Re: [ccp4bb] Crystallization suggestion for antigen-Fc complex

[Enrico Stura]

Ankita,

Fc are different from Fabs. Even if glycosylated their solubility is lower.
Fc stands for constant fragment but also for Fragment crystallizable:
https://en.wikipedia.org/wiki/Fragment_crystallizable_region

From its name, they should be easy to crystallize. If you have problems to  
not hesitate to ask questions.


Enrico.

On Tue, 07 Mar 2017 07:14:34 +0100, Ankita Srivastava  
 wrote:



Dear CCP4 members,

I am trying to crystallize an antigen-bound to the Fc fragment of an
antibody.
Is Fc fragment difficult to crystallize compared to Fab? Are there any
commercial screens widely known to crystallize Fc  fragment or  
antibodies?


I would appreciate if you can share any experience with crystallizing Fc
fragment or Fc-complex with other proteins.

Many thanks in advance!
Ankita



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Proxima-2A, Soleil Synchrotron. Tel: 33 (0)1 69 35 8180 Beamline
http://scholar.google.com/citations?hl=en=Kvm06WIoPAsC=100=pubdate

Re: [ccp4bb] Crystallization with no precipitant

[Enrico Stura]

Dear ccp4bb,

1. Why would you need a precipitant to crystallize a protein?
The principle of salting-in/salting-out means that as you remove the salt  
that
keeps a protein soluble, the protein will tend to come out of solution and  
given

the right conditions, crystallize.

2. From the solubility curve as you increase protein concentration, you  
will reach a point at which
your protein will become supersaturated and possibly crystallize. You just  
evaporate the water.


3. Protein solubility is temperature dependent:
Huang M., Syed R., Stura E.A., Stone M.J., Stefanko R.S., Ruf W.,  
Edgington T.S. & Wilson I.A. (1998) The mechanism of an inhibitory  
antibody on TF-initiated blood coagulation revealed by the crystal  
structures of human tissue factor, Fab 5G9 and TF.5G9 complex. J Mol Biol.  
275:873-894.

Crystallized in the NMR tube when it was stored in the fridge
 De Halleux S., Stura E.A., VanderElst L., Carlier V., Jacquemin M. &  
Saint-Remy J.-M. (2006) Three-dimensional structure and IgE-binding  
properties of mature fully active Der p 1, a clinically relevant major  
allergen. J. Allergy Clin. Immunol.  11: 571-576.
The unglycosylated protein has low solubility - Concentration to 3.5 mg/ml  
and waiting for it to crystallize in the fridge.
Stura E.A., Visse R., Vera L., Cuniasse P., Dive V. & Nagase H. (2013)  
Crystals structure of full-length human collagenase 3 (MMP-13) with  
peptides in the active site defines exosites in the catalytic domain.  
FASEB J. 27:4395-4405.
A bridging peptide and low temperature induces crystallization - No  
precipitant.


4. A precipitant is an agent that is used by protein crystallogenists to  
avoid using too much protein
in the experiment. If salting-out does not work, PEG is used as a common  
crowding-out agent.

Ideally without a precipitant your protein will be in its most pure state.

5. Maïga A., Vera L., Marchetti C., Lorphelin A., Bellanger L., Mourier  
G., Servent D., Gilles N. & Stura E.A. (2013) Crystallization of  
recombinant Green mamba ρ-Da1a toxin during lyophilization procedure and  
its structure determination. Acta Cryst. F 69: 704-709

What about crystallization during lyophilization?

If you check throughout the literature, you  will finnd many more examples.

Enrico.

--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Proxima-2A, Soleil Synchrotron. Tel: 33 (0)1 69 35 8180 Beamline
http://scholar.google.com/citations?hl=en=Kvm06WIoPAsC=100=pubdate

Re: [ccp4bb] Lysozyme soaked with GlcNac?

[Enrico Stura]

Dear Eike,

What is the interest of soaking experiments when you can grow HEWL crystals
in less time (15 min) than it would take to do a soaking experiment?
https://www.hamptonresearch.com/product_detail.aspx?cid=28=173=524

Soaking will work as long as you do it correctly. A 20min soak or longer  
should work.
You should do it directly in the cryosolution with high sugar  
concentrations as most diols might compete with the sugar.
Remember, as soon as you put crystals with bound GlcNac in a cryosolution,  
you will start to back-soak the ligand.
Always add the GlcNac to the cryoprotectant even for co-crystals for the  
best ligand density.


You may find the following reference useful to select a cryosolution that  
does not dissolve your crystals:
Ciccone L., Vera L., Tepshi L., Rosalia L., Rossello A. & Stura E.A.  
(2015) Multicomponent mixtures for cryoprotection and ligand  
solubilization. Biotechnology Reports 7:120—127.


Enrico.

On Mon, 20 Feb 2017 20:59:23 +0100, Schulz, Eike-Christian  
 wrote:



Dear all,

There are several structures in the PDB that show Lysozyme (mutants) in  
complex with GlcNac (or similar compounds). However, all of those  
structures seem to originate from co-crystallization experiments. I was  
wondering whether anybody knew a successful case of complex formation by  
soaking. But maybe the solvent channels are too small …


I would be very happy if anybody could point me to a reference but I  
could also live with anecdotal evidence.


With best regards

Eike






--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Proxima-2A, Soleil Synchrotron. Tel: 33 (0)1 69 35 8180 Beamline
http://scholar.google.com/citations?hl=en=Kvm06WIoPAsC=100=pubdate

Re: [ccp4bb] Tris buffer in cryo protectant

[Enrico Stura]

Ursula,

After extensive testing, I have found out that in most cases pH changes  
during flash freezing does
not pose a problem. In some cases it can  be beneficial. I encourage you  
to try +/- 2 pH units

from your crystallization pH.

Sometimes a sub-optimal pH is chosen just because the crystals were  
obtained the first time at that pH,
sometimes it is for strategic reasons, for example to prevent excessive  
nucleation.
The best pH to obtain BIG crystals is not always the best pH to get the  
best diffraction.

Test various pH if you have enough crystals to do that.

Enrico.


On Fri, 12 Jun 2015 22:47:10 +0200, Ursula Schulze-Gahmen  
uschulze-gah...@lbl.gov wrote:



Does anyone have experience with Tris buffer in cryo protectants? I would
expect the pH of the cryosolution to increase a lot during flash freezing
which could perhaps destroy the diffraction. I rarely use Tris for
crystallization but the current protein really prefers Tris. I would
appreciate any comments.

Ursula




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] how to reduce protein solubility

[Enrico Stura]

Ursula,

Most compounds used for cryosolutions glycerol, ethylene glycol, propane  
diol increase protein solubility.
A warning, these compounds are also hygroscopic, you need to change your  
vapour diffusion methodology.
Vera L., Czarny B., Georgiadis D., Dive V., Stura E.A. (2011) Practical  
Use of Glycerol in Protein Crystallization. Cryst. Growth  Des. 11:  
2755–2762.

http://pubs.acs.org/doi/abs/10.1021/cg101364m
The lack of success in crystallizing proteins in glycerol are due to the  
reasons decribed in the paper.


Enrico.

On Fri, 20 Feb 2015 00:33:48 +0100, Ursula Schulze-Gahmen  
uschulze-gah...@lbl.gov wrote:



Hi Enrico,

How are you? I see you are now in France.

I have also a question about protein complex solubility. I have a
multi-protein complex that also binds RNA. This Protein-RNA complex can  
be

concentrated to 5- 10 mg/ml, but starts precipitating after storage at 4
degrees for several hours ( and can often be resolubilized at room
temperature). The current buffer is 20 mM HEPES 7.3, 0.2 M NaCl, 0.05 M
KCl, 3 mM MgCl2, TCEP. I don't want to increase salt concentration. What
are your suggestions to try to improve the solubility?

Best

Ursula

On Tue, Feb 17, 2015 at 2:00 AM, Enrico Stura est...@cea.fr wrote:


Francesca,

The most common failure is to have an excessive amount of salt (salting
in/ salting out), glycerol or other solubilizing
ingredient in your protein solution. I would suggest that you change the
pH and reduce the salt in your protein solution,
by microdialysis if you do not have much protein, and screen again.
If share with ccp4bb the exact formulation of your protein solution you
might get more suggestions.

Enrico.



On Tue, 17 Feb 2015 05:23:05 +0100, Mattiroli,Francesca 
francesca.mattir...@colostate.edu wrote:

 Hi all,


I am struggling with a protein complex that is too soluble. I have
reached about 20 mg/ml but I still observe very little precipitation  
(clear
drops in 90-95% of the tested conditions). The proteins are expressed  
in
insect cells and going to higher concentration is not easily  
achievable.
I have tried different buffer conditions (salt concentration and pH)  
and

I am testing temperatures. I am at a loss with what to try next.
Do you think PTMs (phosphorylation, acetylation) might be causing this?
Any input on how to decrease solubility?

