Extending the pool of compatible peptide hydrogels for protein crystallization
Abstract
:1. Introduction
2. Materials and Methods
2.1. Hydrogel Preparation
2.2. Crystallization Experiments
2.3. X-ray Data Collection and Analysis
3. Results
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Hydrogel | (% w/v) | Protein | Concentration (mg/mL) | Precipitant |
---|---|---|---|---|
Fmoc-MF Fmoc-Y Agarose | 0.2, 0.5, 0.75 and 1.0 0.2, 0.5, 0.75 and 1.0 0.5 | Lysozyme | 80 | 6.0% (w/v) NaCl, 50 mM of Na acetate pH 4.5 |
Fmoc-MF Agarose | 0.5 and 0.75 0.5 | Thaumatin | 50 | 45% (w/v) KNa tartrate pH 7.6 |
Fmoc-MF Agarose | 0.5 and 0.75 0.5 | Glucose isomerase | 50 | 10% (v/v) PEG 1000, 0.2 M of MgCl2, 0.1 M of Hepes pH 7.0 |
Agarose (% w/v) | Fmoc-MF (% w/v) | ||||
---|---|---|---|---|---|
Concentration | 0.5 | 0.2 | 0.5 | 0.75 | 1.0 |
Data Acquisition | |||||
ESRF | ID23-2 | ID23-2 | ID23-2 | ID23-2 | ID23-2 |
Detector type | PILATUS | PILATUS | PILATUS | PILATUS | PILATUS |
Wavelength (Å) | 0.87290 | 0.87290 | 0.87290 | 0.87290 | 0.87290 |
Distance (mm) | 215.97 | 215.97 | 215.97 | 215.97 | 215.97 |
Exposure time (s) | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 |
Oscillation (°) | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
Data Statistics | |||||
Space group | P 43 21 2 | P 43 21 2 | P 43 21 2 | P 43 21 2 | P 43 21 2 |
Unit cell: a = b, c (Å) | 79.00, 37.26 | 77.33, 38.01 | 77.53, 37.81 | 77.52, 37.90 | 77.56, 37.86 |
Resolution (Å) (High-shell) | 39.50–1.20 (1.22–1.20) | 38.66–1.30 (1.32–1.30) | 38.77–1.20 (1.22–1.20) | 38.76–1.15 (1.17–1.15) | 38.78–1.25 (1.27–1.25) |
Unique reflections | 37471 (1805) | 28991 (1425) | 36659 (1800) | 41636 (2009) | 32570 (1586) |
R-merge (%) | 5.9 (79.8) | 10.5 (95.3) | 4.7 (87.6) | 5.2 (82.6) | 5.1 (83.9) |
I/σ(I) | 20.7 (3.3) | 11.1 (1.9) | 27.8 (3.2) | 23.6 (3.2) | 26.7 (3.6) |
Completeness (%) | 100.0 (100.0) | 100.0 (100.0) | 100.0 (100.0) | 100.0 (100.0) | 100.0 (100.0) |
Redundancy | 13.6 (13.8) | 13.9 (14.4) | 13.7 (13.8) | 13.7 (13.2) | 13.7 (12.8) |
B-factor (Å2) | 9.5 | 11.5 | 10.1 | 8.8 | 10.6 |
Mosaicity | 0.18 | 0.19 | 0.20 | 0.12 | 0.21 |
Thaumatin | Glucose Isomerase | |||||
---|---|---|---|---|---|---|
Agarose (% w/v) | Fmoc-MF (% w/v) | Agarose (% w/v) | Fmoc-MF (% w/v) | |||
Concentration | 0.5 | 0.5 | 0.75 | 0.5 | 0.5 | 0.75 |
Data Acquisition | ||||||
ESRF | ID23-2 | ID23-2 | ID23-2 | ID23-2 | ID23-2 | ID23-2 |
Detector type | PILATUS | PILATUS | PILATUS | PILATUS | PILATUS | PILATUS |
Wavelength (Å) | 0.87290 | 0.87290 | 0.87290 | 0.87290 | 0.87290 | 0.87290 |
Distance (mm) | 215.97 | 215.97 | 215.97 | 215.97 | 215.97 | 215.97 |
Exposure time (s) | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 |
Oscillation (°) | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
Data Statistics | ||||||
Space group | P 41 21 2 | P 41 21 2 | P 41 21 2 | I222 | I222 | I222 |
Unit cell: a, b, c (Å) | 57.98, 57.98, 150.61 | 58.17, 58.17, 151.14 | 58.14, 58.14, 150.63 | 93.41, 99.29, 103.09 | 93.18, 98.67, 102.88 | 93.20, 98.36, 102.91 |
Resolution (Å) (High shell) | 45.94–1.05 (1.07–1.05) | 46.10–1.10 (1.12–1.10) | 46.02–1.15 (1.17–1.15) | 46.71–1.15 (1.17–1.15) | 49.33–1.10 (1.12–1.10) | 46.60–1.05 (1.07–1.05) |
