Re: [ccp4bb] how to get protein crystal

[amala mathimaran]

Dear all..

Wow so many answers.. really thanks to all I will fellow your ways one by
one..








Thanks and regards
Amala

On Sat, Dec 28, 2019 at 9:31 AM Newman, Janet (Manufacturing, Parkville)
 wrote:

> Hi,
>
>
>
> To add to this thread, there are a few more easy things to try –
>
>
>
> Try doing matrix microseeding and try doing limited proteolysis. Even
> though the following link describes how we do this in our laboratory, (the
> C3 Facility in Melbourne) the pages give a quick overview to both matrix
> microseeding and proteolysis, and provides links to a recipe for making
> seeds etc.
>
>
>
> https://research.csiro.au/crystal/user-guide/soluble-proteins/
>
>
>
> Then you might want to try setting up crystallisation at different
> temperatures, and using DSF or some other technique to find a different
> formulation for your protein, as that can significantly alter the behaviour
> of your protein in crystallisation trials.
>
>
>
> Cheers, and may your new year’s resolution be better than 2A
>
>
>
> Janet
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *amala
> mathimaran
> *Sent:* Saturday, 28 December 2019 12:30 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] how to get protein crystal
>
>
>
> Dear all,
>
>
>
> Can you suggest me how to get protein crystal???
>
>
>
> I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final
> purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial
> screening was done using hanging drop method but no crystal. So 2mM NADP
> cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal
> screen, Index) and Molecular dimensions conditions etc. I got precipitate
> like formation the image was attached below. From this formation what I can
> do… mean while I increase the protein concentration and did screening for
> that selected conditions again I got same kind of formations. I am new to
> protein crystallography kindly suggesting me. And how much concentration is
> suitable for protein crystallization?? How to find which concentration is
> enough for our target protein crystallization?? Thanks in advance
>
>
> --
>
> 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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Re: [ccp4bb] how to get protein crystal

[Newman, Janet (Manufacturing, Parkville)]

Hi,

To add to this thread, there are a few more easy things to try –

Try doing matrix microseeding and try doing limited proteolysis. Even though 
the following link describes how we do this in our laboratory, (the C3 Facility 
in Melbourne) the pages give a quick overview to both matrix microseeding and 
proteolysis, and provides links to a recipe for making seeds etc.

https://research.csiro.au/crystal/user-guide/soluble-proteins/

Then you might want to try setting up crystallisation at different 
temperatures, and using DSF or some other technique to find a different 
formulation for your protein, as that can significantly alter the behaviour of 
your protein in crystallisation trials.

Cheers, and may your new year’s resolution be better than 2A

Janet

From: CCP4 bulletin board  On Behalf Of amala mathimaran
Sent: Saturday, 28 December 2019 12:30 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] how to get protein crystal

Dear all,

Can you suggest me how to get protein crystal???

I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final 
purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial 
screening was done using hanging drop method but no crystal. So 2mM NADP 
cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal screen, 
Index) and Molecular dimensions conditions etc. I got precipitate like 
formation the image was attached below. From this formation what I can do… mean 
while I increase the protein concentration and did screening for that selected 
conditions again I got same kind of formations. I am new to protein 
crystallography kindly suggesting me. And how much concentration is suitable 
for protein crystallization?? How to find which concentration is enough for our 
target protein crystallization?? Thanks in advance



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Re: [ccp4bb] how to get protein crystal

[zaigham khan]

Dear Amala,

You have already received a lot of good advices from expert
crystallographers. Apart from the notables, i would still read about the
protein. Is this an enzyme? If yes, what is the ligand or a co-factor? The
protein conformation would be different in apo form versus bound form.
Often, the protein adopts a more stable/rigid conformation upon binding to
its ligand/co-factor, and this addition of ligand/co-factor eventually
helps in crystallization (a floppy region in a protein is
recalcitrant towards crystallization, hence a ligand will do!). Likewise,
is the tag cleaved? Also, is there any free cysteine present in your
protein that may engage in a disulfide bond? You may want to add a reducing
agent to protect the cysteine. Alternatively, one may harness the power of
(non-physiological) disulfide bridge to stabilize the protein of interest.
For reference, you may read my manuscript, Khan et al, (2014) J. Virol. (
- DOI:
- 10.1128/JVI.03455-13

