Table 1.
Data Collection and Refinement Statistics.
| HePTP0e | HePTP24e | HePTP240e | |
|---|---|---|---|
| PDB ID | 3O4S | 3O4T | 3O4U |
| Data Collection | |||
| Space Group | C2 | C2 | C2 |
| Unit Cell | |||
| a, b, c (Å) | 117.8, 39.1, 83.4 | 118.0, 39.0, 83.7 | 119.0, 39.2, 84.0 |
| α, β, γ (°) | 90.0, 124.3, 90.0 | 90.0, 124.2, 90.0 | 90.0, 124.2, 90.0 |
| Wavelength (Å) | 1.0 | 1.54 | 1.1 |
| Resolution (Å) | 50.0-1.90 (1.93-1.90)a |
50.0-2.60 (2.64-2.60)a |
50.0-2.25 (2.29-2.25)a |
| No. protein molecules/ASU | 1 | 1 | 1 |
| Total/unique reflections | 92396/25241 | 20892/8972 | 64467/15443 |
| Redundancy | 3.7 (3.6)a | 2.3 (2.0)a | 4.2 (3.0)a |
| Completeness (%) | 99.7 (99.9)a | 90.3 (86.9)a | 99.0 (87.7)a |
| Rmerge (%)b | 9.2 (51.2)a | 8.8 (29.6)a | 11.3 (56.2)a |
| Mean I/σ(I) | 13.8 (3.5)a | 11.4 (3.7)a | 14.5 (2.6)a |
| Refinement | |||
| Resolution range | 20.00-1.90 | 20.00-2.60 | 20.00-2.25 |
| No. reflections (total) | 23923 | 8530 | 14619 |
| No. reflections (test) | 1287 | 440 | 772 |
| Rwork (%)c | 16.2 | 19.9 | 19.0 |
| Rfree (%)d | 21.2 | 25.3 | 24.3 |
| RMS deviations from ideal geometry | |||
| Bonds (Å) | 0.012 | 0.010 | 0.008 |
| Angles (°) | 1.31 | 1.10 | 1.08 |
| Ramachandran plot | |||
| Residues in allowed regions (%) | 99.7 | 99.6 | 99.6 |
| Residues in disallowed regions (%) | 0.3 | 0.4 | 0.4 |
| Mean B Value | |||
| Protein | |||
| Total | 21.3 | 24.2 | 32.0 |
| Active Sitee | 12.9 | 21.8 | 30.2 |
| Water | |||
| Active Site Sulfate | 16.6 | 25.4 | N/A |
| Active Site Tartrate | N/A | N/A | 44.8 |
| Glycerol Molecules | 44.3 | 44.6 | 43.1 |
| No. Atoms | |||
| Protein | 2340 | 2237 | 2189 |
| Water | 277 | 181 | 143 |
| Sulfate molecules | 6 | 1 | 0 |
| Tartrate molecules | 0 | 1 | 2 |
| Glycerol molecules | 5 | 2 | 2 |
Values in parentheses are for the highest resolution shell.
Rmerge = ∑|Ii−<Ii>|/∑|Ii| where Ii is the scaled intensity of the ith measurement, and <Ii> is the mean intensity for that reflection.
Rwork = ∑||Fobs|−|Fcalc||/∑|Fobs| where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively.
Rfree = as for Rwork, but for 5.0% of the total reflections chosen at random and omitted from refinement.
