Re: [ccp4bb] Secrets of ZYMIT

[Bonsor, Daniel]

Could you take a solution of it, buffer exchange/size exclusion and then 
perform MS fingerprinting to identify the protease if you don't have any luck 
with the companies?

Best

Dan

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From: CCP4 bulletin board  on behalf of Jorg Stetefeld 

Sent: Sunday, September 8, 2019 6:01:49 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Secrets of ZYMIT


Hi everyone,


this is Joerg Stetefeld.


I would like to approach the community in a peculiar case of a specific 
proteolytic cleavage during crystallization attempts.

Performing several crystallization screens we figured out that ZYMIT 
(https://www.ipcol.com/cleaners/zymit-low-foam) is the reason for a highly 
specific, but undesireable cleavage of protein components in our setups. Most 
remarkably, the cleavage site of our crystallisation target is not described in 
any protease database, and is highly inaccessible.



According to a publication by Naschberger et al, 2014 
(doi:10.1107/S2053230X14026053) the authors very nicely describe the 
phenomenon. They also describe a very elegant protease removal protocol, which 
works in our hands very well.

At this point, however, I would like to know what is “behind” ZYMIT? 
Naschberger et al say “Zymit contains an unspecified‘protease enzyme’ as a 
trade secret (http://www.ipcol.com/pdfs/Zymit_msds.pdf)”.


Does anyone has more insight into the secrets of ZYMIT? Our attempts to contact 
several vendors here in Canada/ abroad failed and a further characterisation of 
its nature would help us to understand this particular phenomenon of 
proteolytic cleavage.



Thank you very much in advance- Your advise is appreciated.


js




Jörg Stetefeld


https://stetefeldlab.ca/

[X]



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[ccp4bb] Secrets of ZYMIT

[Jorg Stetefeld]

Hi everyone,


this is Joerg Stetefeld.


I would like to approach the community in a peculiar case of a specific 
proteolytic cleavage during crystallization attempts.

Performing several crystallization screens we figured out that ZYMIT 
(https://www.ipcol.com/cleaners/zymit-low-foam) is the reason for a highly 
specific, but undesireable cleavage of protein components in our setups. Most 
remarkably, the cleavage site of our crystallisation target is not described in 
any protease database, and is highly inaccessible.



According to a publication by Naschberger et al, 2014 
(doi:10.1107/S2053230X14026053) the authors very nicely describe the 
phenomenon. They also describe a very elegant protease removal protocol, which 
works in our hands very well.

At this point, however, I would like to know what is “behind” ZYMIT? 
Naschberger et al say “Zymit contains an unspecified‘protease enzyme’ as a 
trade secret (http://www.ipcol.com/pdfs/Zymit_msds.pdf)”.


Does anyone has more insight into the secrets of ZYMIT? Our attempts to contact 
several vendors here in Canada/ abroad failed and a further characterisation of 
its nature would help us to understand this particular phenomenon of 
proteolytic cleavage.



Thank you very much in advance- Your advise is appreciated.


js




Jörg Stetefeld


https://stetefeldlab.ca/

[X]



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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.)
>
>
> --
>
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Re: [ccp4bb] One protein, two data sets

[Kay Diederichs]

Dear Prerana,

the structures of the protein belonging to these two crystal forms are, 
strictly speaking, different. 
Exactly _how_ different they are can only be stated after solving them. You 
will then be able to compare _three_ structures of the protein - two in crystal 
form 1., one in crystal form 2..  
Probably, the root mean square differences of coordinates in pairwise 
comparisons will be low, i.e. on the order of half an Angstrom. But it is 
possible that one of the structures has a different conformation than the other 
two, or even that all three are quite different. 

best,
Kay


On Sun, 8 Sep 2019 16:45:29 +0530, Prerana G.  wrote:

>Dear all,
>
>We have two data sets of a protein with the following parameters:
>1. Space group P212121 a=61.0, b=100.34, c=133.23 No. of molecules in ASU -
>2
>2. Space group P212121 a=58.33, b=62.86, c=109.45 No. of molecules in ASU -
>1
>
>Can we use them as two different structures?
>
>Regards,
>Prerana
>
>
>
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>



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[ccp4bb] General interest

[John R Helliwell]

Dear Colleagues,
The recent IUCr Committee on Data Satellite Workshop at ECM32 Vienna on “Data 
science skills in publishing” speaker presentations, overall report and photos 
from the day are now available via the IUCr CommDat Public Forum weblink:-  
https://forums.iucr.org/viewtopic.php?f=39&t=422&sid=f14add194ce5e9c146b51b6ace1ad72a
 
Best wishes,
John 
Emeritus Professor John R Helliwell DSc
Chairman of IUCr Committee on Data





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Re: [ccp4bb] One protein, two data sets

[Jonathan Cooper]

I take it that the datasets are from different crystals. Are the 
crystallisation conditions the same? I can't spot any signs of a difference in 
indexing (but I may be wrong) so they look like different crystal forms. Are 
the processing statistics good for both? Is this a case for molecular 
replacement or experimental phasing? You could work with best diffracting one 
and solve that one first and then use it to solve the other by molecular 
replacement. Hope some of this helps.

Sent from Yahoo Mail on Android 
 
  On Sun, 8 Sep 2019 at 12:16, Prerana G. wrote:   Dear all,

We have two data sets of a protein with the following parameters:1. Space group 
P212121 a=61.0, b=100.34, c=133.23 No. of molecules in ASU - 2
2. Space group P212121 a=58.33, b=62.86, c=109.45 No. of molecules in ASU - 1
Can we use them as two different structures?
Regards,Prerana


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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.)




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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&A=1
>


-- 
Regards
Chitra



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[ccp4bb] One protein, two data sets

[Prerana G.]

Dear all,

We have two data sets of a protein with the following parameters:
1. Space group P212121 a=61.0, b=100.34, c=133.23 No. of molecules in ASU -
2
2. Space group P212121 a=58.33, b=62.86, c=109.45 No. of molecules in ASU -
1

Can we use them as two different structures?

Regards,
Prerana



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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&A=1 
> 




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