Crystal growth: Protein-DNA complexes: Difference between revisions
(New page: Tips suggested by the CCP4 community as the best way to crystallize a protein/DNA complex 1. One of the biggest variables is to screen different lengths of DNA 2. Good to try both blunt...) |
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7. Other useful tips? | 7. Other useful tips? | ||
Protein DNA crystals can be very fragile so good idea to screen with cryoprotectant in the screen to save manipulations | * Protein DNA crystals can be very fragile so good idea to screen with cryoprotectant in the screen to save manipulations | ||
Test crystals with a gel to see if DNA is present | * Test crystals with a gel to see if DNA is present | ||
Use of SAXS to get molecular envelop of complex | * Use of SAXS to get molecular envelop of complex | ||
* general guidelines to prepare protein/DNA complexes suitable for crystallization trials: [http://dx.doi.org/10.1007/978-1-59745-209-0_11 Hollis T (2007) Crystallization of protein-DNA complexes. Methods Mol. Biol. 363: 225–237] | |||
* design and composition of a specialized screen for protein/DNA complexes: [http://dx.doi.org/10.1107/S1744309112025316 Pryor EE Jr, Wozniak DJ & Hollis T (2012) Crystallization of Pseudomonas aeruginosa AmrZ protein: development of a comprehensive method for obtaining and optimization of protein-DNA crystals. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68: 985–993] | |||
== Some references: == | |||
Schultz, S. C., Shields, G. C. & Steitz, T. A. (1990). | Schultz, S. C., Shields, G. C. & Steitz, T. A. (1990). | ||
Latest revision as of 10:54, 9 September 2015
Tips suggested by the CCP4 community as the best way to crystallize a protein/DNA complex
1. One of the biggest variables is to screen different lengths of DNA
2. Good to try both blunt and sticky ends. Perhaps as a first attempt try sticky ends with complementary ends
3. Could try purification by gel filtration if the complex is tight. Probably easier, quicker, cheaper and just as successful just to mix and pray!
4. Screen for crystals using a wide variety of crystal screens Natrix, Nucleix, PEGS, Index, JCSG core, MPD screen, PEG ion screen, home made PEG salt screen (+/- Mg++)
5. DNA can be ordered from a wide range of suppliers. Could try using un-purified DNA for screening, purify your own or order HPLC purified DNA
6. Good to try a wide range of ratio’s for protein/DNA. Most popular seems to be a ratio of 1/1.2
7. Other useful tips?
- Protein DNA crystals can be very fragile so good idea to screen with cryoprotectant in the screen to save manipulations
- Test crystals with a gel to see if DNA is present
- Use of SAXS to get molecular envelop of complex
- general guidelines to prepare protein/DNA complexes suitable for crystallization trials: Hollis T (2007) Crystallization of protein-DNA complexes. Methods Mol. Biol. 363: 225–237
- design and composition of a specialized screen for protein/DNA complexes: Pryor EE Jr, Wozniak DJ & Hollis T (2012) Crystallization of Pseudomonas aeruginosa AmrZ protein: development of a comprehensive method for obtaining and optimization of protein-DNA crystals. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68: 985–993
Some references:[edit | edit source]
Schultz, S. C., Shields, G. C. & Steitz, T. A. (1990). Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. J. Mol.Biol. 213, 159–166.
Tan et al, Crystallization of the Yeast MATa2/MCM1/DNA Ternary Complex: General Methods and Principles for Protein/DNA Cocrystallization J. Mol. Biol. (2000) 297, 947-959
Cannone et al, Crystallization of bFGF-DNA aptamer complexes using a Sparse Matrix designed for protein–nucleic acid complexes Journal of Crystal Growth, 2001 232 (2001) 409–417
Book chapter 8 from Crystallization of nucleic acids and proteins: a practical approach. Edited by A.Ducruix and R. Giege (Oxford University Press)