Seeded rescreening with robot?

From: Watson, Randy
Date: 28 October 2011 19:17

Hi all,

I am trying to optimize crystals and have heard of a technique where one can prepare a seedstock from existing crystals and use it to broadly re-screen from scratch for hits in new conditions.

I have access to a mosquito robot and was wondering if anyone has a protocol or recommendations on how to go about doing this using a robot for screening.

Thank you!
Randy Watson

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From: Artem Evdokimov

I would be glad to share ours.

Artem

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From: Artem Evdokimov

By popular request here's my favorite version of the in-screen seeding. We use a Mosquito but it doesn't have to be a specific robot as long as it can dispense relatively tiny volumes of seed stock.

 

Caveats: (1) if I am desperate enough to do this, then the situation is pretty bad indeed and I don't mind wasting some protein (2) my success rate is not hugely favorable but this does work on occasion when other things have failed

 

(1) identify a few likely conditions. Ideally they have microcrystals but desperation has made me try 'lovable precipitates' in the past, with a modest degree of success.

(2) harvest entire drop using a few ul of mother liquor as diluent

(3) break the existing crystals using your favorite method (sead beed, etc.) mine involves swirling a pipette tip in the mixture, running it along the walls, with rapid pipetting up and down. Dilute seed stock to useful volume (enough for screening).

(4) I do not normally centrifuge the resulting seed stock, but some people do

(5) dispense your screen as always with the usual protein/reservoir ratio. Let's say you like drops of the 0.2ul+0.2ul variety - add 25 nanoliters of the seed stock *last*. Optional mixing of the condition is a fun thing to try but it seems not to matter very much. Note that I typically use the same tips to dispense seed stock, fully aware that this causes cross-contamination of conditions. I don't mind :)

  (5a) variation - add seed stock to protein, then dispense ASAP. Surprisingly not a bad option, practically speaking.

  (5b) variation - crosslink seed stock very gently in solution (with trace of glutaraldehyde) before use. Buffers/additives with primary or secondary amine groups do interfere, of course.

  (5c) variation - mix seeds from SEVERAL initial hit conditions, then use as one seed stock. Be ready for fireworks as they may not be compatible!

(6) endure nail-biting wait for results :)

As noted earlier, it's not a sure-fire way to get new hit conditions but it does seem to work and it's a fun way to put to use a remainder of otherwise useless protein (when you've tried all other tricks you like to try).

Comments and suggestions are always welcome!

Artem

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From: Pius Padayatti


I have few personal remarks about revere matrix seeding protocol suggested here
just an addition to Artems suggested protocol
harvesting the whole drop of interest invite guaranteed
salt crystals in the second round especially if one is
using the same screen back.
If one found crystals all over your second trial go back to your
first seed drop and try to get the microcrystals and do the seeding again
you will see the difference. And in this case if you found smaller but
significant improvement in the same condition with better crystals in
other conditions.
(but check in those conditions that can form salt crystals when you
mix initial drop and the current conditions, by setting up control
drops )) I would say keep going for several rounds and you are
definitely in the right track.Or else forget about the whole seed you
looking at in those particular experiment.

In short if you found microcrystals (15 to 20 um)
harvesting them is what i found is more useful and better success than
harvesting the whole drops.

Gluteraldehyde added to the seeds is a good one
but what effect you see may not essentailly due to seeding
but combineing approaches like mentioned is definitely inovel and
thanks for sharing.

Does anybody here have tried this in mesophase and
detergent solubilized vapor diffusion and had success?

Thanks
Padayatti PS
Polgenix Inc

--
Pius S Padayatti,PhD,


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From: Bosch, Juergen

Hm,

I wouldn't call these micro crystals anymore, I mount those things and get datasets from them (sometimes). In my world 50 µm is defined as big.

Jürgen

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Jürgen Bosch

Johns Hopkins University