Thank you very much in advance,

Francesca





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100;
sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90  
71









--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] how to reduce protein solubility

[Enrico Stura]

On Fri, 20 Feb 2015 16:07:18 +0100, Pietro Roversi  
pietro.rove...@bioch.ox.ac.uk wrote:



Dear Enrico,

I wonder if trying different protein:precipitant ratio is also a valid  
strategy to crystallise very soluble proteins.


Please let me know if my reasoning is flawed and if so why!


Yes and No.

I you are using salts and your protein is still soluble when the salt is  
saturated. You have an excellent
way of concentrating the protein. You will then need a co_precipitant,  
like dioxane, to push it over the

edge.

With PEG this is an option for refinement, but not for serious protein  
concentration.
PEG has become the most often used precipipitant, is because it work by  
volume exclusion,

leaving the protein less space and effectively concentrating the protein.
http://dx.doi.org/10.1016/S0022-2836(75)80107-0
In terms of being able to extract water from the drop it is less effective  
than

salts, glycerol ... etc.
So with PEG it will not work as you would think. What I do is to use  
booster solutions.
In the boster solution you may have: 5M NaCl and if you want to change the  
pH because
you know that acidification can increase precipitation, you also have  
acetic acid in your boost.

An example is given in:
 Ciccone L., Tepshi L., Nencetti, S.  Stura E.A. (2015) Transthyretin  
complexes with
curcumin and bromo-estradiol: Evaluation of solubilizing multicomponent  
mixtures New Biotech. 32:54–64

http://dx.doi.org/10.1016/j.nbt.2014.09.002
You can concentrate and crystallize a very soluble protein by starting  
with a low concentration high MW PEG
and by sitting drop vapour diffusion you do several boosts untill you get  
a precipitate.
When you get the precipitate, you do not know where you are in  
crystallization space!
But you can do precipitate transfers to other drops. The speed at which  
the precipitate resolubilizes

allows you to work out approximately where you are.
Complicated, yes I agree, but it uses very very little protein.

Enrico.



Let [Prot]_0 and [ML]_0 be the initial concentrations of protein and  
mother liquor solutions, mixed in volumes V0_prot and V0_ML,  
respectively, to form the initial drop of volume (V0_prot+V0_ML)


In the following, let us assume that the vapour diffusion process  
proceeds based on chemical potential of the ML and let us talk of  
concentrations instead of chemical potentials.


The vapour diffusion process will stop when the concentration of ML in  
the drop is equal to the one it has in the mother liquor, so that the  
drop will shrink till its volume at equilibrium is V0_ML, i.e. the  
volume of ML that was used to make the drop initially.


The final concentration of protein is therefore:
[Prot]eq = ([Prot]0*V0_prot) / V0_ML

and the concentration factor [Prot]eq/[Prot]0 is:

[Prot]eq / [Prot]0 = V0_prot / V0_ML

which show that by increasing V0_prot / V0_ML one can concentrate the  
protein as much as one wants.



Please let me know if my reasoning is flawed and if so why!

Best regards

Pietro




Sent from my Desktop

Dr. Pietro Roversi
Oxford University Biochemistry Department - Glycobiology Division
South Parks Road
Oxford OX1 3QU England - UK
Tel. 0044 1865 275339

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Enrico  
Stura [est...@cea.fr]

Sent: 20 February 2015 14:36
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] how to reduce protein solubility

Ursula,

Most compounds used for cryosolutions glycerol, ethylene glycol, propane
diol increase protein solubility.
A warning, these compounds are also hygroscopic, you need to change your
vapour diffusion methodology.
Vera L., Czarny B., Georgiadis D., Dive V., Stura E.A. (2011) Practical
Use of Glycerol in Protein Crystallization. Cryst. Growth  Des. 11:
2755–2762.
http://pubs.acs.org/doi/abs/10.1021/cg101364m
The lack of success in crystallizing proteins in glycerol are due to the
reasons decribed in the paper.

Enrico.

On Fri, 20 Feb 2015 00:33:48 +0100, Ursula Schulze-Gahmen
uschulze-gah...@lbl.gov wrote:


Hi Enrico,

How are you? I see you are now in France.

I have also a question about protein complex solubility. I have a
multi-protein complex that also binds RNA. This Protein-RNA complex can
be
concentrated to 5- 10 mg/ml, but starts precipitating after storage at 4
degrees for several hours ( and can often be resolubilized at room
temperature). The current buffer is 20 mM HEPES 7.3, 0.2 M NaCl, 0.05 M
KCl, 3 mM MgCl2, TCEP. I don't want to increase salt concentration. What
are your suggestions to try to improve the solubility?

Best

Ursula

On Tue, Feb 17, 2015 at 2:00 AM, Enrico Stura est...@cea.fr wrote:


Francesca,

The most common failure is to have an excessive amount of salt (salting
in/ salting out), glycerol or other solubilizing
ingredient in your protein solution. I would suggest that you change  
the

pH and reduce the salt in your protein solution,
by microdialysis if you do not have much protein

Re: [ccp4bb] how to reduce protein solubility

[Enrico Stura]

Francesca,

The most common failure is to have an excessive amount of salt (salting  
in/ salting out), glycerol or other solubilizing
ingredient in your protein solution. I would suggest that you change the  
pH and reduce the salt in your protein solution,

by microdialysis if you do not have much protein, and screen again.
If share with ccp4bb the exact formulation of your protein solution you  
might get more suggestions.


Enrico.


On Tue, 17 Feb 2015 05:23:05 +0100, Mattiroli,Francesca  
francesca.mattir...@colostate.edu wrote:



Hi all,

I am struggling with a protein complex that is too soluble. I have  
reached about 20 mg/ml but I still observe very little precipitation  
(clear drops in 90-95% of the tested conditions). The proteins are  
expressed in insect cells and going to higher concentration is not  
easily achievable.
I have tried different buffer conditions (salt concentration and pH) and  
I am testing temperatures. I am at a loss with what to try next.

Do you think PTMs (phosphorylation, acetylation) might be causing this?
Any input on how to decrease solubility?

Thank you very much in advance,

Francesca





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] crystallization with hydrophobic ligands

[Enrico Stura]

Using mixed solvents is another approach:

Ciccone L., Tepshi L., Nencetti, S.  Stura E.A. (2014)
Transthyretin complexes with curcumin and bromo-estradiol: Evaluation of  
solubilizing multicomponent mixtures New Biotech. 32:54–64

http://dx.doi.org/10.1016/j.nbt.2014.09.002

Volatile solvents are a problem when you try to harvest the crystals.

Enrico.



On Wed, 15 Oct 2014 20:35:33 +0200, Jurgen Bosch jbos...@jhu.edu wrote:

An alternative is to dissolve your compound in MeOH and dispense it  
either manually or via robot, let the plate sit sometime in the hood for  
faster evaporation and then add your protein + reservoir.

Jürgen
..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742tel:%2B1-410-614-4742
Lab:  +1-410-614-4894tel:%2B1-410-614-4894
Fax:  +1-410-955-2926tel:%2B1-410-955-2926
http://lupo.jhsph.edu

On Oct 15, 2014, at 5:18 PM, Keller, Jacob  
kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org wrote:


Since you mentioned EtOH, why not do this:

-Make a tray with the appropriate mother liquors
-Make a drop for each well containing mother liquor and  
high-concentration ligand in EtOH (you could vary the ratios here as  
needed.)
-Equilibrate by vapor diffusion until EtOH all goes into the well soln  
(couple of hours at the most?)

-Add protein to these drops

You could skip right to the protein step if your protein doesn't mind  
EtOH at fairly high concentrations, and anyway it will be gone fairly  
quickly, esp at RT


Jacob Keller


From: CCP4 bulletin board  
[CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of  
Monica Mittal  
[monica.mitta...@gmail.commailto:monica.mitta...@gmail.com]

Sent: Wednesday, October 15, 2014 2:13 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystallization with hydrophobic ligands

Dear All

Can anyone give suggestions for handling the solubility problem of  
highly hydrophobic compounds, during co-crystallization or inhibition  
assays?
The ligands I am using are almost insoluble in aquous medium. In DMSO or  
95% Ethanol, the solubility is higher.
Besides crystallization, this solubility is also a hindrance for  
in-vitro or in-vivo assays requiring higher conc. of ligand.


Thanks in advance !
Monica




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Cryo

[Enrico Stura]

 Vitul,

0.8 M  Lithium sulphate is not excessively high salt (a 2X precipitant is  
possible)

You have many options available:
2M for lithium sulphate is one of them see:
http://pubs.acs.org/doi/full/10.1021/cg301531f
for various combinations of mixed cryoprotectants.