Unique reflections | 120047 (5702) | 106068 (5180) | 92658 (4534) | 167421 (8287) | 190602 (9407) | 214576 (9993) |
R-merge (%) | 5.6 (68.7) | 6.0 (96.1) | 7.2 (90.1) | 6.8 (77.1) | 6.3 (73.6) | 5.4 (83.8) |
I/σ(I) | 24.6 (3.5) | 22.6 (2.8) | 19.6 (3.0) | 11.9 (1.9) | 12.6 (1.9) | 15.1 (1.7) |
Completeness (%) | 99.7 (97.8) | 100.0 (100.0) | 100.0 (100.0) | 99.3 (99.9) | 99.9 (100.0) | 98.4 (93.4) |
Redundancy | 13.6 (10.4) | 13.9 (13.0) | 14.0 (13.3) | 5.2 (4.8) | 5.1 (4.7) | 5.1 (4.4) |
B-factor (Å2) | 5.6 | 6.9 | 7.2 | 8.2 | 6.3 | 6.0 |
Mosaicity | 0.05 | 0.06 | 0.06 | 0.1 | 0.12 | 0.09 |
Agarose (% w/v) | Fmoc-Y (% w/v) | ||||
---|---|---|---|---|---|
Concentration | 0.5 | 0.2 | 0.5 | 0.75 | 1.0 |
Data Acquisition | |||||
ALBA | XALOC | XALOC | XALOC | XALOC | XALOC |
Detector type | PILATUS 6M | PILATUS 6M | PILATUS 6M | PILATUS 6M | PILATUS 6M |
Wavelength (Å) | 0.980 | 0.979154 | 0.979154 | 0.979154 | 0.979154 |
Distance (mm) | 160.35 | 128.0 | 128.0 | 128.0 | 128.0 |
Exposure time (s) | 0.2seg | 0.2 | 0.2 | 0.2 | 0.2 |
Oscillation (°) | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
Data Statistics | |||||
Space group | P 43 21 2 | P 43 21 2 | P 43 21 2 | P 43 21 2 | P 43 21 2 |
Unit cell: a = b, c (Å) | 77.25, 37.90 | 78.68, 37.04 | 78.57, 37.08 | 78.52, 37.12 | 78.63, 37.22 |
Resolution (Å) (High shell) | 38.62–1.15 (1.17–1.15) | 39.34–1.00 (1.02–1.00) | 39.28–1.05 (1.07–1.05) | 39.26–1.05 (1.07–1.05) | 39.31–1.05 (1.07–1.05) |
Unique reflections | 41317 (1997) | 63226 (3165) | 51967 (2377) | 54672 (2658) | 54848 (2599) |
R-merge * (%) | 8.0 (67.4) | 3.9 (87.9) | 4.4 (61.9) | 4.3 (72.0) | 3.8 (95.4) |
I/σ(I) | 22.0 (4.5) | 43.7 (4.2) | 41.9 (6.1) | 40.1 (4.4) | 43.9 (4.0) |
Completeness (%) | 100.0 (100.0) | 100.0 (100.0) | 95.3 (89.8) | 100.0 (100.0) | 99.8 (98.0) |
Redundancy | 23.7 (23.2) | 24.2 (23.0) | 25.6 (26.6) | 24.3 (23.5) | 24.5 (23.9) |
B-factor (Å2) | 9.7 | 10.705 | 10.298 | 11.701 | 11.680 |
Mosaicity | 0.22 | 0.08 | 0.09 | 0.10 | 0.08 |
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Escolano-Casado, G.; Contreras-Montoya, R.; Conejero-Muriel, M.; Castellví, A.; Juanhuix, J.; Lopez-Lopez, M.T.; Álvarez de Cienfuegos, L.; Gavira, J.A. Extending the pool of compatible peptide hydrogels for protein crystallization. Crystals 2019, 9, 244. https://doi.org/10.3390/cryst9050244
Escolano-Casado G, Contreras-Montoya R, Conejero-Muriel M, Castellví A, Juanhuix J, Lopez-Lopez MT, Álvarez de Cienfuegos L, Gavira JA. Extending the pool of compatible peptide hydrogels for protein crystallization. Crystals. 2019; 9(5):244. https://doi.org/10.3390/cryst9050244
Chicago/Turabian StyleEscolano-Casado, Guillermo, Rafael Contreras-Montoya, Mayte Conejero-Muriel, Albert Castellví, Judith Juanhuix, Modesto T. Lopez-Lopez, Luis Álvarez de Cienfuegos, and José A. Gavira. 2019. "Extending the pool of compatible peptide hydrogels for protein crystallization" Crystals 9, no. 5: 244. https://doi.org/10.3390/cryst9050244
APA StyleEscolano-Casado, G., Contreras-Montoya, R., Conejero-Muriel, M., Castellví, A., Juanhuix, J., Lopez-Lopez, M. T., Álvarez de Cienfuegos, L., & Gavira, J. A. (2019). Extending the pool of compatible peptide hydrogels for protein crystallization. Crystals, 9(5), 244. https://doi.org/10.3390/cryst9050244