)

Further, i would invest a little time in database search. You may aim to
find the structure(s) of the homologous protein(s) in the protein data
bank. This will allow you to gain confidence in the (1) domain boundaries
of the expression cassette; (2) buffer composition of the final protein
prep; (3) selection of the ligand/co-factor; final protein concentration;
and (4) crystallization condition (homologous proteins often tend to
crystallize in the similar range of the pH and precipitant). Eventually,
you may procure a more focused crystallization screen (such as JBS kinase
screen for kinases).

happy voyages!

z


Best wishes

-Z


Zaigham Khan, PhD

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York


On Fri, Dec 27, 2019 at 8:31 AM amala mathimaran 
wrote:

> Dear all,
>
> Can you suggest me how to get protein crystal???
>
>
>
> I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final
> purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial
> screening was done using hanging drop method but no crystal. So 2mM NADP
> cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal
> screen, Index) and Molecular dimensions conditions etc. I got precipitate
> like formation the image was attached below. From this formation what I can
> do… mean while I increase the protein concentration and did screening for
> that selected conditions again I got same kind of formations. I am new to
> protein crystallography kindly suggesting me. And how much concentration is
> suitable for protein crystallization?? How to find which concentration is
> enough for our target protein crystallization?? Thanks in advance
>
> --
>
> 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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Re: [ccp4bb] how to get protein crystal

[Kevin Jin]

Due the resolution of your images, it is a little bit difficult to identify
crystallization under these conditions. However, F3, F6 and F5 are
crystals, but the condition needs to be optimized.  Likely, you may try
different concentration of NaCl, instead of MgCl2 and KBr, respectively.
Good luck.

On Fri, Dec 27, 2019 at 5:31 AM amala mathimaran 
wrote:

> Dear all,
>
> Can you suggest me how to get protein crystal???
>
>
>
> I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final
> purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial
> screening was done using hanging drop method but no crystal. So 2mM NADP
> cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal
> screen, Index) and Molecular dimensions conditions etc. I got precipitate
> like formation the image was attached below. From this formation what I can
> do… mean while I increase the protein concentration and did screening for
> that selected conditions again I got same kind of formations. I am new to
> protein crystallography kindly suggesting me. And how much concentration is
> suitable for protein crystallization?? How to find which concentration is
> enough for our target protein crystallization?? Thanks in advance
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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Re: [ccp4bb] how to get protein crystal

[Edward Snell]

Dear Amala,


There are some good suggestions coming your way from here. I would also 
recommend checking out the High-Throughput Crystallization Screening Center at 
the Hauptman-Woodward Medical Research Institute or other similar services. 
They run most of the common types of screens in an efficient and cost effective 
manner.


There is good advice available on how to interpret the outcomes.? Our Center 
(http://getacrystal.org) has a section on crystallization research 
https://hwi.buffalo.edu/high-throughput-crystallization-center/crystallization-research/
 and there are links to papers that describe how to make some sense of results 
from initial crystallization screening and how to optimize those results. I'm 
biased but the advice is good and the references many of those papers contain 
are comprehensive. I've been involved many of these studies and have copies of 
some of the full papers on my own website at 
https://hwi.buffalo.edu/scientist-directory/snell/ - just click on the 
Crystallization Methods and Analysis section.


I would recommend looking into the solubility phase diagram for 
crystallization.  That would give you an idea of the influence of changing 
different parameters. You do have some common outcomes in the results you 
showed which define a pH range, salt, and exclusion agent concentrations. Also 
small changes in pH can have a dramatic outcome. Increasing the protein 
concentration may push you further into the nucleation region and may result in 
far more but smaller crystals - which could be mistaken for precipitate. Screen 
around each condition and try to understand the impact of each axis and the 
overall combination of those axes. I think the results you showed bode well and 
it shouldn't take to much work to optimize those conditions.  I would also take 
a UV image to confirm protein or spin some of the crystals down in a capillary 
and take a powder pattern to confirm protein crystals and not the ligand or 
crystallization screen component.


