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Horizontal Tabs
PDB ID |
Structure Details |
Apo structure of the phosphatase and C2 domains |
|
Phosphatase and C2 domains with magnesium and phosphate bound |
Fragments and PDB depositions
Note: only fragments in site 3 are seen as relevant for further development.
PDBID |
Ligand |
Binding Location |
Binding Pocket |
Resolution (Å) |
x0019 |
Site 1 |
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1.22 |
|
x0021 |
Site 1 |
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1.31 |
|
x0030![]() |
Site 1 |
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1.32 |
|
x0039![]() |
Site 1 |
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1.23 |
|
x0040 |
Site 1 |
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1.27 |
|
x0054 |
Site 1 |
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1.45 |
|
x0084 |
Site 1 |
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1.46 |
|
x0097 |
Site 1 |
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1.26 |
|
x0100 |
Site 1![]() |
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1.32 |
|
x0118 |
Site 1![]() |
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1.48 |
|
x0121![]() |
Site 1 |
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1.43 |
|
5RX2 | x0169![]() |
Site 1![]() |
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1.27 |
5RX6 |
x0183 |
Site 1![]() |
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1.45 |
5RXD | x0243![]() |
Site 1![]() |
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1.33 |
5RXE | x0251 ![]() |
Site 1![]() |
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1.25 |
5RXF | x0300![]() |
Site 1![]() |
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1.26 |
5RXO | x0342![]() |
Site 1![]() |
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1.71 |
5RXP | x0345![]() |
Site 1![]() |
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1.53 |
5RXQ | x0350![]() |
Site 1![]() |
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1.65 |
5RXT | x0361![]() |
Site 1![]() |
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1.63 |
5RXW | x0382![]() |
Site 1![]() |
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1.34 |
5RXZ | x0417![]() |
Site 1![]() |
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1.56 |
5RY0 | x0423![]() |
Site 1![]() |
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1.98 |
5RY2 | x0449![]() |
Site 1![]() |
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1.54 |
5RY3 | x0469![]() |
Site 1![]() |
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1.50 |
5RY4 | x0479 ![]() |
Site 1![]() |
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1.50 |
5RY6 | x0494![]() |
Site 1![]() |
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1.74 |
5RY8 | x0518 ![]() |
Site 1![]() |
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1.43 |
5RYD | x0558 ![]() |
Site 1![]() |
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1.6 |
5RYE | x0560![]() |
Site 1![]() |
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1.7 |
5RYL | x0609![]() |
Site 1![]() |
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1.55 |
5RW3 | x0020![]() |
Site 2![]() |
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1.37 |
5RWA | x0058![]() |
Site 2![]() |
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1.29 |
5RWC | x0067![]() |
Site 2![]() |
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1.40 |
5RWE | x0075 ![]() |
Site 2![]() |
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1.34 |
5RWF | x0083![]() |
Site 2![]() |
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1.35 |
5RWH | x0088![]() |
Site 2![]() |
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1.56 |
5RWI | x0092![]() |
Site 2![]() |
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1.29 |
5RWO | x0114![]() |
Site 2![]() |
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1.29 |
5RWQ | x0119![]() |
Site 2![]() |
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1.32 |
5RWS | x0126![]() |
Site 2![]() |
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1.28 |
5RWT | x0129![]() |
Site 2![]() |
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1.43 |
5RWV | x0140![]() |
