Crystallographic Characterization of Sodium Ions in a Bacterial Leucine/Sodium Symporter
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Purification, Characterization, and Crystallization
2.2. Crown-5 Soaking
2.3. Diffraction Data Collection
2.4. Data Reduction and Assembly
2.5. Structure Determination and Low-Z Element Identification
3. Results
3.1. Anomalous Diffraction of Na+ in LeuT
3.2. Speciation of Anomalous Scattering Elements in LeuT
3.3. Characterization of Na+ in LeuT
3.4. Conformational Change upon Na+ and Substrate Depletion
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Data Collection | LeuT (Multi-Crystals) | LeuT w Crown-5 |
---|---|---|
Beamline | FMX (NSLS-II) | FMX (NSLS-II) |
Wavelength (Å) | 2.48 | 2.48 |
Space group | C2 | C2 |
Unit cell (Å) | ||
a, b, c (Å) | 87.11, 86.38, 80.73 | 86.44, 87.38, 80.80 |
a, b, γ (°) | 90.0, 95.3, 90.0 | 90.00, 95.1, 90.00 |
Solvent content (%) | 53.2 | 53.5 |
Bragg spacings (Å) | 38.04–4.001 (4.144–4.001) | 39.44–3.8 (3.936–3.8) |
Total reflections | 595,518 (60,371) | 19,702 (1975) |
Bijvoet unique reflections | 10,240 (1028) | 11,251 (594) |
Completeness (%) | 99.65 (100.00) | 98.50 (98.99) |
<I/σ(I)> | 26.6 (21.3) | 2.3 (0.7) |
Rsplit | 0.027 | 0.272 |
Multiplicity | 116 (117.9) | 3.3 (3.3) |
CC1/2 | 0.997 (0.996) | 0.982 (0.535) |
Refinement | ||
Resolution (Å) | 4 | 3.8 |
No. reflections | 5110 (514) | 5911 (589) |
Rwork/Rfree | 0.169/0.201 | 0.272/0.315 |
No. atoms | 4263 | 4056 |
Wilson B factor (Å2) | 60.79 | 122.14 |
Average B factor (Å2) | 55.7 | 125.05 |
R.m.s. deviations | ||
Bond length (Å) | 0.004 | 0.002 |
Bond angle (°) | 0.69 | 0.49 |
PDB code | 8FT4 | 8FT5 |
Occupancy | f”(Na1) | f”(Na2) | f”(Cl) | f”(Peak11) | f”(S) |
---|---|---|---|---|---|
1 | 0.16 | 0.20 | 1.33 | 0.51 | 1.05 |
0.9 | 0.32 | 0.33 | 1.21 | 0.52 | 1.13 |
0.8 | 0.37 | 0.36 | 1.25 | 0.62 | 1.21 |
0.7 | 0.43 | 0.39 | 1.32 | 0.69 | 1.27 |
0.6 | 0.50 | 0.45 | 1.45 | 0.80 | 1.42 |
0.5 | 0.57 | 0.55 | 1.61 | 0.91 | 1.58 |
0.4 | 0.71 | 0.54 | 1.84 | 1.09 | 1.94 |
0.3 | 0.89 | 0.56 | 2.15 | 1.36 | 2.30 |
0.2 | 0.88 | 0.61 | 2.24 | 1.48 | 2.69 |
0.1 | 0.41 | 0.38 | 1.22 | 0.79 | 2.97 |
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Karasawa, A.; Liu, H.; Quick, M.; Hendrickson, W.A.; Liu, Q. Crystallographic Characterization of Sodium Ions in a Bacterial Leucine/Sodium Symporter. Crystals 2023, 13, 183. https://doi.org/10.3390/cryst13020183
Karasawa A, Liu H, Quick M, Hendrickson WA, Liu Q. Crystallographic Characterization of Sodium Ions in a Bacterial Leucine/Sodium Symporter. Crystals. 2023; 13(2):183. https://doi.org/10.3390/cryst13020183
Chicago/Turabian StyleKarasawa, Akira, Haijiao Liu, Matthias Quick, Wayne A. Hendrickson, and Qun Liu. 2023. "Crystallographic Characterization of Sodium Ions in a Bacterial Leucine/Sodium Symporter" Crystals 13, no. 2: 183. https://doi.org/10.3390/cryst13020183