Calculated for residues 270–276 of the HePTP PTP loop. HePTP (residues 44–339) containing the S72D mutation was subcloned into a derivative of the pET28a bacterial expression vector (Novagen) containing an N-terminal expression and hexahistidine purification tag (MGSDKIHHHHHH).30 Protein expression and purification was carried out using standard protocols.10;17 HePTP44–339 S72D initially formed clusters of small, one-dimensional ‘needle’ crystals in 1.8 M ammonium sulfate pH 5.0 using the sitting drop vapor diffusion method at 4°C. These initial crystals were used as seed for microseeding. This led to the formation larger, two-dimensional ‘plate’ crystals by microseeding into 1.7–1.9 M ammonium sulfate pH 5.0 using the sitting drop vapor diffusion method at 4°C. HePTP0: unsoaked HePTP44–339 S72D crystals (HePTP0) were cryoprotected in 1.28 M ammonium sulfate pH 5.0, 25% (v/v) glycerol for 30 seconds prior to diffraction screening and data collection. HePTP24: a subset of HePTP44–339 S72D crystals were transferred from the crystallization drop to 0.2 M ammonium tartrate pH 6.6, 20% (w/v) PEG 3,350 for 30 seconds at 4°C, then to a second drop of this solution for 30 seconds at 4°C, and subsequently to a third drop of this solution for 24 hours at 4°C, after which they were cryoprotected in 0.16 M ammonium tartrate pH 6.6, 16% (w/v) PEG 3,350, 20% (v/v) glycerol for 20 seconds prior to diffraction screening and data collection. HePTP240: another subset of HePTP44–339 S72D crystals were transferred from the crystallization drop through five drops of 0.2 M ammonium tartrate pH 6.6, 20% (w/v) PEG 3,350 for 30 seconds/drop at 4°C, then to a second drop of this solution for 142 hours at 4°C, subsequently to a third drop of this solution for 72 hours at 4°C, and finally a fourth drop of this solution for 26 hours at 4°C, after which they were cryoprotected in 0.15 M ammonium tartrate pH 6.6, 15% (w/v) PEG 3,350, 25% (v/v) glycerol for 20 seconds prior to diffraction screening and data collection. Crystallographic data for the HePTP0/HePTP24/HePTP240 crystals were collected at Brookhaven National Laboratory National Synchrotron Light Source (BNL-NSLS) Beamlines X6A and X25 at 100K using an ADSC QUANTUM 270 CCD detector or at Brown University at 100K using a Rigaku MicroMax-007 X-ray generator and R-AXIS IV++ imaging plate detector. All crystallographic data were indexed, scaled and merged using HKL2000 0.98.692i.31 The structures were solved by rigid body refinement using the program RefMac 5.2.001932 and the structure of HePTP44–339 D236A/C270S/Q314A (PDB ID: 2QDM) or HePTP0 as input models, after omitting solvent molecules, resulting in an initial Rfree = 31.2% and FOM = 0.75% for HePTP0, Rfree = 27.1% and FOM = 0.79 for HePTP24 and Rfree = 30.4% and FOM = 0.78 for HePTP240. All models were completed by cycles of manual building using the program Coot 6.0.233 coupled with structure refinement using RefMac 5.2.0019 against the datasets. The structure of HePTP0 was determined to 1.90 Å resolution and refined to Rwork = 16.2% and Rfree = 21.2%, and contains 1 molecule of HePTP, 277 water molecules, 6 sulfate molecules, and 5 glycerol molecules per asymmetric unit (HePTP residues 337–339 were not observed in the electron density map and so were not modeled). The structure of HePTP24 was determined to 2.60 Å resolution and refined to Rwork = 19.9% and Rfree = 25.3%, and contains 1 molecule of HePTP, 181 water molecules, 1 sulfate molecule, 1 tartrate molecule, and 2 glycerol molecules per asymmetric unit (HePTP residues 176–182, 235–239 and 337–339 were not observed in the electron density map and so were not modeled). The structure of HePTP240 was determined to 2.25 Å resolution and refined to Rwork = 19.0% and Rfree = 24.3%, and contains 1 molecule of HePTP, 143 water molecules, 2 tartrate molecules, and 2 glycerol molecules per asymmetric unit (HePTP residues 122–123, 175–183, 236–240 and 336–339 were not observed in the electron density map and so were not modeled). The stereochemical quality of the models was analyzed using MolProbity,34 which performs Ramachandran plot, Cβ deviation, and rotamer analyses. The agreement of the models to the diffraction data was analyzed using SFCheck 7.2.02.35 Atomic coordinates of the final models and experimental structure factors for the HePTP structures presented herein have been deposited with the Protein Data Bank (PDB) as entries 3O4S, 3O4T, 3O4U.
Calculated for ligand bound at the HePTP PTP loop.