Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II
Abstract
:1. Introduction
2. Results
2.1. Single Crystal Vector Data Collection
2.1.1. Akt1
2.1.2. PI3Kα
2.1.3. CDP-Chase
2.2. Multicrystal Data Collection
2.2.1. CDP-Chase
2.2.2. H108A-PHM
3. Discussion
3.1. Biological Insights
3.2. Data Collection Strategies
4. Materials and Methods
4.1. Protein Expression, Purification, and Crystallization
4.2. Data Collection
4.3. Accession Codes
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Expression vectors for PI3K (p110α-16644; p85α-niSH2-17744) and CDP-Chase (73161) have been deposited in Addgene. The expression vector for Akt1 is available from P.A.C ([email protected]). |
Akt1 6NPZ | PI3Kα 6NCT | CDP-Chase/ADP-Ribose (Single) 6NCI | CDP-Chase/ADP-Ribose (Multi) 6NCH | H108A-PHM 6NCK | |
---|---|---|---|---|---|
Data Collection | |||||
Diffraction source | NSLS-II X17-ID-1 | NSLS-II X17-ID-1 | NSLS-II X17-ID-2 | NSLS-II X17-ID-2 | NSLS-II X17-ID-1 |
Wavelength (Å) | 0.99962 | 0.919909 | 0.97934 | 0.97934 | 0.918394 |
Beam size (µm) | 8 × 6 | 7 × 5 | 5 × 6 | 5 × 6 | 7 × 5 |
Temperature (K) | 100 | 100 | 100 | 100 | 100 |
Detector | Dectris Eiger 9M | Dectris Eiger 9M | Dectris Eiger 16M | Dectris Eiger 16M | Dectris Eiger 9M |
Rotation range per image (°) | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 |
Total rotation range (°) | 180 | 140 | 280 | 119 | 140 |
Space group | p21 | p212121 | p212121 | p212121 | p212121 |
a, b, c (Å) | 86.32, 56.09, 92.02 | 115.36, 117.72, 151.33 | 61.77, 67.01, 111.43 | 61.71, 67.20, 111.29 | 59.31, 65.88, 69.75 |
α, β, γ (°) | 90.00, 104.56, 90.00 | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.00 |
Resolution range (Å) | 29.82–2.12 (2.17–2.12) | 49.54–3.35 (3.47–3.35) | 29.47–2.08 (2.13–2.08) | 19.76–2.00 (2.05–2.00) | 47.89–2.70 (2.80–2.70) |
Total no. of observations | 326,363 | 159,060 | 369,062 | 213,498 | 38,364 |
No. of unique observations | 48,316 | 29,228 | 28,297 | 30,674 | 7872 |
Completeness (%) | 98.5 (81.8) | 96.4 (97.2) | 98.8 (84.5) | 96.6 (96.8) | 99.6 (99.3) |
Redundancy | 6.8 (6.6) | 5.4 (5.4) | 13.0 (11.2) | 7.0 (7.0) | 4.9 (4.2) |
〈I/σ(I)〉 | 11.3 (2.0) | 11.8 (1.5) | 14.4 (2.6) | 4.3 (1.8) | 8.2 (2.8) |
Rmerge | 0.101 (0.760) | 0.104 (0.935) | 0.136 (0.942) | 0.357 (1.11) | 0.130 (0.580) |
Rp.i.m. | 0.042 (0.314) | 0.046 (0.418) | 0.039 (0.280) | 0.137 (0.425) | 0.062 (0.309) |
CC1/2 | 0.996 (0.849) | 0.998 (0.685) | 0.998 (0.818) | 0.941 (0.608) | 0.991 (0.830) |
Refinement | |||||
Resolution range (Å) | 89.07–2.12 (2.17–2.12) | 49.54–3.35 (3.44–3.35) | 29.47–2.08 (2.13–2.08) | 19.76–2.00 (2.05–2.00) | 44.08–2.70 (2.77–2.70) |
No. of reflections, working set | 45,932 | 27,765 | 26,741 | 29,211 | 7477 |
No. of reflections, test set | 2364 | 1462 | 1502 | 1461 | 394 |
Rwork/Rfree | 0.185/0.241 (0.249/0.316) | 0.199/0.270 (0.314/0.343) | 0.171/0.222 (0.241/0.291) | 0.200/0.261 (0.276/0.334) | 0.219/0.294 (0.322/0.342) |
No. of non-H atoms | |||||
Protein | 5406 | 10,462 | 3288 | 3311 | 2350 |
Ligand/ion | 178 | 31 | 52 | 25 | 2 |
Water | 475 | 0 | 255 | 366 | 20 |
R.m.s. deviations | |||||
Bonds (Å) | 0.016 | 0.013 | 0.009 | 0.008 | 0.008 |
Angles (°) | 2.06 | 1.95 | 1.51 | 1.45 | 1.54 |
Average B factors (Å2) | |||||
Protein | 41.7 | 133.8 | 35.7 | 27.6 | 58.9 |
Ligand/ion | 56.2 | 189.2 | 78.0 | 51.8 | 109.4 |
Water | 45.8 | n/a | 40.9 | 34.7 | 41.6 |
Ramachandran (%) | |||||
Favorable | 96.4 | 95.1 | 97.2 | 96.9 | 95.6 |
Allowed | 2.0 | 4.5 | 2.6 | 3.1 | 4.4 |
Disallowed | 1.6 | 0.4 | 0.2 | 0 | 0 |
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Miller, M.S.; Maheshwari, S.; Shi, W.; Gao, Y.; Chu, N.; Soares, A.S.; Cole, P.A.; Amzel, L.M.; Fuchs, M.R.; Jakoncic, J.; et al. Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II. Molecules 2019, 24, 496. https://doi.org/10.3390/molecules24030496
Miller MS, Maheshwari S, Shi W, Gao Y, Chu N, Soares AS, Cole PA, Amzel LM, Fuchs MR, Jakoncic J, et al. Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II. Molecules. 2019; 24(3):496. https://doi.org/10.3390/molecules24030496
Chicago/Turabian StyleMiller, Michelle S., Sweta Maheshwari, Wuxian Shi, Yuan Gao, Nam Chu, Alexei S. Soares, Philip A. Cole, L. Mario Amzel, Martin R. Fuchs, Jean Jakoncic, and et al. 2019. "Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II" Molecules 24, no. 3: 496. https://doi.org/10.3390/molecules24030496