Crystal growth: Protein-DNA complexes

2009-07-20T11:51:43Z

Cwilliam: 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...

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

Several references below:

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)

Crystals

2009-07-20T11:31:49Z

Cwilliam:

== Crystal Characterization ==
* [[Birefringence]]

== Crystal Imperfections ==
* [[Diffuse Scattering]]
* [[Incommensurate Lattices]]

== Crystal Growth ==

* [[Modifying the protein to crystallize better]]
* [[Crystallization screens and methods]]
* [[I have crystals, but are they salt?]]
* [[Conditions prone to salt crystallization]]
* [[Biophysical methods]] for evaluation of crystallization tendency
* [[Improving crystal quality]]
* [[Robots for crystallization setup]]
* [[Visualization of crystal growth]]
* [[Crystal mounting]]
* [[Robots for crystal mounting]]
* [[Robots for crystallization monitoring]]
* [[Crystal growth: Tips and Tricks]]
* [[Crystal growth: Protein-DNA complexes]]
* [[BMCD|Biomolecular crystallization database]]

== Crystal handling ==
* [[Sticky crystals]]
* [[Soaking with heavy atoms]]

== Data collection ==
* [[X-ray generators and detectors]] (experiences)
* [[Synchrotrons]]
* [[Cryo]]
* [[Strategies]]
* [[Data quality]]
* [[Radiation damage]]
* [[Data collection: Tips and Tricks]]

== References ==

* crystallization of soluble proteins (see also [[Improving crystal quality]])

* crystallization of membrane proteins (see also [[Improving crystal quality#Improving diffraction quality of membrane protein crystals]])
# Wiener MC (2004) A pedestrian guide to membrane protein crystallization. Methods 34(3), 364-372.
# Loll PJ (2003) Membrane protein structural biology: the high throughput challenge. J. Struct. Biol. 142(1), 144-153.
# Methods and Results in Crystallization of Membrane Proteins Iwata S (Ed.), San Diego, CA: International University Line. (2003)
# Macromolecular Crystallography Protocols, Volume 1: Preparation and Crystallization of Macromolecules. Doublie S (Ed.), Totowa, NJ: Humana Press. (2007)
# Structural Biology of Membrane Proteins. Grisshammer R, & Buchanan SK (Eds.), Cambridge, UK: Royal Society of Chemistry. (2006)

CCP4 wiki - User contributions [en]

Crystal growth: Protein-DNA complexes

Crystals