Site 2![]() |
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1.25 |
5RWW | x0141![]() |
Site 2![]() |
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1.16 |
5RWY | x0152![]() |
Site 2![]() |
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1.35 |
5RWZ | x0154![]() |
Site 2![]() |
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1.42 |
5RX1 | x0163![]() |
Site 2![]() |
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1.31 |
5RX3 | x0174![]() |
Site 2![]() |
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1.45 |
5RX4 | x0176![]() |
Site 2![]() |
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1.35 |
5RX7 | x0194![]() |
Site 2![]() |
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1.36 |
5RX8 | x0195![]() |
Site 2![]() |
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1.34 |
5RX9 | x0200![]() |
Site 2![]() |
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1.29 |
5RXA | x0201![]() |
Site 2![]() |
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1.24 |
5RXB | x0221![]() |
Site 2![]() |
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1.58 |
5RXC | x0239![]() |
Site 2![]() |
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1.59 |
5RXG | x0302![]() |
Site 2![]() |
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1.52 |
5RXH | x0303![]() |
Site 2![]() |
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1.42 |
5RXI | x0311![]() |
Site 2![]() |
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1.74 |
5RXJ | x0316![]() |
Site 2![]() |
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1.52 |
5RXK | x0320![]() ![]() |
Site 2![]() |
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1.46 |
5RXM | x0340![]() |
Site 2![]() |
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1.46 |
5RXR | x0358![]() |
Site 2![]() |
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1.40 |
5RXU | x0368![]() |
Site 2![]() |
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1.64 |
5RXX | x0401![]() |
Site 2![]() |
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1.43 |
5RXY | x0403![]() |
Site 2![]() |
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1.40 |
5RY5 | x0490![]() |
Site 2![]() |
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1.54 |
5RY7 | x0510![]() |
Site 2![]() |
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1.60 |
5RYA | x0527![]() |
Site 2![]() |
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1.32 |
5RYF | x0573![]() |
Site 2![]() |
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1.49 |
5RYH | x0588![]() |
Site 2![]() |
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1.72 |
5RYI | x0590![]() |
Site 2![]() |
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1.45 |
5RYK | x0604![]() |
Site 2![]() |
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1.55 |
5RWD | x0073![]() |
Site 3![]() |
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1.29 |
5RWL | x0101![]() |
Site 3![]() |
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1.37 |
5RXV | x0378![]() |
Site 3![]() |
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1.50 |
5RY9 | x0524![]() |
Site 3![]() |
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1.52 |
5RYC | x0543![]() |
Site 3![]() |
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1.56 |
5RW5 | x0027![]() |
Site 4![]() |
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1.38 |
5RWN | x0106![]() |
Site 4![]() |
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1.38 |
5RWU | x0133![]() |
Site 4![]() |
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1.37 |
5RX0 | x0161![]() |
Site 4![]() |
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1.43 |
5RXL | x0325![]() |
Site 4![]() |
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1.49 |
5RYG | x0577![]() |
Site 4![]() |
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1.47 |
5RYJ | x0596![]() |
Site 4![]() |
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1.42 |
5RWB | x0062![]() |
Site 5![]() |
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1.25 |
5RWM | x0103![]() |
Site 7![]() |
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1.36 |
5RWX | x0148![]() |
Site 8![]() |
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1.34 |