Enrico.




.On Thu, 09 Oct 2014 11:55:49 +0200, Nicolas Soler  
nso...@mrc-lmb.cam.ac.uk wrote:



Hi Vitul,

Having a final concentration of 2M for lithium sulphate worked for me
(from a 2.5M stock solution).

Nicolas

On 09/10/2014 10:27, vitul jain wrote:

Hello everybody,

can anyone please suggest a good Cryo for condition having 0.8 M
Lithium sulphate and 0.1 M  Sodium acetate 4.6.

Thanks in advance

Vitul


--
Vitul Jain
PhD student
C/O Dr. Amit Sharma
Structural and Computational Biology lab
International Center for Genetic Engineering and Biotechnology
Aruna Asaf Ali road, New Delhi
110067.

Mb. no: +91-9818004350, 8860942543
E-mail: vituljain...@gmail.com mailto:vituljain...@gmail.com /
vi...@icgeb.res.in mailto:vi...@icgeb.res.in






--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] 3 letter code

[Enrico Stura]

Dear ccp4bb,

Another easy method to find and/or generate ligands is via the smiles code:

Wikipedia has smiles codes for many compounds:
http://en.wikipedia.org/wiki/Para-Nitrophenylphosphate
look up the smiles code:
smiles: C1=CC(=CC=C1[N+](=O)[O-])OP(=O)(O)O

You can now generate the cif file with phenix.elbow
$ phenix.elbow smiles=C1=CC(=CC=C1[N+](=O)[O-])OP(=O)(O)O

At the PDB select -ligand and enter the smiles code.
select the exact match if it exists.

The smiles code will also help you search for similar molecules

Enrico.



On Thu, 02 Oct 2014 15:45:36 +0200, Faisal Tarique  
faisaltari...@gmail.com wrote:



Thank you every one..

On Thu, Oct 2, 2014 at 5:27 PM, Robbie Joosten  
robbie_joos...@hotmail.com

wrote:


Hi Faisal,

You can also look in LigandExpo http://ligand-expo.rcsb.org/index.html

If you don't find a result immediately, you can also search by formula,
even
without hydrogens.

Cheers,
Robbie

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Paul Emsley
 Sent: Thursday, October 02, 2014 13:31
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] 3 letter code

 On 02/10/14 11:50, Faisal Tarique wrote:
 
 
  I request you to please tell me the 3 letter code for p nitrophenyl
  phosphate..
 

 No.  But here's how to find it yourself:

 Go to rcsb.org
 nitrophenyl phosphate - Search
 Top hit in Chemical Name table.

 or similarly File - Search Monomer Library in Coot.

 Paul.








--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Calcium soaking

[Enrico Stura]

Masaki,

If your crystals crack when you add calcium it implies that calcium  
binding induces a conformational

change.
You should try co-crystallization with an epitaxial jump approach:
 Stura, E. A., Charbonnier, J.-B.  Taussig, M. J.(1999) Epitaxial jumps.  
J. Cryst. Growth 196: 250-260.


Since it is likely that calcium binding destroys only one crystal contact,  
you should screen for co-crystals
with seeds of your calcium-free crystals. One of the planes of your  
calcium-free form should be able to stimulate
growth of crystals with calcium but your crystallization conditions are  
likely to be different.
Without seeding you are likely to be hindered by the nucleation barrier  
which seeding overcomes.


Enrico.

On Thu, 20 Feb 2014 11:29:47 +0100, Masaki UNNO unn...@mx.ibaraki.ac.jp  
wrote:



Dear all

Apologies for the off-topic question:
We are studying an enzyme that is activated by Ca2+. We obtained the
crystals of the substrate and Ca2+-free form and solved the structure at  
2.7
A resolution. However, the active site electron density map was not  
clear,

although other regions are clear. We would like to determine the
substrate-complex with Ca2+, which will elucidate the active site  
structure

at a higher resolution.
Now we have a problem that the crystals of a mutant which can bind the
substrate and Ca2+ always have cracks in soaking to the crystallization
solution containing CaCl2. Co-crystallization does not work at this  
time. We

estimate the structural change in Ca2+-binding is not so big because the
isozyme structure did not change very much when binding Ca2+. An isozyme
structure in complex with the substrate was determined by soaking Ca2+  
(and

the substrate).

How should we overcome this problem?

