2bsw
From Proteopedia
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==Overview== | ==Overview== | ||
| - | The success of structural studies performed on an individual target in | + | The success of structural studies performed on an individual target in small scale or on many targets in the system-wide scale of structural genomics depends critically on three parameters: (i) obtaining an expression system capable of producing large quantities of the macromolecule(s) of interest, (ii) purifying this material in soluble form, and (iii) obtaining diffraction-quality crystals suitable for x-ray analysis. The attrition rate caused by these constraints is often quite high. Here, we present a strategy that addresses each of these three parameters simultaneously. Using DNA shuffling to introduce functional sequence variability into a protein of interest, we screened crude lysate supernatants for soluble variants that retain enzymatic activity. Crystallization trials performed on three WT and eight shuffled enzymes revealed two variants that crystallized readily. One of these was used to determine the high-resolution structure of the enzyme by x-ray analysis. The sequence diversity introduced through shuffling efficiently samples crystal packing space by modifying the surface properties of the enzyme. The approach demonstrated here does not require guidance as to the type of mutation necessary for improvements in expression, solubility, or crystallization. The method is scaleable and can be applied in situations where a single protein is being studied or in high-throughput structural genomics programs. Furthermore, it should be readily applied to structural studies of soluble proteins, membrane proteins, and macromolecular complexes. |
==About this Structure== | ==About this Structure== | ||
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[[Category: ]] | [[Category: ]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Castle, L | + | [[Category: Castle, L A.]] |
[[Category: Gorton, R.]] | [[Category: Gorton, R.]] | ||
| - | [[Category: Keenan, R | + | [[Category: Keenan, R J.]] |
| - | [[Category: Siehl, D | + | [[Category: Siehl, D L.]] |
[[Category: CAO]] | [[Category: CAO]] | ||
[[Category: GOL]] | [[Category: GOL]] | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:41:18 2008'' |
Revision as of 14:41, 21 February 2008
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CRYSTAL STRUCTURE OF A GLYPHOSATE-N-ACETYLTRANSFERASE OBTAINED BY DNA SHUFFLING.
Overview
The success of structural studies performed on an individual target in small scale or on many targets in the system-wide scale of structural genomics depends critically on three parameters: (i) obtaining an expression system capable of producing large quantities of the macromolecule(s) of interest, (ii) purifying this material in soluble form, and (iii) obtaining diffraction-quality crystals suitable for x-ray analysis. The attrition rate caused by these constraints is often quite high. Here, we present a strategy that addresses each of these three parameters simultaneously. Using DNA shuffling to introduce functional sequence variability into a protein of interest, we screened crude lysate supernatants for soluble variants that retain enzymatic activity. Crystallization trials performed on three WT and eight shuffled enzymes revealed two variants that crystallized readily. One of these was used to determine the high-resolution structure of the enzyme by x-ray analysis. The sequence diversity introduced through shuffling efficiently samples crystal packing space by modifying the surface properties of the enzyme. The approach demonstrated here does not require guidance as to the type of mutation necessary for improvements in expression, solubility, or crystallization. The method is scaleable and can be applied in situations where a single protein is being studied or in high-throughput structural genomics programs. Furthermore, it should be readily applied to structural studies of soluble proteins, membrane proteins, and macromolecular complexes.
About this Structure
2BSW is a Single protein structure of sequence from [1] with , and as ligands. Known structural/functional Site: . Full crystallographic information is available from OCA.
Reference
DNA shuffling as a tool for protein crystallization., Keenan RJ, Siehl DL, Gorton R, Castle LA, Proc Natl Acad Sci U S A. 2005 Jun 21;102(25):8887-92. Epub 2005 Jun 10. PMID:15951425 [[Category: ]]
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