The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae.
<h4>Background</h4>Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other...
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Public Library of Science (PLoS)
2008-09-01
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| author | Maninder K Sohi Dennis A Veselkov Ivan Laponogov Xiao-Su Pan L Mark Fisher Mark R Sanderson |
| author_facet | Maninder K Sohi Dennis A Veselkov Ivan Laponogov Xiao-Su Pan L Mark Fisher Mark R Sanderson |
| author_sort | Maninder K Sohi |
| collection | DOAJ |
| description | <h4>Background</h4>Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other anti-pneumonococcal drugs such as quinolones that target the enzymes DNA gyrase and topoisomerase IV. During crystallization and in the avenues to finding a method to determine phases for the structure of the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae, obstacles were faced at each stage of the process. These problems included: majority of the crystals being twinned, either non-diffracting or exhibiting a high mosaic spread. The crystals, which were grown under conditions that favoured diffraction, were difficult to flash-freeze without loosing diffraction. The initial structure solution by molecular replacement failed and the approach proved to be unviable due to the complexity of the problem. In the end the successful structure solution required an in-depth data analysis and a very detailed molecular replacement search.<h4>Methodology/principal findings</h4>Crystal anti-twinning agents have been tested and two different methods of flash freezing have been compared. The fragility of the crystals did not allow the usual method of transferring the crystals into the heavy atom solution. Consequently, it was necessary to co-crystallize in the presence of the heavy atom compound. The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized. Ultimately, molecular replacement was used to solve the structure by sorting through a large number of solutions in space group P1 using CNS.<h4>Conclusions/significance</h4>The main objective of this paper is to describe the obstacles which were faced and overcome in order to acquire data sets on such difficult crystals and determine phases for successful structure solution. |
| format | Article |
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| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2008-09-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-a0a08757fa574262a43df4f00321df7d2025-08-20T02:00:50ZengPublic Library of Science (PLoS)PLoS ONE1932-62032008-09-0139e320110.1371/journal.pone.0003201The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae.Maninder K SohiDennis A VeselkovIvan LaponogovXiao-Su PanL Mark FisherMark R Sanderson<h4>Background</h4>Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other anti-pneumonococcal drugs such as quinolones that target the enzymes DNA gyrase and topoisomerase IV. During crystallization and in the avenues to finding a method to determine phases for the structure of the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae, obstacles were faced at each stage of the process. These problems included: majority of the crystals being twinned, either non-diffracting or exhibiting a high mosaic spread. The crystals, which were grown under conditions that favoured diffraction, were difficult to flash-freeze without loosing diffraction. The initial structure solution by molecular replacement failed and the approach proved to be unviable due to the complexity of the problem. In the end the successful structure solution required an in-depth data analysis and a very detailed molecular replacement search.<h4>Methodology/principal findings</h4>Crystal anti-twinning agents have been tested and two different methods of flash freezing have been compared. The fragility of the crystals did not allow the usual method of transferring the crystals into the heavy atom solution. Consequently, it was necessary to co-crystallize in the presence of the heavy atom compound. The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized. Ultimately, molecular replacement was used to solve the structure by sorting through a large number of solutions in space group P1 using CNS.<h4>Conclusions/significance</h4>The main objective of this paper is to describe the obstacles which were faced and overcome in order to acquire data sets on such difficult crystals and determine phases for successful structure solution.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0003201&type=printable |
| spellingShingle | Maninder K Sohi Dennis A Veselkov Ivan Laponogov Xiao-Su Pan L Mark Fisher Mark R Sanderson The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae. PLoS ONE |
| title | The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae. |
| title_full | The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae. |
| title_fullStr | The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae. |
| title_full_unstemmed | The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae. |
| title_short | The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae. |
| title_sort | difficult case of crystallization and structure solution for the parc55 breakage reunion domain of topoisomerase iv from streptococcus pneumoniae |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0003201&type=printable |
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