Best regards

~~~
Masaki UNNO

Graduate School of Science and Engineering, Ibaraki University, Japan



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Recovering crystals from dry drops

[Enrico Stura]

 Debasish

Assuming you do not need seeds, just lift the coverslip, add a small  
amount of water
to the reservoir, close and  allow the drop to equilibrate for 20min, or  
untill you are sure that you have
enough liquid to avoid that the drops becomes solid while you pick up the  
crystals with
a loop and transfer to a suitable cryosolution very rich in cryocomponents  
with very little water.


Improved diffraction? 10% chance!

Enrico.

On Thu, 20 Feb 2014 17:21:53 +0100, Zhijie Li zhijie...@utoronto.ca  
wrote:



Hi Debasish,

I would first use some of those crystals to make seeds and grow some new  
crystals so that I would not lose the crystal. Dehydration, even done  
systematically (eg,  
http://www.mitegen.com/mic_catalog.php?c=jenCrystaloptdehydrate), may or  
may not improve the diffraction. Like most other things in biological  
crystallography, it varies from case to case. I do not think other  
people’s experience really means anything for your crystals.


Zhijie


From: Debasish Chattopadhyay
Sent: Thursday, February 20, 2014 10:59 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Recovering crystals from dry drops

Would you please share your experience and comments on recovering  
protein crystals from dry (or almost dry hanging drops) for data  
collection.



I found some beautiful crystals in hanging drops that were set up three  
years ago; from the color of the crystals ( the protein binds a colored  
substrate) and the their shape I know these are crystals of my protein.   
I had collected data using some of these crystals when they were fresh;  
resolution was poor and the overall I/sigma was low.  I am curious if  
the dehydration would improve the diffraction now.



Thanks


Debasish




Ph: (205)934-0124; Fax: (205)934-0480





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] High Salt Cryo

[Enrico Stura]

Dear All,

I would like to point out that the conditions 1.8 - 2.0 M NaCl are not  
considered High Salt as
NaCl is soluble to 5M and a 2X solution (i.e. 4M NaCl) is possible. Also  
NaCl contrary to
ammonium sulfate, citrate, phosphate, etc. is compatible with polyethylene  
glycol without phase

separation problems.

This means that with 1.8 - 2.0 M NaCl you have an vast repertoire of  
possible ways
to cryo-protect crystals and with the vast repertoire you gain a good  
possibility of finding

conditions that enhance diffraction:
see:
Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.  
Crystal Growth  Design,

http://pubs.acs.org/doi/full/10.1021/cg301531f

For me the definition for High Salt is that 2X for the precipitant  
component is not possible.


Enrico.

On Wed, 19 Feb 2014 16:06:27 +0100, Karolina Michalska  
dzi...@amu.edu.pl wrote:




4M NaCl should work too. It worked for the conditions with 1.8 - 2.0 M
NaCl.

Karolina

W dniu 2014-02-19 06:38, Mooers, Blaine H.M. (HSC) napisał(a):

For crystals grown out of a 2 uL drop of 1.2-1.8 M LiSO4 or 1.6-2.4 M  
AmmSO4, we do in situ cryoprotection with sodium malonate. We add 2-4  
uL of 1.9 M Na malonate to the crystallization drop, wait 10 seconds  
and add 2-4 uL of 2.4 M sodium malonate, repeat with 2.8 M and then 3.4  
M. We do not bother withdrawing aliquots to maintain a fixed volume.  
You may need to tweak the volumes to optimize the resulting  
diffraction. You can also break the additions at given concentration  
into smaller aliquots to reduce the osmotic shock. This approach is  
much gentler than transferring the crystal directly to 3 M sodium  
malonate. Do not leave the drop exposed to the air for more than 3  
minutes or so because salt crystals will start to grow. When there are  
multiple crystals in a drop, often the unused crystals in the very high  
salt solution will still diffract well up to a year later if the  
crystallization chamber is resealed well; their diffraction might even  
improve with the prolonged exposure

to high salt.


Blaine Mooers
Assistant Professor
Department of Biochemistry and Molecular Biology
University of Oklahoma Health Sciences Center
S.L. Young Biomedical Research Center Rm. 466

Shipping address:
975 NE 10th Street, BRC 466
Oklahoma City, OK 73104-5419

Letter address:
P.O. Box 26901, BRC 466
Oklahoma City, OK 73190

office: (405) 271-8300 lab: (405) 271-8313 fax: (405) 271-3910
e-mail: blaine-moo...@ouhsc.edu

Faculty webpage:  
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-  
[1]


X-ray lab webpage:  
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory  
[2]


Small Angle Scattering webpage:  
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0  
[3]


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of  
Katherine Sippel [katherine.sip...@gmail.com]

Sent: Tuesday, February 18, 2014 12:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] High Salt Cryo

Hi all,

I'm looking for a cryo condition for high NaCl (3+ M) crystallization  
condition. I would do it the proper way, but our beam/cryostream is  
down.


I've tried a bunch of things at the moment. Ethylene glycol and PEG 400  
nuke the crystals immediately even at low concentrations. Prolonged  
exposure to glycerol and sucrose starts to break them down so I'm  
thinking that the diffraction will probably suffer. I can't find any  
reports of NaCl's viability as a cryosalt. I've got Paratone/Paraffin  
oil/Mitegen's LV cryo oil on tap but I was hoping to not put all my  
eggs in one basket.


I tried the ISRDB database through  
archive.comhttps://urldefense.proofpoint.com/v1/url?u=http://archive.comk=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0As=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e  
[4] without any luck (no search function). I've gone to the PDB  
searching for similar crystallization conditions and looked up the  
papers for their cryos, but they are all glycerol. Google gives me the  
same.


I thought I'd see if anyone on the bb has an anecdotal this worked for  
us story. I would love to hear it.


Thank you for your time,
Katherine

--
Nil illegitimo carborundum - Didactylos



Links:
--
[1]
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-
[2]
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory
[3]
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0
[4]

Re: [ccp4bb] השב: Re: [ccp4bb] Sister CCPs

[Enrico Stura]

George,

Crystallization, cryoprotection are wet but important steps before data  
collection. Which topics

would you like to exclude?

Enrico.


On Wed, 12 Feb 2014 17:32:47 +0100, Boaz Shaanan bshaa...@bgu.ac.il  
wrote:


Dear George, I'm not sure it's a good idea since the wet work and  
coputational
work are quite often linked. For example preparation of heavy atom  
derivatives
and handling the data, to which bb would you send questions related to  
this
subject? To both bb's? Wouldn't that make life even more complicated in  
terms of

handling more bb's. Just wondering.


Cheers, Boaz


 הודעה מקורית 
מאת George Sheldrick gshe...@shelx.uni-ac.gwdg.de
תאריך: 12/02/2014 18:03 (GMT+02:00)
אל CCP4BB@JISCMAIL.AC.UK
נושא Re: [ccp4bb] Sister CCPs


It would be so nice to have a 'sister CCP' for questions aboud wet-lab
problems that have nothing to do with CCP4 or crystallographic computing,
The is clearly a big need for it, and those of us who try to keep out of
wet-labs would not have to wade though it all.

George


On 02/12/2014 03:05 PM, Martyn Winn wrote:

As advertised at the Study Weekend, CCPBioSim is the sister CCP in
biomolecular simulation. The annual conference will take place in  
Edinburgh in

May, see details below.


Cheers
Martyn








--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] suggestions for cryoprotectant

[Enrico Stura]

Dear All,

Since 80% saturated Li2SO4 has not been mentioned, I will do so. It is a  
good cryosalt and I have often used

it even without any buffer added.
see:
Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.  
Crystal Growth  Design, e-print

http://pubs.acs.org/doi/full/10.1021/cg301531f

The problem with phosphate precipitants is the formation of salt crystals  
in the cryoprotectant solution.
Cryoprotecting molecules that normally work well with high ionic strength  
conditions do not always

work well with phosphates.

800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic
is well matched by 80% saturated lithum sulphate in terms of ionic  
strength.


Enrico.


On Fri, 07 Feb 2014 06:20:58 +0100, Tanner, John J.  
tanne...@missouri.edu wrote:



Try L-proline.  It works well with high ionic strength conditions:

http://www.ncbi.nlm.nih.gov/pubmed/22868767

Sent from Jack's iPad

On Feb 6, 2014, at 10:40 PM, Deepak Thankappan Nair  
deepaktn...@gmail.commailto:deepaktn...@gmail.com wrote:


Hello,
Does anybody know what would be a good cryoprotectant for the following  
condition:
800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic  
100 mM Sodium acetate/Aceticacid pH4.5


Thanks
Deepak




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Room temperature data collection

[Enrico Stura]

Dear Joern and other BBers,

While I fully agree that it is important to test a few images at room  
temperature, to know the crystal's
potential, I think that almost always it will be possible to achieve  
better diffraction using cryogenic

data collection.
Those rare cases, as the one you mention below are worthy of critical  
investigation  as

to why there is a loss of order on cryo-cooling:
Unsuitable cryoprotectant is my first guess.
The rate of cooling in liquid N2 is slow, liquid ethane could be a better  
choice.


Enrico


On Thu, 06 Feb 2014 11:19:47 +0100, Joern Krausze jk...@helmholtz-hzi.de  
wrote:



Dear Theresa,

We recently collected a room temperature data set from one single  
crystal at Petra III. The beam line was equipped with a Pilatus  
detector. Data were good to 2.7 A. In contrast, at 100 K similar  
crystals diffracted very poorly. So, it is perfectly possible to obtain  
useful room temperature data sets from synchrotron sources. I have to  
admit that in our case it certainly helped that the crystal belonged to  
a high-symmetry space and full completeness was achieved after 40  
degrees angular range.


Regards,

Joern

Sent from my iPad


On 06.02.2014, at 10:51, Theresa Hsu theresah...@live.com wrote:

Dear crystallographers

Just out of curiosity, is it possible to collect datasets from crystals  
