Re: [ccp4bb] What could these crystals be?

[Harry Powell]

Hi

I can’t see any (obvious) suggestions in the current list of answers to try 
room temperature diffraction before introducing cryoprotectants and cryocooling 
your crystals (but I might just have missed it). 

Often, just the act of changing the conditions that your crystals sit in will 
destroy internal order and stop them being crystals. Cooling the crystals to 
cryogenic temperatures can also destroy crystallinity (that’s why people spend 
so much time and effort in optimising cryoprotectant strategies).

Over the last couple of days, people on ccp4bb have suggested in-situ data 
collection, i.e. with the crystals sitting in the plate they grew in - this is 
a great way to avoid any physical damage. There may well be a facility 
relatively close to you that you could get access to in order to try this. As 
pointed out by some of the correspondents in the “Fragile Crystals” thread, 
some places will also grow your crystals for you on site, using your 
conditions. Unfortunately, I don’t know where “yahoo” is, so I can’t make a 
suggestion as to which might be closer/easier for you to access!

best wishes

Harry

> On 24 Nov 2023, at 08:59, careinaedgo...@yahoo.com 
> <02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> 
> I wanted to thank everyone for their suggestions and ideas regarding the 
> eyeball shaped crystals that I got at the beginning of the month.
> I can confirm that these crystals do indeed contain protein and DNA which is 
> good news. 
> I have tried a number of different buffers and salts since then. I have also 
> tried seeding but nothing I have tried has changed the morphology of the 
> crystals. They remain thin, flat and eyeball/pumpkin seed shaped.
> It is not difficult to make the crystals, they form quite easily under quite 
> a few conditions. The difficulty is in the diffraction. By changing the 
> buffer conditions we can now see some very weak diffraction (previously there 
> was no diffraction at all).
> Are there any suggestions as to how to improve diffraction of these crystals? 
> I did try different cryoprotectants, parabar, glycerol, PEG but no 
> difference. I think perhaps the problem is heterogeneity considering my 
> sample contains both protein and DNA. 
> Any suggestions or thoughts are welcome.
> Thank you
> Careina 
> 
> On Wednesday, November 8, 2023 at 06:18:46 PM GMT+2, careinaedgo...@yahoo.com 
> <02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> 
> 
> The reservior solution is 0.2 M NaCl2, 0.1 M HEPES pH 7.5, and 25% 3350 PEG
> 
> Protein buffer is 300 mM NaCl, 20 mM HEPES pH 7.5, 3mM TCEP
> 
> The drop contains 1.5ul protein-DNA complex and 1.5 ul reservior solution 
> 
> 
> On Wednesday, November 8, 2023 at 05:12:25 PM GMT+2, 
> stephen.c...@rc-harwell.ac.uk  wrote:
> 
> 
> Hi Careina,
> 
> Without knowing what's in your protein buffer or crystallisation condition 
> it's hard to comment.
> 
> Best wishes,
> Steve
> 
> Dr Stephen Carr
> Research Complex at Harwell (RCaH)
> Rutherford Appleton Laboratory
> Harwell Oxford
> Didcot
> Oxon OX11 0FA
> United Kingdom
> Email stephen.c...@rc-harwell.ac.uk
> tel 01235 567717
> From: CCP4 bulletin board  on behalf of 
> careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
> Sent: 08 November 2023 15:00
> To: ccp4bb 
> Subject: [ccp4bb] What could these crystals be?
>  
> Hi all
> We have been trying with no success to crystalize a protein. Recently we got 
> these strange shape "crystals". They are hard and flat but they do not 
> diffract at all. Any ideas as to what could cause this?
> Careina
> 
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Re: [ccp4bb] What could these crystals be?

[Antony Oliver]

Dear Careina,

Don't forget that the DNA you use is also a variable — you may need to make 
minor or indeed major tweaks to this is order to improve your crystals; e.g. 
subtract or add a base / base-pair.  Add a 1 base overhang etc..

Good luck!

Antony



- - - - - - - - - -
Dr Antony W. Oliver
Principal Research Fellow
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

antony.oli...@sussex.ac.uk (he/him)
https://tinyurl.com/aw-oliver

(office): +44 (0)1273 678349
(lab): +44 (0)1273 677512






From: CCP4 bulletin board  on behalf of 
careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Sent: 24 November 2023 08:59
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] What could these crystals be?


I wanted to thank everyone for their suggestions and ideas regarding the 
eyeball shaped crystals that I got at the beginning of the month.
I can confirm that these crystals do indeed contain protein and DNA which is 
good news.
I have tried a number of different buffers and salts since then. I have also 
tried seeding but nothing I have tried has changed the morphology of the 
crystals. They remain thin, flat and eyeball/pumpkin seed shaped.
It is not difficult to make the crystals, they form quite easily under quite a 
few conditions. The difficulty is in the diffraction. By changing the buffer 
conditions we can now see some very weak diffraction (previously there was no 
diffraction at all).
Are there any suggestions as to how to improve diffraction of these crystals? I 
did try different cryoprotectants, parabar, glycerol, PEG but no difference. I 
think perhaps the problem is heterogeneity considering my sample contains both 
protein and DNA.
Any suggestions or thoughts are welcome.
Thank you
Careina

On Wednesday, November 8, 2023 at 06:18:46 PM GMT+2, careinaedgo...@yahoo.com 
<02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:



The reservior solution is 0.2 M NaCl2, 0.1 M HEPES pH 7.5, and 25% 3350 PEG

Protein buffer is 300 mM NaCl, 20 mM HEPES pH 7.5, 3mM TCEP

The drop contains 1.5ul protein-DNA complex and 1.5 ul reservior solution


On Wednesday, November 8, 2023 at 05:12:25 PM GMT+2, 
stephen.c...@rc-harwell.ac.uk  wrote:


Hi Careina,

Without knowing what's in your protein buffer or crystallisation condition it's 
hard to comment.

Best wishes,
Steve

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

From: CCP4 bulletin board  on behalf of 
careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Sent: 08 November 2023 15:00
To: ccp4bb 
Subject: [ccp4bb] What could these crystals be?

Hi all
We have been trying with no success to crystalize a protein. Recently we got 
these strange shape "crystals". They are hard and flat but they do not diffract 
at all. Any ideas as to what could cause this?
Careina



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Any views or opinions presented are solely those of the author and do not 
necessarily represent those of the Research Complex at Harwell.



There is no guarantee that this email or any attachments are free from viruses 
and we cannot accept liability for any damage which you may sustain as a result 
of software viruses which may be transmitted in or with the message.



We use an electronic filing system. Please send electronic versions of 
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