Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system.
IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2(1)2(1)2(1), with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 A, and data were collected to 2.9 A resoluti...
| Main Authors: | , , , , , , , |
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| פורמט: | Journal article |
| שפה: | English |
| יצא לאור: |
2006
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| _version_ | 1826266557694607360 |
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| author | Johnson, S Roversi, P Espina, M Deane, J Birket, S Picking, W Blocker, A Picking, W Lea, S |
| author_facet | Johnson, S Roversi, P Espina, M Deane, J Birket, S Picking, W Blocker, A Picking, W Lea, S |
| author_sort | Johnson, S |
| collection | OXFORD |
| description | IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2(1)2(1)2(1), with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 A, and data were collected to 2.9 A resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 A resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 A, beta = 107.9 degrees . An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit. |
| first_indexed | 2024-03-06T20:40:46Z |
| format | Journal article |
| id | oxford-uuid:34337bdf-f3fb-4831-b462-e094f46c63eb |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T20:40:46Z |
| publishDate | 2006 |
| record_format | dspace |
| spelling | oxford-uuid:34337bdf-f3fb-4831-b462-e094f46c63eb2022-03-26T13:24:35ZExpression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:34337bdf-f3fb-4831-b462-e094f46c63ebEnglishSymplectic Elements at Oxford2006Johnson, SRoversi, PEspina, MDeane, JBirket, SPicking, WBlocker, APicking, WLea, SIpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2(1)2(1)2(1), with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 A, and data were collected to 2.9 A resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 A resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 A, beta = 107.9 degrees . An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit. |
| spellingShingle | Johnson, S Roversi, P Espina, M Deane, J Birket, S Picking, W Blocker, A Picking, W Lea, S Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system. |
| title | Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system. |
| title_full | Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system. |
| title_fullStr | Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system. |
| title_full_unstemmed | Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system. |
| title_short | Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system. |
| title_sort | expression limited proteolysis and preliminary crystallographic analysis of ipad a component of the shigella flexneri type iii secretion system |
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