at room temperature at synchrotron? Are fast detectors like Pilatus  
useful for this?


Thank you.

Theresa



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Room temperature data collection

[Enrico Stura]

Dear all,

Thermal motion reduction and lower radiation damage are reasons for  
improved data associated
with low temperature data collection. The liquid to glass transition of  
the solvent allows us to
have a better idea of the hydration shell around the protein. This is  
something that room temperature
data collection with current techniques does not allow. There can be valid  
biological reasons to purse
RT data collection (without the aim to achieve higher resolution that with  
cryo methods).
Historically, the transition from routine RT data collection to 100K was  
quite painful at the start, but now we think
nothing of it. Every case that presents a new challenge is an opportunity  
to improve our techniques.


But my comment was more related to the idea that with a better  
undertsanding of the physical parameters
that allow crystal formation and stabilization we can find a way to  
collect better data on our current and future

crystals.

Post-crystallization interventions (dehydration, soaking in various  
compounds annealing, etc.)
have been used to improve diffraction. The room temperature result are  
important to define
the starting point, from here the we have the challenge to find out how to  
improve the crystal

and the data that can be extracted from it.

I accept a biological interest in also collecting RT data, but to achieve  
higher resolution data,

low temperature has many advantages. I hope my comment is clearer now,

Enrico.



Tim

On 02/06/2014 12:50 PM, James Foadi wrote:

Dear Enrico,

almost always it will be possible to achieve better diffraction
using cryogenic data collection.

I would say almost always until now. Times change,
instrumentation improves and data collection techniques are
becoming cunning. It's right time people start exploring the new
possibilities offered by modern synchrotrons.

All the best,

J


Dr James Foadi PhD Membrane Protein Laboratory Diamond Light Source
Ltd. Diamond House Harwell Science and Innovation Campus Didcot
Oxfordshire OX11 0DE United Kingdom


office email: james.fo...@diamond.ac.uk alternative email:
j.fo...@imperial.ac.uk


personal web page: http://www.jfoadi.me.uk



On Thursday, 6 February 2014, 11:35, Enrico Stura est...@cea.fr
wrote:

Dear Joern and other BBers,

While I fully agree that it is important to test a few images at
room temperature, to know the crystal's potential, I think that
almost always it will be possible to achieve better diffraction
using cryogenic data collection. Those rare cases, as the one you
mention below are worthy of critical investigation  as to why there
is a loss of order on cryo-cooling: Unsuitable cryoprotectant is my
first guess. The rate of cooling in liquid N2 is slow, liquid
ethane could be a better choice.

Enrico


On Thu, 06 Feb 2014 11:19:47 +0100, Joern Krausze
jk...@helmholtz-hzi.de wrote:


Dear Theresa,

We recently collected a room temperature data set from one single
 crystal at Petra III. The beam line was equipped with a Pilatus
 detector. Data were good to 2.7 A. In contrast, at 100 K similar
 crystals diffracted very poorly. So, it is perfectly possible to
obtain useful room temperature data sets from synchrotron
sources. I have to admit that in our case it certainly helped
that the crystal belonged to a high-symmetry space and full
completeness was achieved after 40 degrees angular range.

Regards,

Joern

Sent from my iPad


On 06.02.2014, at 10:51, Theresa Hsu theresah...@live.com
wrote:

Dear crystallographers

Just out of curiosity, is it possible to collect datasets from
crystals at room temperature at synchrotron? Are fast detectors
like Pilatus useful for this?

Thank you.

Theresa





- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Icedove - http://www.enigmail.net/

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cCzjfbXBCT/HuiMGzPoX8l0=
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--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Intergrown crystals

[Enrico Stura]

Klaus,

 You say that  crystallises readily

So you have solved your own problem. You need to control the rate at which  
the crystals grow.
Among all the things you have tried already, you may have the answer  
regarding how you can
control the crystal growth rate so as to slow it down enough as to have  
large single crystals that
are not intertwinned. This is achievable unless your sample has impurities  
or aggregates.


Enrico.


On Wed, 22 Jan 2014 16:01:44 +0100, Klaus Fütterer k.futte...@bham.ac.uk  
wrote:



Dear ccp4bb contributors,

We are dealing with the problem of a protein (~ 50 kDa) that  
crystallises readily, but has an annoying habit of forming highly  
intergrown rods or needles.
Even when the crystals look optically homogenous under the microcsope,  
diffraction is so so (3.5 Å or so on the synchrotron), but patterns  
reflect several crystal lattices that the processing software cannot  
resolve properly.


We have tried this:

- additive screens
- switching the His-tag from N- to C-terminus
- cutting the tag
- thermal stability screens in a variety of buffers
- growth in the presence of potential ligands/substrates

Any suggestions for tricks that we haven't thought of so far?

Thank you.

Klaus


===
Dr. Klaus Fütterer

School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham  F: +44-(0)-121-414 5925
Edgbaston  E: k.futte...@bham.ac.uk
Birmingham, B15 2TT, UK   W: http://tinyurl.com/futterer-lab
===








--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Isolation of protein-protein complexes.

[Enrico Stura]

Dear David,

Following the discussion I am starting to wonder if I have been doing  
something wrong all these years.
I always forgot to purify the complexes, I just mixed the two  
macromolecules and did the crystallization experiments.
And I will admit that I did not even worry about getting the right  
stoichiometry. It is true that I got

crystals with the wrong stoichiometry:
Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G.,  
Silverman, G.J.  Charbonnier, J.-B. (2001) Crystallization of  
macromolecular complexes: Stoichiometric variation screening. J. Cryst.  
Growth 232: 580-590.
But then ... I thought that as long as I followed the standard rules for  
crystal growth, even stoichiometry variation could be part of

standard crystallization methodology.

Since you still got all the details of the interaction from the structure  
even with extra molecules, I was not ashamed of my mistake and tried

to publish the structure. The referees did not seem to mind:
 Graille, M., Stura, E. A., Taussig, M. J., Corper, A., Sutton, B. J.,  
Charbonnier, J.-B.  Silverman, G. J. (2000) Crystal structure of a  
Staphylococcus aureus protein A domain complexed with the Fab fragment of  
a human IgM antibody: structural basis for recognition of B-cell receptors  
and superantigen activity. Proc. Natl. Acad. Sci. USA. 97: 5399-5404.


Enrico.


On Tue, 21 Jan 2014 18:02:48 +0100, Eugene Osipov e.m.osi...@gmail.com  
wrote:



May be your complex components interact with column? I mean: you have
calibration plot for column, right? Does the molecular mass of proteins
calculated from elution volume equals to mass calculated by another  
method

(like SDS-PAGE)?


2014/1/21 Keller, Jacob kell...@janelia.hhmi.org

 Maybe there is something required for interaction that was in the  
buffer

used for the other binding studies, but not in your SEC buffer?



JPK



*From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf  
Of *David

Briggs
*Sent:* Tuesday, January 21, 2014 10:52 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Isolation of protein-protein complexes.



Dear all,



sorry for the slightly off topic post,



I have 2 proteins that have been shown to interact, by multiple groups,
and by multiple techniques - namely ELISA, SPR and DPI.



The Kd of the interaction as determined by SPR is on the order of 1 nM.



I would very much like to crystallise this protein-protein complex, and  
as
a first step I attempted to purify the complex by mixing the two  
proteins

(same protein preps and same buffers as the SPR experiment) and then
running them down a gel filtration column (Superose 6 - predicted size  
of

the complex is ~500kDa).



Somewhat irritatingly the two proteins separate beautifully on the  
column

into two distinct peaks. There is no trace of complex formation when the
peaks are analysed by SDS-PAGE.



As far as I am aware, two proteins that interact this strongly should
remain associated during gel filtration, and I was wondering if anyone  
else

has encountered anything similar in the past, and if they managed to
resolve the problem, how they went about it?



Cheers in advance,



Dave


David C. Briggs PhD
http://about.me/david_briggs








--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] crystals with large solvent content -dehydratation

[Enrico Stura]

Dear Andre,

66% solvent is on the high side but not a good reason for poor resolution.
Other with similaa solvent content have achieved resolutions of 1.5 Ang.  
and even better.
I would screen at a lower protein concentration. It will require more  
precipitant

and you should end up with less water in the cell.

Enrico.


On Tue, 29 Oct 2013 16:18:21 +0100, Andre Godoy andre_go...@yahoo.com.br  
wrote:



Dear all

I'm trying to solve a beautiful large crystal that, unfortunately,  
doesn't go further than 5 A resolution. I believe that in this case, the  
lack of resolution is due the high solvent content (about 66%).  
Therefore, my next strategy should be the dehydratation. Yet, I never  
(sucessfully) did that. I read different approachs, were people  
equilibrate crystals in dehydratation solution for days, or do more than  
20 steps, or add solvents. Since i never had sucess in my trials, I was  
thinking that someone can suggest a protocol (should I remove all salt?,  
should I keep the additive concentration?, how much precipitant should I  
add? how many steps?).


crystal condition: 23% PEG 3350, 0.2M NaCl, 0.1M Tris pH 8.5, 3%  
galactose (orthorhombic crystals, with about  0.6 x 0.6 mm)


all the best,

Andre Godoy



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] AW: [ccp4bb] cryoprotection

[Enrico Stura]

Herman,

The trick you suggest is not as valid as you may think. The ice rings can  
originate from the crystal itself.
If you crystallize in a high concentration PEG precipitant you will avoid  
