
Chemical
compound, drug		IP3
(D-myo-inositol
1,4,5-trisphosphate)		Merck Millipore407137
Chemical
compound, drug		diC8-PI(5)PEchelon BiosciencesP-5008
Chemical
compound, drug		diC8-PI(4,5)P2Echelon BiosciencesP-4508
Chemical
compound, drug		diC8-PI(3,5)P2Echelon BiosciencesP-3508
Chemical
compound, drug		diC8-PI(3,4,5)P3Echelon BiosciencesP-3908
Chemical
compound, drug		disodium-4-
nitrophenyl
phosphate (DNPP)		SigmaN-4645
Sequence-based
reagent		Y793C_FThis paperPCR primersCGACTGTGACACCA
GTGAAAAGTGCCG
Sequenced-based
reagent		Y793C_RThis paperPCR primersCTGGTGTCACAG
TCGTCAGAAAACAAG
Sequence-based
reagent		R800C_FThis paperPCR primersGTGCTGCACCCCTG
CCTGGACAGAC
Sequenced-based
reagent		R800C_RThis paperPCR primersGGTGCAGCACTTTT
CACTGGTGTC
Sequence-based
reagent		Y849C_FThis paperPCR primersCACTGTGGAAGAG
CTGAGCTGAAG
Sequenced-based
reagent		Y849C_RThis paperPCR primersCTTCCACAGTGCAGC
AAAGTGCCTGG
Peptide,
recombinant protein		CaptureSelect
Biotin anti-C-tag
conjugate		Thermo Fisher Scientific7103252100
Peptide,
recombinant protein		Streptavidin
Alkaline
Phosphatase		PromegaV5591
Commercial
assay or kit		Malachite Green
Phosphate
Assay kit		GentaurPOMG-25H
Software, algorithmautoPROCVonrhein et al., 2011
(DOI: 10.1107/S0907444911007773)		RRID:SCR_015748
https://www.globalphasing.comautoproc/
Software, algorithmSTARANISOTickle et al., 2018RRID:SCR_018362
http://staraniso.globalphasing.org/cgi-bin/staraniso.cgi
Software, algorithmPhaserMcCoy et al., 2007
(DOI:10.1107/S0021889807021206)		RRID:SCR_014219
https://www.phenix-online.org/documentation/reference/phaser.html
Software, algorithmPhenix.Ligand FitTerwilliger et al., 2006
(DOI:10.1107/S0907444906017161)		https://www.phenix-online.org/documentation/reference/ligandfit.html
Software, algorithmPhenix.RefineAfonine et al., 2012
(DOI: 10.1107/S0907444912001308)		RRID:SCR_016736
https://www.phenix-online.org/documentation/reference/refinement.html
Software, algorithmCootEmsley et al., 2010
(DOI: 10.1107/S0907444910007493)		RRID:SCR_014222
https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Software, algorithmMolProbityChen et al., 2010
(DOI: 10.1107/S0907444909042073)		RRID:SCR_014226
http://molprobity.biochem.duke.edu
Software, algorithmPDB-REDO serverJoosten et al., 2014
(DOI: 10.1107/S2052252514009324)		RRID:SCR_018936
https://pdb-redo.eu/
Software, algorithmPyMOL
(version 2.0)		SchrödingerRRID:SCR_000305
https://pymol.org/2/
Software, algorithmGraphPad Prism
(version 8)		Graphpad SoftwareRRID:SCR_002798
Software, algorithmCCP4 suiteWinn et al., 2011
(DOI: 10.1107/S0907444910045749)		RRID:SCR_007255
http://www.ccp4.ac.uk/
Software, algorithmClustal OmegaMadeira et al., 2019
(DOI: 10.1093/nar/gkz268)		RRID:SCR_001591
http://www.ebi.ac.uk/Tools/msa/clustalo/
Software, algorithmESPriptRobert and Gouet, 2014
(DOI: 10.1093/nar/gku316)		RRID:SCR_006587
http://espript.ibcp.fr/ESPript/ESPript/
Software, algorithmACD/ChemSketch
(version 2019.2.1)		Advanced Chemistry Developmenthttp://www.acdlabs.com
Author response table 1
diC8‐PI(3,4,5)P3‐bound Synj1528‐873
STARANISO
2.73 Å		Without STARANISO
2.73 Å		Without STARANISO
3.00 Å
PDB number7A17
Data collection
Resolution range (Å)a87.39‐2.73
(3.02‐2.73)		87.39‐2.73
(2.83‐2.73)		87.39‐3.00
(3.16‐3.00)
Spherical completeness (%)a76.2 (22.8)99.8 (100)99.8 (99.9)
Ellipsoidal completeness (%)a91.5 (57.3)//
Unique reflections321494201331720
Mean (I)/SD(I)a5.3 (1.4)4.1 (0.7)5.2 (1.4)
CC(1/2)a0.964 (0.474)0.953 (0.195)0.962 (0.448)
Multiplicitya3.5 (3.6)3.5 (3.6)3.5 (3.4)
Rmeas (%)a28.2 (123.7)37.1 (259.9)28.1 (114.4)
Author response table 2
R, Rfree and R‐Rfree statistics for structures in the resolution range 2.50‐2.90 Å as given by phenix.r_factor_statistics.
Histogram of Rwork for models in PDB at resolution 2.50‐2.90 Å :
0.119 ‐ 0.171 : 281 structures
0.171 ‐ 0.223 : 5171 structures
0.223 ‐ 0.274 : 3263 structures
0.274 ‐ 0.326 : 122 structures
0.326 ‐ 0.378 : 4 structures
Histogram of Rfree for models in PDB at resolution 2.50‐2.90 Å:
0.159 ‐ 0.220 : 469 structures
0.220 ‐ 0.281 : 6011 structures
0.281 ‐ 0.343 : 2315 structures
0.343 ‐ 0.404 : 45 structures
0.404 ‐ 0.465 : 1 structures
Histogram of Rfree‐Rwork for all model in PDB at resolution 2.50‐2.90 Å:
0.001 ‐ 0.021 : 408 structures
0.021 ‐ 0.041 : 2370 structures
0.041 ‐ 0.060 : 3841 structures
0.060 ‐ 0.080 : 1809 structures
0.080 ‐ 0.100 : 413 structures
Number of structures considered: 8841

Additional files

Supplementary file 1

(A) Comparison of the 5PPase domain of human Synj1 (Synj1528–873) with the corresponding 5PPase domain of the other human inositol polyphosphate 5-phosphatases and SPSynj. The root-mean-square deviation (rmsd) after superposition of the structures was determined using CCP4 SUPERPOSE. Furthermore, the sequence identity was determined using Clustal Omega. (B) Residues interacting with the diC8-PI(3,4,5)P3 substrate in Synj1528–873 and the corresponding residues in the other nine human 5PPases and SPSynj. Completely conserved residues are written in white with a red background and similar residues are written in red.

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  1. Jone Paesmans
  2. Ella Martin
  3. Babette Deckers
  4. Marjolijn Berghmans
  5. Ritika Sethi
  6. Yannick Loeys
  7. Els Pardon
  8. Jan Steyaert
  9. Patrik Verstreken
  10. Christian Galicia
  11. Wim Versées
(2020)
A structure of substrate-bound Synaptojanin1 provides new insights in its mechanism and the effect of disease mutations
eLife 9:e64922.
https://doi.org/10.7554/eLife.64922