Re: [ccp4bb] Optimization from needle shaped crystals

[Patrick Shaw Stewart]

Hi Chitra

Needles usually work very well for making seed stocks for random Microseed
Matrix Screening (MMS).  Your protein probably crystallizes well, but it is
growing too fast in one direction.

MMS has lots of advantages.  If it's going to work it will almost certainly
work within 12 hours.  Also, it allows you to control the number of
crystals per drop by diluting the seed stock.

Another excellent (open-access) paper that gives lots of examples of
seeding, cross-seeding, dilution, optimization etc within a defined set of
fabs is here:

https://scripts.iucr.org/cgi-bin/paper?nj5193


Good luck, Patrick



On Sun, Sep 8, 2019 at 1:46 PM David Briggs 
wrote:

> 4. Matrix microseeding. Make a seed stock from these crystals and then
> re-run your primary screens.
>
> https://www.ncbi.nlm.nih.gov/m/pubmed/25195878/
>
> --
> Dr David C. Briggs
> Senior Laboratory Research Scientist
> Signalling and Structural Biology Lab
> The Francis Crick Institute
> London, UK
> ==
> about.me/david_briggs
>
> --
> *From:* CCP4 bulletin board  on behalf of chitra
> latka 
> *Sent:* Sunday, September 8, 2019 12:12:28 PM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* Re: [ccp4bb] Optimization from needle shaped crystals
>
> I can share what has worked for my crystals :
>
> 1. You can put a grid across your condition with same or altered drop
> ratios.
>
> 2. You can try micro seeding. (This has given me the best results so far).
>
> 3. You can try Hampton's additive screen.
>
> On Sun, Sep 8, 2019 at 10:09 AM Prem Prakash  wrote:
>
>> Dear all,
>> Sorry for a trivial query. I am trying to Co-crystallize my protein with
>> its substrate (peptide) using commercial screenings. In one condition of
>> JCSG plus (Molecular Dimension) that contains  0.2 M Magnesium chloride
>> hexahydrate,  0.1 M Tris 8.5 50 % v/v Ethylene glycol, I got needle like
>> crystals (picture attached). Does anyone have idea to optimize such needles
>> into better crystals. I would appreciate all your suggestions.
>>
>> Thank you
>> With kind regards,
>> Prem Prakash  (Ph.D.)
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>> <https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7C9e5bd9b3c9e7485ed8af08d7344ee01c%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637035385686224007=635CdZ1OnWv%2BoTW6YrzI%2BJaPg64OrVyWzz7p9HiAt2c%3D=0>
>>
>
>
> --
> Regards
> Chitra
>
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>
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>
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Re: [ccp4bb] Optimization from needle shaped crystals

[Rajiv gandhi.s]

Dear prem,
I am sharing my experience with needle crystal that worked for me.

1.First check that salt or  a protein crystal by SDS PAGE or mass
spectroscopy.
2. For improvement of morphology and size,  you could try microseeding of
your crushed crystal, generally needle crystal appear quickly, further this
could be improved by seeding them again in 96 well plates with commercial
screens. If this yield any improved crystals then Pick the best looking and
larger sized crystal to seed again into 24 well plates, by streaking the
seed solution using cat whisker.

3. If you get any better crystal from seeding, then do a grid screening
around the original condition.

4. Try to do different volume ratios of protein with well solution, and
even keep them at cold room. Also you could vary the protein concentration.

5. Do an additive screen as well, once you figure out  condition that yield
better crystal morphology and with improved  thickness.

Hope this helps

Best

Rajivgandhi Sundaram
Macromolecular crystallography lab
institute of life Sciences
India.

On Sun, Sep 8, 2019, 10:10 AM Prem Prakash  wrote:

> Dear all,
> Sorry for a trivial query. I am trying to Co-crystallize my protein with
> its substrate (peptide) using commercial screenings. In one condition of
> JCSG plus (Molecular Dimension) that contains  0.2 M Magnesium chloride
> hexahydrate,  0.1 M Tris 8.5 50 % v/v Ethylene glycol, I got needle like
> crystals (picture attached). Does anyone have idea to optimize such needles
> into better crystals. I would appreciate all your suggestions.
>
> Thank you
> With kind regards,
> Prem Prakash  (Ph.D.)
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Optimization from needle shaped crystals

[David Briggs]