ice rings,
but if you transfer or soak your crystals in the same solution the high  
molecular weight

PEG will not enter the crystal lattice and you will still get ice rings.
I have a picture of this in:
Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.  
Crystal Growth  Design, e-print

http://pubs.acs.org/doi/full/10.1021/cg301531f PDF: Figure3 Page G.

So cryoprotectants need to penetrate the crystal lattice to prevent ice  
rings, but even in the

presence of ice rings the data can be used.

Regarding optimization:
The main problem you encounter in cryoprotection is that some compounds
like glycerol and ethylene glycol solubilize protein crystals, but if you  
create a
mixture of various compounds that is precipitation-solubilization neutral,  
then

there is no real need for optimization.

Enrico.

On Tue, 27 Aug 2013 08:30:28 +0200, herman.schreu...@sanofi.com wrote:

A trick I like is just to freeze the reservoir solution or would-be  
cryo-solution without a crystal present. If the frozen solution stays  
clear and does not show ice rings on e.g.  a home source, it is worth  
trying. Otherwise, the solution needs optimization.

Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von  
Uday Kumar

Gesendet: Freitag, 23. August 2013 19:52
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] cryoprotection

Hello

Can anyone suggest a cryoprotectant for the following crystallization  
condition


0.2-0.4M sodium formate

~20% PEG 3350

0-25 mM Nickel

0-100 mM Malonate

Thank you

with regards
uday



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] database of crystallization condition

[Enrico Stura]

On Wed, 24 Jul 2013 16:36:17 +0200, Hena Dutta hdutt...@gmail.com wrote:


Hi,
Can anyone tell, if there is any database containing the crystallization
conditions of published structures? I want to see the conditions people
have used for those proteins having some structural similarity. Any
suggestion would be appreciated.
Regards...
Hena


You can use the reports in the PDB database:
1) Sequence search with your sequence
2) Report pull down menu - select Crystallization

How is that going to help? Even a single amino acid change can change the  
manner a protein
crystallizes! The precipitant concentration is affected by the protein  
concentration.

The purity and purification procedure can also affect crystallization.

Enrico.


--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] AW: [ccp4bb] help identifying ligand

[Enrico Stura]

Dear CCP4BB,

The most likely components are those at the highest concentration in the  
crystallization

or cryosolution.

And a few wild ideas to continue the discussion that is very important as  
the ligands are

always very difficult to identify.

Example: If you have 1.5 M ammonium sulfate you should consider hydrated  
ammonium ions

H3O+ + NH3 in equilib. H2O + NH4+
The pH will determine the equilibrium point  and NH4+ would be a good  
ligand for a carboxylate.

Assuming 200mM Li2SO4:

A lithium ion (H20-Li-H20 with a Li-O distance of 2.14 Ang) Li+ is often  
associated with more that two H2O molecules
with an angle of 105° not 180° but cannot be excluded in proximity of a  
carboxylate where the environ ment could be

distorted (not very believable).

(H2O, Na+ and Mg++ 10 electrons) water is always the most probable.
2 H2O in equilib. OH- + H3O+
Carboxylates are often destroyed by radiation damage.

The most probable ligand will be at high concentration in the mother  
liquor the moment

the crystal was flashcooled.
This is rarely the case for typically 0.02% azide (I would made an  
exception in proximity to Cu++, Fe++ or Zn++ ions).
Azide -N=N+=N- is also suspitious as a negative ion is a bad counterion  
for a carboxylate.


Enrico.


On Mon, 08 Jul 2013 11:19:46 +0200, herman.schreu...@sanofi.com wrote:


Dear Ed,

What is the pH of your crystallization buffer? If it is acidic, either  
the azide or the carboxylate may be protonated. Also the local  
environment of the carboxylate can make a hugh difference in PKa. You  
could also use some Bayesian logic: given the elongated linear density,  
what else of the available components of your crystallization drop would  
fit?


Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von  
Edward A. Berry

Gesendet: Sonntag, 7. Juli 2013 22:21
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] help identifying ligand

In a structure I'm refining, there are a couple of oblong blobs  
associated with carboxylates.

(screenshots at http://sb20.lbl.gov/berry/ccp4/azide/)
If I modeled with two waters, they refine too close together for normal  
H-bond,

2.3 to 2.5 A; and their density is connected.

I considered one water with alternate locations, but the distal position  
wouldn't make much sense if the proximal water wasn't there. The density  
is the right size for azide, which was present in the medium, but I  
expect a chemist would find it unreasonable to have anionic azide (pKa  
of hydrazoic acid ~4.6) associating with a carboxylate.

Would that make sense? or does anyone have other suggestions?
(resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma)

Thanks,
Ed



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Crystallisation below 0°C

[Enrico Stura]

Glenn Masson,

We had crystals that appeared by chance at underfined low temparature:
Maïga, A., Vera, L., Marchetti, C., Lorphelin, A., Bellanger, L., Mourier,  
G., Servent, D., Gilles, N.  Stura, E. A. (2013) Crystallization of  
recombinant Green mamba ρ-Da1a toxin during lyophilization procedure and  
its structure determination. Acta Cryst. F 704-709.


There is an intereasting zone for crystallization around zero, but the  
problem is going to be how to observe the crystals and

accurately control the temperature.

Enrico.


On Thu, 30 May 2013 13:26:44 +0200, Glenn Masson glennmas...@gmail.com  
wrote:



Hello CCP4BBers,

I am currently playing with some crystals that seem to enjoy lower
temperatures, and I was thinking of breaking the 0°C threshold.

Looking for examples of this in the literature is problematic, as  
searching

for examples in the PDB (Under the advanced search- crystal properties-
temperature (K)) turns up a large amount of false positives. Many  
otherwise
supremely intelligent people seem unable or unwilling to grasp the  
concept

of Kelvin (it's amazing how many protein structures were solved only 22
degrees above absolute zero...).

I was wondering if anyone has much experience in this area? I see a few
structures e.g. 2Z97 (-5°C) and 4H0W (-2°C), but I was wondering if  
anyone

has a more systematic knowledge, some more examples, and what the
parameters and best practice of this technique are.

Many thanks,

Glenn Masson
MRC-Laboratory of Molecular Biology



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] cryo condition

[Enrico Stura]

80% saturated Li2SO4


On Thu, 23 May 2013 11:42:09 +0200, Faisal Tarique  
faisaltari...@gmail.com wrote:



Dear all

Can anybody tell me the appropriate cryo condition for the crystals
obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10%
glycerol to it but still the ice ring is forming..

Thanx in advance



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] cryocrystals

[Enrico Stura]

careina

Cryocrystals will last several months and more. Make sure that your LN2
is dry.


On Wed, 22 May 2013 14:38:51 +0200, Careina Edgooms  
careinaedgo...@yahoo.com wrote:



Hi
Does anybody know how long one could store a crystal in liquid nitrogen  
for before it will no longer diffract well? I'm talking in the order of  
weeks to months...

careina



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] P212121 and P213

[Enrico Stura]

Polymorphs are another possibility. The packing could be very similar but  
the space

group could be different.

Enrico.


On Thu, 17 Jan 2013 15:30:20 +0100, Eleanor Dodson  
eleanor.dod...@york.ac.uk wrote:


Hard to say without data - but I would generate the 3 symmetry copies of  
the molecule you would have in P213,
then do rigid refinement of   those coordinates with the P212121 cell  
dimensions  and symmetry. There are so many possibilities for origin  
shifts
But you don't say how non-equivalent the axes are;  - if -18.16 ~ 1/4 ~  
21.39 that is a pretty big difference?

Eleanor

On 17 Jan 2013, at 14:08, Nicholas Keep wrote:


I have a structure which normally crystallises in P213 but one data set
the edges became slightly non-equivalent in length by a couple of
angstroms and the data process in P212121

P212121 symmetry operators appears to be a subset of P213

http://img.chem.ucl.ac.uk/sgp/large/019az1.htm
http://img.chem.ucl.ac.uk/sgp/large/198az1.htm

(Not absolutely sure the above are world viewable)

Naively I expected the two structures to roughly align.  However they
don't there appears to be some change of axes between them.
The transform of the P213 onto the P212121 structure is
0.03345 -0.9977 -0.0598
-0.9994 0.03402 -0.08653
0.01066 0.05929  -0.9982
-18.16 -57.18 21.39

This is clearly close to

0 -1 0
-1 0 0
0 0 -1
-0.25 -.75 0.25 in fractionals.

Applying either the exact transformation or the regularised one
with ether indexing of  p213 ie original and k h -l
http://www.ccp4.ac.uk/html/reindexing.html
the rfactors are well above 50% and don't drop.

Any suggestions of what I am doing wrong or is this not going to work
(tried MR into the reindexed data and that does not align either- in  
fact I have tried quite a lot things)


The reason for trying to get the two structures on the same axes is to
compare the electron densities.  The P212121 seems to have some of the
disorder loops better defined.

Using the Coot transform map by LSQ superpose is quite good BUT it would
be better if it were another map other than the refinement map that is
transformed as I then want to refine the unmoved structure into the
unmoved map guided by the moved map and that involved a lot of changes  
of map selection.


Thanks
nick



--
Prof Nicholas H. Keep
Executive Dean of School of Science
Professor of Biomolecular Science
Crystallography, Institute for Structural and Molecular Biology,
Department of Biological Sciences
Birkbeck,  University of London,
Malet Street,
Bloomsbury
LONDON
WC1E 7HX

email n.k...@mail.cryst.bbk.ac.uk
Telephone 020-7631-6852  (Room G54a Office)
 020-7631-6800  (Department Office)
Fax   020-7631-6803
If you want to access me in person you have to come to the
crystallography entrance
and ring me or the department office from the internal phone by the door


--