5RXS | x0359![]() |
Site 8![]() |
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1.37 |
5RX5 | x0182![]() |
Site 9![]() |
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1.28 |
5RY1 | x0438![]() |
Site 10![]() |
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1.52 |
5RYB | x0542![]() |
Site 12![]() |
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1.55 |
Table 1: Details of fragment hits
Cloning and expression
Expression plasmids
INPP5DA-c232 (Phosphatase and C2 domains 396-856). Vector: pFB-HGT-LIC (Baculovirus transfer vector with N-terminal His6-GST-TEV tag. Ampicillin -resistance). The plasmid is recombined with a baculovirus genome using the Bac-to-Bac system (Thermo), and the DNA is transfected into SF9 cells for virus generation and propagation.
Protein sequence with tag (Tag sequence underlined; * TEV protease cleavage site):
MGHHHHHHSSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSSSGVDLGTENLYFQ*SMEQPEPDMITIFIGTWNMGNAPPPKKITSWFLSKGQGKTRDDSADYIPHDIYVIGTQEDPLSEKEWLEILKHSLQEITSVTFKTVAIHTLWNIRIVVLAKPEHENRISHICTDNVKTGIANTLGNKGAVGVSFMFNGTSLGFVNSHLTSGSEKKLRRNQNYMNILRFLALGDKKLSPFNITHRFTHLFWFGDLNYRVDLPTWEAETIIQKIKQQQYADLLSHDQLLTERREQKVFLHFEEEEITFAPTYRFERLTRDKYAYTKQKATGMKYNLPSWCDRVLWKSYPLVHVVCQSYGSTSDIMTSDHSPVFATFEAGVTSQFVSKNGPGTVDSQGQIEFLRCYATLKTKSQTKFYLEFHSSCLESFVKSQEGENEEGSEGELVVKFGETLPKLKPIISDPEYLLDQHILISIKSSDSDESYGEGCIALRLEATETQLPIYTPLTHHGELTGHFQGEIKLQTSQ
Predicted mass: 81077.6
Protein sequence after tag removal:
SMEQPEPDMITIFIGTWNMGNAPPPKKITSWFLSKGQGKTRDDSADYIPHDIYVIGTQEDPLSEKEWLEILKHSLQEITSVTFKTVAIHTLWNIRIVVLAKPEHENRISHICTDNVKTGIANTLGNKGAVGVSFMFNGTSLGFVNSHLTSGSEKKLRRNQNYMNILRFLALGDKKLSPFNITHRFTHLFWFGDLNYRVDLPTWEAETIIQKIKQQQYADLLSHDQLLTERREQKVFLHFEEEEITFAPTYRFERLTRDKYAYTKQKATGMKYNLPSWCDRVLWKSYPLVHVVCQSYGSTSDIMTSDHSPVFATFEAGVTSQFVSKNGPGTVDSQGQIEFLRCYATLKTKSQTKFYLEFHSSCLESFVKSQEGENEEGSEGELVVKFGETLPKLKPIISDPEYLLDQHILISIKSSDSDESYGEGCIALRLEATETQLPIYTPLTHHGELTGHFQGEIKLQTSQ
Predicted mass: 52812.9
DNA sequence (ORF):
ATGGGCCACCATCATCATCATCATTCTTCTATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGAGCTCAGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGAGCAGCCGGAGCCCGACATGATCACCATCTTCATCGGCACCTGGAACATGGGTAACGCCCCCCCTCCCAAGAAGATCACGTCCTGGTTTCTCTCCAAGGGGCAGGGAAAGACGCGGGACGACTCTGCGGACTACATCCCCCATGACATTTACGTGATCGGCACCCAAGAGGACCCCCTGAGTGAGAAGGAGTGGCTGGAGATCCTCAAACACTCCCTGCAAGAAATCACCAGTGTGACTTTTAAAACAGTCGCCATCCACACGCTCTGGAACATCCGCATCGTGGTGCTGGCCAAGCCTGAGCACGAGAACCGGATCAGCCACATCTGTACTGACAACGTGAAGACAGGCATTGCAAACACACTGGGGAACAAGGGAGCCGTGGGGGTGTCGTTCATGTTCAATGGAACCTCCTTAGGGTTCGTCAACAGCCACTTGACTTCAGGAAGTGAAAAGAAACTCAGGCGAAACCAAAACTATATGAACATTCTCCGGTTCCTGGCCCTGGGCGACAAGAAGCTGAGTCCCTTTAACATCACTCACCGCTTCACGCACCTCTTCTGGTTTGGGGATCTTAACTACCGTGTGGATCTGCCTACCTGGGAGGCAGAAACCATCATCCAGAAAATCAAGCAGCAGCAGTACGCAGACCTCCTGTCCCACGACCAGCTGCTCACAGAGAGGAGGGAGCAGAAGGTCTTCCTACACTTCGAGGAGGAAGAAATCACGTTTGCCCCAACCTACCGTTTTGAGAGACTGACTCGGGACAAATACGCCTACACCAAGCAGAAAGCGACAGGGATGAAGTACAACTTGCCTTCCTGGTGTGACCGAGTCCTCTGGAAGTCTTATCCCCTGGTGCACGTGGTGTGTCAGTCTTATGGCAGTACCAGCGACATCATGACGAGTGACCACAGCCCTGTCTTTGCCACATTTGAGGCAGGAGTCACTTCCCAGTTTGTCTCCAAGAACGGTCCCGGGACTGTTGACAGCCAAGGACAGATTGAGTTTCTCAGGTGCTATGCCACATTGAAGACCAAGTCCCAGACCAAATTCTACCTGGAGTTCCACTCGAGCTGCTTGGAGAGTTTTGTCAAGAGTCAGGAAGGAGAAAATGAAGAAGGAAGTGAGGGGGAGCTGGTGGTGAAGTTTGGTGAGACTCTTCCAAAGCTGAAGCCCATTATCTCTGACCCTGAGTACCTGCTAGACCAGCACATCCTCATCAGCATCAAGTCCTCTGACAGCGACGAATCCTATGGCGAGGGCTGCATTGCCCTTCGGTTAGAGGCCACAGAAACGCAGCTGCCCATCTACACGCCTCTCACCCACCATGGGGAGTTGACAGGCCACTTCCAGGGGGAGATCAAGCTGCAGACCTCTCAGTGA
Note: in initial experiments, the same His6-GST-INPP5D(396-856) fusion protein was expressed in E. coli from construct INPP5DA-c132. This protein was used to generate the first crystal structure, 6IBD. This construct was supplanted by the baculovirus construct, which gives much higher yields and purity, in all subsequent experiments and structures (6XY7 and the fragment screen). The protein was expressed in Sf9 cells grown in Sf-900 II SFM. Each litre of cells at a density of 0.2x106/ml was infected with in 3 ml of P2 viruses and allowed to grow for approximately 68 hours at 27 °C. Cells were harvested by centrifugation at 1500g, flash frozen in liquid nitrogen and stored at -80 °C.
