From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Preparation
2.2. Crystallization
2.3. Deuterium Labeling and X-ray Analysis
3. Results & Discussion
3.1. Initial Screening
3.2. Microseed Matrix Screening (MMS)
3.3. Optimization with Seed Concentration
3.4. Deuterated CA IX SV and Large Volume Crystallization
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Precipitant | Buffer | pH | Additive/Extra | Crystallization Set-Up | Correlation to Figure 2 |
---|---|---|---|---|---|
20% PEG 3350 | None | n/a | 0.2 M Ammonium Formate | Hanging drop | 1A 1 |
20–24% PEG 3350 | 0.1 M Tris | 7.0 | None | Sitting drop | n/a |
25% PEG 3350 | 0.1 M HEPES | 7.5 | None | Sitting drop | 2H |
20–24% PEG 3350 | 0.1 M Tris | 8.0 | None | Sitting drop | n/a |
25% PEG 3350 | 0.1 M Tris | 8.5 | None | Sitting drop | 2G |
25% PEG 3350 | 0.1 M Tris | 5.5 | 0.2 M Magnesium Chloride | Sitting drop | 2C |
20–26% PEG 3350 | 0.1 M Tris | 7.0 | 4–10% 2-propanol | Sitting drop | n/a |
25% PEG 3350 | 0.1 M HEPES | 7.5 | 0.2 M Ammonium Acetate | Sitting drop | 2D |
22% PEG 3350 | 0.1 M Tris | 7.5 | 0.2 M Sodium Chloride | Hanging drop, microbatch | 1B 1 |
20–26% PEG 3350 | 0.1 M Tris | 8.0 | 4–10% 2-propanol | Sitting drop | n/a |
18–22% PEG 4000 | 0.1 M Tris | 7.0 | None | Sitting drop | n/a |
18–22% PEG 4000 | 0.1 M Tris | 7.0 | 0.2 M Sodium Chloride | Hanging drop, microbatch | 1C 1 |
20% PEG 4000 | 0.1 M HEPES | 7.5 | 10% 2-propanol | Sitting drop | 2E |
22–26% PEG 4000 | 0.1 M Tris | 8.0 | 10% 2-propanol | Sitting drop | n/a |
30% PEG 4000 | 0.1 M Tris | 8.5 | 0.2 M Sodium Acetate | Sitting drop, microbatch | 2F, 3A–D |
18–22% PEG 6000 | 0.1 M Tris | 7.5 | None | Sitting drop | n/a |
18–22% PEG 6000 | 0.1 M Tris | 7.5 | 0.2 M Sodium Chloride | Hanging drop | 1D 1 |
22.5% PurePEGs Cocktail | 0.1 M Citric Acid | 3.5 | 0.3 M Sodium Formate | Sitting drop | n/a |
22.5% PurePEGs Cocktail | 0.1 M Ammonium Citrate | 5.0 | 0.3 M Cesium Chloride | Sitting drop | 2B |
22.5% PurePEGs Cocktail | 0.1 M Sodium Cacodylate | 6.5 | 0.3 M Magnesium Chloride | Sitting drop | 2A |
1.0 M Sodium Citrate | 0.1 M Sodium Cacodylate | 6.5 | None | Sitting drop | n/a |
H/H | H/D | D/D | |
---|---|---|---|
Wavelength (Å) Beamline, source | 0.979 BM-30 (FIP), ESRF | 0.979 BioMAX, MAX IV laboratory | 0.979 BioMAX, MAX IV laboratory |
Space group, unit cell | P21; a = 44.3, b = 65.1, c = 46.7; β = 115.1 | P21; a = 44.5, b = 65.4, c = 46.7; β = 115.1 | P21; a = 44.4, b = 65.1, c = 46.6; β = 114.7 |
Resolution range (Å) | 50.0–1.77 (1.88–1.77) | 40.0–1.39 (1.42–1.39) | 40.0–1.49 (1.51–1.49) |
Total No. of reflections | 63,296 (9199) | 327,912 (15,926) | 265,165 (13,184) |
No. of unique reflections | 22,187 (3463) | 48,352 (2406) | 39,461 (1948) |
Redundancy | 2.8 (2.6) | 6.8 (6.6) | 6.7 (6.8) |
Completeness (%) | 95.1 (92.9) | 99.8 (99.1) | 99.9 (99.3) |
〈I/σ(I)〉 | 10.5 (2.2) | 19.9 (2.2) | 14.3 (2.2) |
Rmerge † (%) | 6.3 (48.6) | 3.6 (78.1) | 6.0 (78.1) |
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Koruza, K.; Lafumat, B.; Nyblom, M.; Knecht, W.; Fisher, Z. From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography. Crystals 2018, 8, 434. https://doi.org/10.3390/cryst8110434
Koruza K, Lafumat B, Nyblom M, Knecht W, Fisher Z. From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography. Crystals. 2018; 8(11):434. https://doi.org/10.3390/cryst8110434
Chicago/Turabian StyleKoruza, Katarina, Bénédicte Lafumat, Maria Nyblom, Wolfgang Knecht, and Zoë Fisher. 2018. "From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography" Crystals 8, no. 11: 434. https://doi.org/10.3390/cryst8110434