Prof Nicholas H. Keep
Executive Dean of School of Science
Professor of Biomolecular Science
Crystallography, Institute for Structural and Molecular Biology,
Department of Biological Sciences
Birkbeck,  University of London,
Malet Street,
Bloomsbury
LONDON
WC1E 7HX

email n.k...@mail.cryst.bbk.ac.uk
Telephone 020-7631-6852  (Room G54a Office)
 020-7631-6800  (Department Office)
Fax   020-7631-6803
If you want to access me in person you have to come to the  
crystallography entrance

and ring me or the department office from the internal phone by the door



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/ibitecs/simopro/ltmb/cristallogenese
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] side question re crystal dehydration

[Enrico Stura]

Juan,

Humidity variation is what vapour diffusion crystallization achieves.
In your list of all possible dehydration methods you would end up  
classifying all
vapour diffusion experiments as a case of dehydration. After nucleation,  
crystals
continue to grow and the drop continues to become more concentrated in  
precipitant.

I agree you are completely right!

The concept of crystal hydration is very complicated:
Crystals are grown in liquid water and often analysed in vitrified water  
with a solvent-hydrogen bond network that is different from that in the  
liquid state.

What does this mean in terms of hydration?
The use of cryo-protectant alter the solvent hydrogen bonding pattern.  
What does this mean in terms of hydration?
VM, the Matthews coefficient, defined as the crystal volume per unit of  
protein molecular weight is a a measure of hydration?

So if the VM is the same the hydration is the same?

I agree that all the methods that you mention will affect hydration of the  
crystals, but the way that X-ray crystallography is carried out today

cannot avoid it.

Crystal dehydration must be defined as an explicit effort to use  
methodology designed to alter the hydration of crystals, preferably using
a defined measured and controlled relative humidity value. As  you start  
to consider badly defined systems, you will also have badly defined  
hydration.


Enrico.


On Wed, 16 Jan 2013 14:18:05 +0100, Juan Sanchez-Weatherby  
juan.sanchez-weathe...@diamond.ac.uk wrote:



Dear all,

From Leonid's reply earlier you can see a problem some of us have been  
having for a while now, when looking for literature regarding  
dehydration. Most of you that perform dehydration either don't consider  
it happening or don't report it in great detail in your publications.  
This is only understandable because it isn't the focus of your work and  
it only helps you get to where you want to get to.


I'm trying to get an up to date picture of what is out there but I  
haven't got the time or eyes to go through everyone's methods to pick  
the couple of lines that describe your particular method. I really want  
to find out what is being done to be able to give people better advice.


So: Could people out there that think that in their particular projects  
dehydration/hydration had an effect send me a ref. or a short  
description? (can be done outside the BB to not spam everyone) I will  
duly acknowledge everyone!!


By dehydration I mean:

1 Soaking with increasing concentration of precipitants or salts
2 By equilibrating against a new precipitant or salt (by vapour  
diffusion or dialysis)

3 By letting the drops dry (controlled or uncontrolled)
4 by using an FMS/HC1/MicroRT or any other gadget
5 By some other magical trick you may have

Thank you all for your help,

Regards

Juan


Juan Sanchez-Weatherby, PhD
Beamline Scientist - I02
Macromolecular Crystallography Group
 Diamond Light Source Ltd
Diamond House DR1.64
Harwell Science and Innovation Campus
RAL, Chilton, Didcot
Oxfordshire
OX11 0DE
United Kingdom
 Tel: +44 (0)1235 778661
Mob:+44 (0)7795 641259
Fax:+44 (0)1235 778052
 juan.sanchez-weathe...@diamond.ac.uk
 http://www.diamond.ac.uk


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of  
Leonid Sazanov

Sent: 15 January 2013 19:32
To: ccp4bb
Subject: Re: [ccp4bb] crystal dehydration

In case if dehydration needs to be done slowly and under tight control  
of all parameters, one possibility is to use micro-dialysis  buttons.


We used it for a large membrane protein complex and diffraction improved  
from ~7 to 2.7 A. The crystal is fished out and put into mother liquor  
solution in the button, sealed with dialysis membrane and the button is  
then placed into about 5 mls of mother liquor with slightly higher PEG  
concentration. Then you just exchange outside buffer every day or so for  
solutions containing higher concentrations of PEG. We went from ~9 to 30  
% PEG4000 in about a week. You can easily observe crystal under  
microscope and if it cracks - you went too far/too quickly with PEG and  
need to use a bit less next time. Also, this method allows you to  
control all other components of the dehydrating solution - we needed to  
decrease salt concentration at the same time as increasing PEG. You can  
also introduce/increase cryo-protectant concentration at the same time.  
With these crystals, otherwise excellent dehydration machines already  
mentioned did not work, possibly because the process had to be really  
slow. The reference is here: http://www.ncbi.nlm.nih.gov/pubmed/21822288


Best wishes.



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/ibitecs/simopro/ltmb/cristallogenese

Re: [ccp4bb] Ammonium phosphate as cryoprotectant

[Enrico Stura]

Joy,

Try 80% saturated Lithium sulfate (2.5M) instead.
Just transfer the crystals and if it does not work
let me have more precise crystallization conditions and I will post you
other options.


Enrico.



On Thu, 03 Jan 2013 18:33:05 +0100, Joy joybeiy...@gmail.com wrote:


Dear All,


May I  have your expert opinion on the following question: what is the
minimum concentration of ammonium dibasic phosphate if I would like to  
use

it as cryoprotectant?


Happy New Year!


Joy




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Ammonium phosphate as cryoprotectant

[Enrico Stura]

On Mon, 07 Jan 2013 18:30:32 +0100, Joy joybeiy...@gmail.com wrote:


Dear Bryan,


Thank you very much for the comments, I have tried glycerol and it did  
affect the resolution, at the same time, through reading former posts, I  
find that sucrose might not be a good choice for crystals grown in salt,  
I will definitely try adding glycerol stepwise, but at the same time,


Glycerol is a solubilizer. A step wise approach will work only if as you
increase the glycerol concentration you also increase the precipitant
concentration. Be careful of salt crystals appearing as you take this path.

I  am wondering if high conc. ammonium phosphate could be used as  
cryoprotectant, if so, I would be able to achieve dehydration and cryo  
at the same time.
All salts will prevent ice formation at high concentration, but some reach  
the crystallization point
before.  There is also the possibility of making a salt mixture by adding  
for example

ammonium formate, an excellent cryosalt.
If you try the experiment and let the BB know the result.

Enrico.




From: Prince, D Bryan [mailto:dbryan.pri...@astrazeneca.com]
Sent: 2013年1月3日 17:17
To: Joy
Subject: RE: [ccp4bb] Ammonium phosphate as cryoprotectant


Dear Joy,


I do not believe that ammonium dibasic phosphate will make a good  
cryosalt. I would recommend a lithium cryosalt (formate, acetate or  
sulfate) or glycerol if you really need to stay with the phosphate.



Hope this helps!


Bryan


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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Joy
Sent: Thursday, January 03, 2013 12:33 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ammonium phosphate as cryoprotectant


Dear All,


May I  have your expert opinion on the following question: what is the  
minimum concentration of ammonium dibasic phosphate if I would like to  
use it as cryoprotectant?



Happy New Year!


Joy




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Expert opinion for optimizing ethylene glycol crystallization condition

[Enrico Stura]

Dear Sankaranarayanan Srinivasan

Try replacing both glycerol and ethylene glycol by propylene glycol in part
or completely.

Enrico.


On Wed, 02 Jan 2013 18:59:21 +0100, Sankaranarayanan Srinivasan  
texs...@gmail.com wrote:



Dear all,

A very happy new year to all.

I would appreciate some expert advice on optimizing a crystallization
condition in which the initial hits were obtained with ethylene glycol as
the main precipitant. Here is the summary of things tried.

We have a protein, size (31Kda) and the starting protein buffer is 0.1M
Tris pH7.5, 0.1M NaCl, 10% glycerol.
The initial crystal hit was obtained from the emerald cryo kit condition
that has 0.1M imidazole pH 8.0 , 50% (v/v) ethylene glycol. The crystals
were tiny (10-20um). A crystallization matrix to obtain better crystals  
by
varying the imidazole pH and ethylene glycol concentrations was tried  
from

which the best condition obtained was 0.1M imidazole pH 6.5 , 30% (v/v)
ethylene glycol. The crystals were slightly bigger 50um.
On trying the additive screen, bigger crystals (200um) were obtained, but
putting them under the x-ray beam with direct freezing did not yield any
diffraction spots.
Trying other cryo-conditions like glycerol and 50-50 paratone/oil mixture
also yielded similar results.
Low resolution spots near the beam stop were also not seen. Similarly  
spots

indicative of salt was also not seen. It just had hazy ice rings kind of
stuff. (The beam was definitely on the crystal)
To check if what we have was salt, a control condition with no protein  
was

tried. Also the crystals were run on a gel after thorough washing. Both
these tests, show that they are definitely protein crystals and not salt.
Seeding also did not yield any improved crystals.
I was suggested using di-ethylene glycol, propane diol as alternatives.
I would greatly appreciate if you can give your opinion on using other
di-alcohols as precipitants or other ways to improve these crystals.
I tried searching the PDB to see if someone had actually used ethylene
glycol as a precipitant, most of them were used as a cryo condition than
actually as a precipitant.

Thank you very much in advance.

Regards
Shankar Srinivasan



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Binding constants/kinetics for crystallisation

[Enrico Stura]

Dear  Roger,

I disagree with Ganesh. Knowing the stoichiometry is not necessary.  
Stoichiometry
