Structure and Function of the T4 Spackle Protein Gp61.3
Abstract
1. Introduction
2. Materials and Methods
2.1. Construction of Expression Vectors
2.2. Protein Expression
2.3. Purification of Gp61.3
2.4. Purification of Gp5*
2.5. Purification of Gp51-372 and Gp5G322D1-372
2.6. Purification of T4L
2.7. Purification of Gp61.3-Gp5Lys Complex
2.8. Crystallization and Data Collection
2.9. Structure Determination
2.10. Lysozyme Halo Assay
2.11. Analytical Ultracentrifugation
2.12. Cell Lysis Assay
2.13. Molecular Dynamics
2.14. Sequence Alignments and Molecular Graphics Generation
3. Results
3.1. Gp61.3 is Translocated into the Periplasm and Its Signal Sequence Is Cleaved
3.2. Gp61.3 Forms a 1:1 Complex with Gp5*
3.3. Gp61.3 Inhibits Lysozyme Activity of Gp5 but Does Not Affect the Activity of T4L In Vitro
3.4. The Crystal Structure of Gp61.3 Shows That It Has a Novel Fold
3.5. The Crystal Structure of the Gp61.3–Gp5Lys Complex
3.6. The Critical Location of G322D Mutation in Gp5Lys
3.7. Gp5 Can Penetrate the E. coli Inner Membrane from the Inside on Its Own
4. Discussion
Supplementary Materials
Note Added during the Proofreading
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Crystal | Gp61.3 SeMet | Gp61.3-gp5Lys |
---|---|---|
Data collection | ||
Wavelength (Å) | 0.97941 | 1.0000 |
Number of frames | 325 | 360 |
Frame width (°) | 1.0 | 1.0 |
Space group | P21 | P212121 |
Cell dimensions a, b, c (Å) | 30.376, 46.921, 75.277 | 46.72, 69.18, 83.82 |
Resolution (Å) | 39.8–1.60 (1.64–1.60) * | 17.92–1.15 (1.21–1.15) |
Rmeas | 0.064 (0.158) | 0.109 (1.885) |
<I>/<σI> | 16.0 (5.8) | 12.2 (1.8) |
Completeness (%) | 94.2 (73.7) | 99.7 (99.8) |
Multiplicity | 3.3 (3.1) | 13.5 (12.6) |
Anomalous signal # | 4.91 (2.42) | N.A. $ |
Refinement | ||
Resolution | 19.90–1.60 | 17.92–1.15 |
No. reflections used in refinement | 51,366 | 97,671 |
No. atoms (non-H) | 2272 | 2630 |
Protein | 1870 | 2195 |
Ligand/ion | 3 | 53 |
Water | 399 | 382 |
Rwork/Rfree | 0.179/0.228 | 0.107/0.122 |
Average B factor (Å2) | 18.0 | 16.3 |
Protein | 15.6 | 13.5 |
Ligand/Ion | 33.1 | 32.7 |
Water | 29.1 | 30.1 |
R.m.s. deviations | ||
Bond lengths (Å) | 0.002 | 0.008 |
Bond angles (°) | 0.493 | 1.066 |
Ramachandran plot statistics | ||
Favored (%) | 99.01 | 97.39 |
Allowed (%) | 0.91 | 1.61 |
Outliers (%) | 0.00 | 0.00 |
PDB accession code | 7CN6 | 7CN7 |
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Kanamaru, S.; Uchida, K.; Nemoto, M.; Fraser, A.; Arisaka, F.; Leiman, P.G. Structure and Function of the T4 Spackle Protein Gp61.3. Viruses 2020, 12, 1070. https://doi.org/10.3390/v12101070
Kanamaru S, Uchida K, Nemoto M, Fraser A, Arisaka F, Leiman PG. Structure and Function of the T4 Spackle Protein Gp61.3. Viruses. 2020; 12(10):1070. https://doi.org/10.3390/v12101070
Chicago/Turabian StyleKanamaru, Shuji, Kazuya Uchida, Mai Nemoto, Alec Fraser, Fumio Arisaka, and Petr G. Leiman. 2020. "Structure and Function of the T4 Spackle Protein Gp61.3" Viruses 12, no. 10: 1070. https://doi.org/10.3390/v12101070
APA StyleKanamaru, S., Uchida, K., Nemoto, M., Fraser, A., Arisaka, F., & Leiman, P. G. (2020). Structure and Function of the T4 Spackle Protein Gp61.3. Viruses, 12(10), 1070. https://doi.org/10.3390/v12101070