Crystal behave funny

From: Prem kumar
Date: 13 April 2012 11:51

Hi all,

I got some Protein + DNA complex crystals (image attached) recently.
They are needle shape some times splitted chromosome type crystals. When we pick long needles they bend so much than normal crystal but they dont break. The small needle dissolve very fast as try to open the drop's film. we try to diffract the long needle crystals and they diffract up to 20 A resolution. Any suggestion how to improve those crystal packing.

Thanks in advance!

-Prem

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From: Gregory Bowman

Hi Prem,

The first thing I would do is to make certain that you really have prot+DNA crystals, and not DNA alone. If you can isolate enough crystals (you may need 15 or 30, depending on how large they are), SDS page would be informative. Run protein and DNA alone and together in the same gel as controls. This could save you a lot of time/effort since you don't want to optimize DNA-only crystals.

In any case, if it is the complex and still doesn't diffract well, I'd just start changing the DNA, both in length and sequence (the bases that don't matter for protein binding).

Good luck,

Greg

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Department of Biophysics

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From: Phoebe Rice
I'd suggest:

- Find a dinosaur from my generation who can suck one into a capillary and check diffraction at room T.

- Try using those loops that look like miniature tennis paddles to give the crystal a little more support

- To minimize strain on the crystal when pulling it out of the drop, try to get its long dimension perpendicular to the air-water interface (usually easier said than done).

- Try to find conditions where the crystals don't start to redissolve while you mount them

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From: aaleshin

> - Find a dinosaur from my generation who can suck one into a capillary and check diffraction at room T.

> - Try to find conditions where the crystals don't start to redissolve while you mount them

As a matter of fact, people begin to forget that capillaries are good not only for checking the diffraction, but also for the data collection.
However, the laws of Nature do not change with time, and the old dinosaur ways may still work. We just collected at SSRL a room T. data set from crystals that consisted by 75% v/v of water and 25% of a 2500 aa protein complex. The crystals did not like cryoprotectants (including Paratone), but were happy in the capillaries.

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From: <mjvdwoerd
Hi Prem,

In addition to other remarks made:
- You could dissolve one or more crystals in water and have mass spec done to verify that your crystals are a complex. It takes many crystals (20-30) to make sure on an SDS-PAGE. You will probably need to silver stain the gel to enhance the sensitivity. And optimize for visualizing DNA (of course protein and DNA would each be control lanes).
- Apart from optimizing your DNA length and overhang as suggested, you could also try to see what a detergent does for you. My experience is that they can dramatically improve the crystal quality for protein-DNA complexes.

But you first need to know if the crystal consists of both protein and DNA. I am optimistic about the probability.

Mark (who apparently is also a dinosaur because he practices room temperature crystallography)

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From: Bosch, Juergen

Considering myself a meso-dinosaur,

I would suggest to simply grow your crystals in capillaries and then wick off the liquid and shoot them like this without much handling.

A very efficient and simple trick is to stick a capillary in an Eppendorf tube with the lid on using the Eppendorf tube as a reservoir.

I admit this method was used before I was born but works well, not quite high throughput though and need some training in handling etc.

As a tip use a syringe to poke a hole into the Eppendorf tube and then wiggle until the capillary fits.

Jürgen

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Jürgen Bosch