Protein purification
Purifications were performed on cell pellets from an expression volume of 1 or 2 litres. Pellets were resuspended in lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole, 5% glycerol, 1 mM TCEP) to a volume of 120 ml per litre expression. EDTA-free protease inhibitors were also added. The cells were lysed by sonication on ice with 5 seconds on, 10 seconds off for a total of 15 minutes. The lysate was mixed halfway through. Once lysis was complete, lysate was cleared by centrifugation at 75,000g for 20 minutes. 3 ml of nickel beads prewashed in lysis buffer were added to the supernatant, split between 50 ml falcon tubes, which were rotated in a cold room for 1 hour. The beads were pelleted by centrifugation at 700g for 5 minutes, resuspended in 50 ml lysis buffer, pelleted, then in resuspended in 50 ml and pelleted again. The beads were resuspended in 20 ml lysis buffer and put on a gravity column. The column was washed with 10 ml wash buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 40 mM imidazole, 5% glycerol, 1 mM TCEP) and the protein was eluted with three 10 ml washes with elution buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 300 mM imidazole, 5% glycerol, 1 mM TCEP). TEV protease was added to the eluate at a concentration (mg/ml) of 5:1 target protein:TEV (This concentration was required for INPP5DA-c232 and INPPL1A-c232, but not for other inositol phosphatases in the selectivity panel. The mixture was dialysed overnight into dialysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 1 mM TCEP) at 4 °C. The TEV was removed by passing the sample back down the nickel column, which was washed with 10 ml lysis buffer. The flow through and wash fractions were combined and concentrated to a volume of 1 ml. This was passed down a Superdex 200 16/60 column in gel filtration buffer (50 mM HEPES pH 7.5, 250 mM NaCl, 5% glycerol, 1 mM TCEP). Selected fractions were combined and concentrated.
Crystallisation
Crystallisation conditions were screened at 20 °C by sitting drop vapour diffusion using 150 nl drops with ratios of 2:1, 1:1 and 1:2. Screens were set up around identified conditions. Apo SHIP1 crystals grew from protein at 6 mg/ml in 0.1M bis-tris pH 7.0, 14% PEG2KMME, 12% PEG3350. The crystals were cryoprotected by addition of 1 μl reservoir to the drop. These crystals were also used for seeding. Crystals from 6 drops were resuspended in a total of 30 μl reservoir solution and conditions were rescreened with 20 nl seeds added to 150 nl drops. Crystals were observed in Molecular Dimensions Morpheus C1 (30 mM sodium phosphate, ammonium sulphate, sodium nitrate, 100 mM MES/imidazole pH 6.5, 10% PEG 20,000, 20 % PEG 500 MME) after around a week. Crystals grown in this way were used for X-ray fragment screens at Diamond Light Source using the standard X-Chem protocol with fragment soaks at 75 mM (15% DMSO) (19). Crystals were harvested after approximately 2.5 to 5 hours. Alternatively, they were cryoprotected by addition of 1 μl reservoir solution to the drop with 2mM MgCl2 added to obtain the magnesium and phosphate bound structure. SHIP2 crystals were obtained in the same form as the published SHIP2 structure (5OKM)
Data collection and processing
Data collection: For the apo structure 6IBD, data were collected at Diamond Light Source on beamline I03 to a resolution of 1.48 Å. For the magnesium and phosphate bound structure 6XY7, data were also collected on I03 to a resolution of 1.09 Å. Data processing: The data were integrated with Dials and scaled with Aimless. For the magnesium and phosphate bound structure 6XY7, an anisotropic high-resolution limit of 1.09-1.34 Å was applied using Staraniso. Both structures were determined by molecular replacement with Phaser using the phosphatase domains of SHIP2 (5OKM, chain B) as a model. The structures were refined using Refmac to final R / Rfree of 17.3%/20.0% and 13.6%/15.9% for 6IBD and 6XY7, respectively.
Malachite green assay
The assay was performed in a 384-well clear-bottomed, black-walled microtitre plate (Greiner Bio One, # - 781906). Additions of INPP5D enzyme dilution, test compound and INPP5D substrate phosphatidylinositol (3,4,5)-trisphosphate (Echelon Biosciences, # - P-3908) were made to a final assay volume of 40 µL/well. The [final] for substrate was 50 µM. The known INPP5D catalytic activity inhibitors 3AC (Calbiochem, # - 565835) or K118 (Echelon Biosciences, # - B-0344) were used as positive controls for inhibition at a [final] of ≥ 100 µM. After 30 min incubation at room temperature, the assay was terminated and detection performed using the Malachite Green Phosphate Assay Kit (Sigma-Aldrich, # - MAK307). Detection solution was prepared to the required volume by combining 100 volumes of Reagent A to 1 volume of Reagent B. Detection solution was added to test wells 10 µL/well, followed by 30 min incubation at room temperature. The signal was determined by measuring Abs620nm using the PHERAstar FS (BMG Labtech).
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