Best of luck,


Eddie




From: CCP4 bulletin board  on behalf of amala mathimaran 

Sent: Friday, December 27, 2019 8:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] how to get protein crystal

Dear all,

Can you suggest me how to get protein crystal???

I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final 
purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial 
screening was done using hanging drop method but no crystal. So 2mM NADP 
cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal screen, 
Index) and Molecular dimensions conditions etc. I got precipitate like 
formation the image was attached below. From this formation what I can do... 
mean while I increase the protein concentration and did screening for that 
selected conditions again I got same kind of formations. I am new to protein 
crystallography kindly suggesting me. And how much concentration is suitable 
for protein crystallization?? How to find which concentration is enough for our 
target protein crystallization?? Thanks in advance



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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Re: [ccp4bb] how to get protein crystal

[Rajesh Kumar]

Hi Amala,

In addition to Limbini´s suggestions.

1. You can always do MALS, SLS, Native Page or cross linking experiments to
check whether your protein is monodisperse in the solution or not. This is
very important step.

2. When you set up crystallization and if you have 40-50% drops
precipitated that would be ideal concentration for protein crystallization.

3. As Lumbini said fresh gel filtration passed protein samples behave much
better than frozen one.

4. You can also try sitting drop OR under oil method of crystallization too
because they follow different crystallization phase diagram.

5. Adding ligands and cofactor would be good idea as well.

6. Add some DTT if you think you have several Cys residues, which may be
surface exposed.

You can always contact me for more help.

Best wishes,
  ---x
With regards
Rajesh K. Harijan, Ph.D.
Albert Einstein College of Medicine
1300 Morris Park Ave., Bronx, NY 10461
Tel: 718.430.2777  |  Fax: 718.430.8565



On Fri, Dec 27, 2019 at 8:30 AM amala mathimaran 
wrote:

> Dear all,
>
> Can you suggest me how to get protein crystal???
>
>
>
> I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final
> purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial
> screening was done using hanging drop method but no crystal. So 2mM NADP
> cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal
> screen, Index) and Molecular dimensions conditions etc. I got precipitate
> like formation the image was attached below. From this formation what I can
> do… mean while I increase the protein concentration and did screening for
> that selected conditions again I got same kind of formations. I am new to
> protein crystallography kindly suggesting me. And how much concentration is
> suitable for protein crystallization?? How to find which concentration is
> enough for our target protein crystallization?? Thanks in advance
>
> --
>
> 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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Re: [ccp4bb] how to get protein crystal

[Lumbini Yadav]

Dear Amala,
1. Hampton provides a precrystallisation screening kit which can be used to
determine the protein concentration to be used for crystallisation.

2. Also if you observe the screening crystallisation plate, according to
what I follow,  at least there should be minimum 60 % of conditions where
you should see protein precipitate.

3. Always prefer using homogeneous, freshly prepared protein at least for
screening because certain protein don't behave well once freezed or stored
for longer duration. Size exclusion chromatography can enhance your success
rate.

4. Observe the screening plate atleast
 for 3 months after setting the trial.

5. Not all protein will give you crystals in initial screening.  You will
have to pick the promising conditions and optimise it further. Prefer
optimising atleast  6-7 promising hits few of which may give you
diffractable quality crystal.

After all getting crystals for difficult proteins that diffract, requires
lot of optimization, dedication, efforts, perseverance and luck.

BEST OF LUCK.


On Fri, 27 Dec 2019, 19:00 amala mathimaran,  wrote:

> Dear all,
>
> Can you suggest me how to get protein crystal???
>
>
>
> I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final
> purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial
> screening was done using hanging drop method but no crystal. So 2mM NADP
> cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal
> screen, Index) and Molecular dimensions conditions etc. I got precipitate
> like formation the image was attached below. From this formation what I can
> do… mean while I increase the protein concentration and did screening for
> that selected conditions again I got same kind of formations. I am new to
> protein crystallography kindly suggesting me. And how much concentration is
> suitable for protein crystallization?? How to find which concentration is
> enough for our target protein crystallization?? Thanks in advance
>
> --
>
> 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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