4. Matrix microseeding. Make a seed stock from these crystals and then re-run 
your primary screens.

https://www.ncbi.nlm.nih.gov/m/pubmed/25195878/

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of chitra latka 

Sent: Sunday, September 8, 2019 12:12:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Optimization from needle shaped crystals

I can share what has worked for my crystals :

1. You can put a grid across your condition with same or altered drop ratios.

2. You can try micro seeding. (This has given me the best results so far).

3. You can try Hampton's additive screen.

On Sun, Sep 8, 2019 at 10:09 AM Prem Prakash 
mailto:prem...@gmail.com>> wrote:
Dear all,
Sorry for a trivial query. I am trying to Co-crystallize my protein with its 
substrate (peptide) using commercial screenings. In one condition of JCSG plus 
(Molecular Dimension) that contains  0.2 M Magnesium chloride hexahydrate,  0.1 
M Tris 8.5 50 % v/v Ethylene glycol, I got needle like crystals (picture 
attached). Does anyone have idea to optimize such needles into better crystals. 
I would appreciate all your suggestions.

Thank you
With kind regards,
Prem Prakash  (Ph.D.)




To unsubscribe from the CCP4BB list, click the following link:
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--
Regards
Chitra



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Re: [ccp4bb] Optimization from needle shaped crystals

[chitra latka]

I can share what has worked for my crystals :

1. You can put a grid across your condition with same or altered drop
ratios.

2. You can try micro seeding. (This has given me the best results so far).

3. You can try Hampton's additive screen.

On Sun, Sep 8, 2019 at 10:09 AM Prem Prakash  wrote:

> Dear all,
> Sorry for a trivial query. I am trying to Co-crystallize my protein with
> its substrate (peptide) using commercial screenings. In one condition of
> JCSG plus (Molecular Dimension) that contains  0.2 M Magnesium chloride
> hexahydrate,  0.1 M Tris 8.5 50 % v/v Ethylene glycol, I got needle like
> crystals (picture attached). Does anyone have idea to optimize such needles
> into better crystals. I would appreciate all your suggestions.
>
> Thank you
> With kind regards,
> Prem Prakash  (Ph.D.)
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Regards
Chitra



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Re: [ccp4bb] Optimization from needle shaped crystals

[Savvas Savvides]

Dear Prem,

before moving on with any optimization rounds it would be good to first confirm 
that these crystals are actually protein crystals, and in particular the 
protein of interest.

For example, a Silver-stained SDS-PAGE gel can be very informative.
You can run a couple of your crystalline clusters after 2 washing steps on 
SDS-PAGE. Make sure you also load the washing steps, the content of clear drops 
(from the same screen), and protein as purified,- as controls.
We have found that such a simple diagnostic early in the process can be 
crucially important.

best wishes
Savvas



> On 8 Sep 2019, at 06:39, Prem Prakash  wrote:
> 
> Dear all, 
> Sorry for a trivial query. I am trying to Co-crystallize my protein with its 
> substrate (peptide) using commercial screenings. In one condition of JCSG 
> plus (Molecular Dimension) that contains  0.2 M Magnesium chloride 
> hexahydrate,  0.1 M Tris 8.5 50 % v/v Ethylene glycol, I got needle like 
> crystals (picture attached). Does anyone have idea to optimize such needles 
> into better crystals. I would appreciate all your suggestions. 
> 
> Thank you 
> With kind regards,
> Prem Prakash  (Ph.D.) 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 




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Re: [ccp4bb] Optimization from needle shaped crystals

[Arunabh Athreya]

You could always make a 96 or a 24 well narrow spectrum screen around this 
condition, and then check if the morphology changes or not. What I have seen is 
that they work better if you tweak your drop ratios too. After such procedures, 
you could try using Hamptons Additive screen on top of your optimized condition.

Get Outlook for Android<https://aka.ms/ghei36>


From: CCP4 bulletin board  on behalf of Prem Prakash 

Sent: Sunday, September 8, 2019 10:09:29 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Optimization from needle shaped crystals

Dear all,
Sorry for a trivial query. I am trying to Co-crystallize my protein with its 
substrate (peptide) using commercial screenings. In one condition of JCSG plus 
(Molecular Dimension) that contains  0.2 M Magnesium chloride hexahydrate,  0.1 
M Tris 8.5 50 % v/v Ethylene glycol, I got needle like crystals (picture 
attached). Does anyone have idea to optimize such needles into better crystals. 
I would appreciate all your suggestions.

Thank you
With kind regards,
Prem Prakash  (Ph.D.)




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https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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