may need adjusting to reflect the relative solubility of the interacting  
partners

under the various crystallization conditions.
See also:
Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G.,  
Silverman, G.J.  Charbonnier, J.-B. (2001) Crystallization of  
macromolecular complexes: Stoichiometric variation screening. J. Cryst.  
Growth 232: 580-590.


Affinity is very important when the affinity is better than nanomolar, you  
do not need stoichiometric

compensation.

Enrico.

On Fri, 07 Dec 2012 16:44:48 +0100, Ganesh Natrajan  
ganesh.natra...@ibs.fr wrote:



Dear Roger,

In my humble opinion, the qualitative knowledge that the complex
actually forms (established through pull down assays, gel filtration
etc) is probably far more important than the Kd values in solution. In
any case, the crystallization is done at very high concentrations,  far
above the Kd values of the interacting partners. So having a thumb rule
is not necessary.  The knowledge of the stoichiometry of the binding
partners may actually be more important to get a more homogenious  
complex.


In a recent complex I've worked on, we determined the Kd only after
solving the structure.

.

cheers

Ganesh



Le 07/12/12 16:11, Roger Pickman a écrit :

Dear all - is there a rule of thumb for favourable values of Kd, kon
and koff of  protein-protein or protein-dna complexes for protein
crystallisation?  Are these measurements useful in crystallisation, or
should one just put it down a gel filtration column, hope for a
complex and not worry?  If anyone can point toward a reference, i'd
appreciate it.

Roger





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] vitrification vs freezing

[Enrico Stura]

As a referee I also dislike the word freezing but only if improperly  
used:
The crystals were frozen in LN2 is not acceptable because it is the  
outside

liquor that is rapidly cooled to cryogenic temperatures.

But the use of freezing used as the opposite of melting is fine and  
does not

imply a crystalline state. Ice is not always crystalline either:
http://en.wikipedia.org/wiki/Amorphous_ice


--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] cryo conditions

[Enrico Stura]

Dear Ed,

Glycerol is a solubilizing agent. Unless something is done to conteract  
such effect
the crystals will dissolve. Laura Vera from my lab made a presentation at  
ICCBM-14

on this subject.
We have developed a set of balanced solutions, mixed solubilizers like  
glycerol and ethylene
glycol with precipitants like MPD, DMSO and neutral compounds like  
di-ethylene glycol
and propanediol that with PEG and salts crystallization conditions take  
out the guesswork

from designing cryo-conditions.
The mixed conpounds are marketed by Molecular Dimensions
google search: CryoProtX

Before ordering the kit check that the crystals do not dissolve with MPD.  
If crystals crack
but do not dissolve with MPD (it may be worth ordering the kit). Cracking  
is good since it means
that the crystals do indeed dissolve in glycerol because of glycerol's  
solubilizing properties.


Now try Propylene glycol (1,2-propanediol).
This is a neutral single component. It is a good starting point to work on  
your cryo-conditions
which you will then be able to improve when you have all the various  
cryo-components and

premixed combinations.

The paper for the mixed cryosolutions will be submitted to the ICCBM-14  
special issue

of the Crystal Growth and Design probably this week.

polyacrylic acid is a novell precipitant, make sure that you keep  
magnesium in your
cryosolution. I would also check if increasing the magnesium concentration  
in the

cryoprotectant solution might not be beneficial.

Enrico.

On Wed, 17 Oct 2012 06:01:08 +0200, Edward A. Berry ber...@upstate.edu  
wrote:



Would someone suggest a cryoprotectant for index screen #59? It contains
0.02 M MgCl2
0.1 M HEPES pH 7.5
22% polyacrylic acid 5100, Na salt

Adding glycerol tends to dissolve the crystals.

Thanks,
Ed



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Professor Dame Louise Johnson

[Enrico Stura]

Professor Dame Louise Johnson was my thesis supervisor and I am saddened by
her departure.

I would like to encourage the crystallographic community to contribute to:
http://en.wikipedia.org/wiki/Louise_Johnson
so that her achievemens can be remenbered and can continue to inspire
future generations of crystallographers.

Those that have access to a copyright -free photograph can upload it to  
Wikipedia Commons:

http://commons.wikimedia.org/wiki/Main_Page

I would like to extend my condolences her family and all her friends.

Enrico.



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Are these xtal conditions worth optimizing?

[Enrico Stura]

Dear CCP4bb,

1) Check the bottom of the tube from which the protein was taken. Round
objects could be sephadex beads.

2) Touch the object with a cat wisker. Does it break up easily?
Yes: probably protein crystals. If it is an oil instead of a solid object  
you have

your answer.

3) If these objects break up easily can you use them as seeds?
If so, it is well worth optimizing!!

Birefringence is also useful, but you need to know your salts. Some are  
difficult
to distinguish from small (less than 60 aa) proteins that can also be  
strongly birefringent.
Birefringence is useful in particular if you check how fast the color  
changes as you rotate the
cross-polarizer. Portein crystals tend not to change color as fast as salt  
crystals do. Sephadex

beads are not birefringent.

Enrico.


On Thu, 07 Jun 2012 00:10:47 +0200, Jacob Keller  
j-kell...@fsm.northwestern.edu wrote:



Birefringence?

JPK

On Wed, Jun 6, 2012 at 4:52 PM, Harman, Christine 
christine.har...@fda.hhs.gov wrote:



Hi All,
I have these very weird drops that I found from screening (please find
pictures attached).  I am not sure if they are worth optimizing.  I am  
very
interesting to know your opinions of what you think of these drops  
could be

(are these what you call spherulites?).  If you think these conditions
are worth optimizing, I welcome any ideas on how to optimize.  I have  
done

some optimization and still get the same result which is many of these
weird things growing throughout the drop and with no sharp edges, and
sometimes a skin forms after 2weeks (with the sodium malonate conditions
only).   I have also opened the drop and poked around to see if it is  
phase
separation and this things are definitely solid and slightly-very  
mushy.  I

haven't had a chance to check for diffraction, but will be very soon.
Both of these drops contain the same protein preparation of a  
Fab/peptide

complex @ ~5mg/mL in buffer containing 0.1M Sodium Acetate pH 5, 150mM
NaCl. I appreciate any advice, thoughts or comments that you could  
provide.


Peace,
Christine











--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Criteria for Ligand fitting

[Enrico Stura]

Naveed,

You mention:
The active site hydrophobic crown  had been
reported to re-orient and a charged residue is known to position for
forming a salt-bridge with similar ligands.

When you induce structural changes on ligand binding, lattice forces that  
stabilize
one particular conformation may be able to fight against ligand binding.  
The result
is not always absence of ligand. You will get a statistical sampling of  
various viable solutions
but the answer in not satisfactory. You should try co-crystallization  
instead of soaking!


For hydrophobic ligands the problem is different. (I assume that the  
acidic tail
gives your ligand excellent water solubility so I will not mention what to  
do in that case).


Enrico.



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of  
Naveed A Nadvi

Sent: Tuesday, April 24, 2012 12:02 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Criteria for Ligand fitting

Dear Crystallographers,

We have obtained a 1.7 A dataset for a crystal harvested from  
crystallization drop after 2 weeks of soaking with inhibitor. The  
inhibitor has an aromatic ring and also an acidic tail derived from  
other known inhibitors. The active site hydrophobic crown  had been  
reported to re-orient and a charged residue is known to position for  
forming a salt-bridge with similar ligands. When compared to apo  
strucutres, we can clearly see the re-orientation of these protein  
residues.


However, there are no clear density visible for the ligand in the Fo-Fc  
map. Some density is visible in the 2Fo-Fc map with default settings in  
COOT. We were expecting co-valent modifcations between the inhbitor,  
co-factor and protein residues. In fact, the Fo-Fc map suggested the  
protein residue is no longer bonded to the co-factor (red negative  
density) and a green positive density is observed nearby for the protein  
residue. These observations, along with the extended soaking and the  
pre-determined potency convince us that the inhibitor is present in the  
complex.


When I lower the threshold of the blue 2Fo-Fc map (0.0779 e/A^3; 0.2  
rmsd) we can see the densities for the aromatic ring and the overall  
structural features. These densities were observed without the cofactor  
and the inhibtor in the initial MR search model. The R/Rfree for this  
dataset without inhibitor was 0.20/0.24 (overall Bfactor 17.4 A^2). At  
50% occupancy, modeling the inhibtor showed no negative desities upon  
subsequent refinement. With the inhibtor, the R/Rfree was 0.18/0.22   
(overall Bfactor 18.8 A^2). The temp factors of the inhibitor atoms (50%  
occ) were 15-26 A^2.


My understanding is phase from the MR search model may influence Fo-Fc  
maps, and the 2Fo-Fc map minimizes phase bias. Since the inhibitor was  
absent from the MR search model, can these observations be used to  
justify the fitting of the ligand in the map? Given the low map-level  
used to 'see' the ligand, would this be considered noise? Can I justfiy  
the subsequent fall in R/Rfree and the absence of negative density upon  
ligand fitting as proof of correct inhibtor modeling? I would appreciate  
if you could comment on this issue. Or tell me that I'm dying to see the  
inhibitor and hence imagining things!


Kind Regards,

Naveed Nadvi.



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Substitution to glycerol during crystallogenesis

[Enrico Stura]

Glycerol is known to be able to reduce nucleation. This might be countered
by an increase in protein concentration.
Vera, L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011)  
Practical Use of Glycerol in Protein Crystallization. Cryst. Growth  Des.  
11: 2755–2762.


Enrico.

On Tue, 03 Apr 2012 13:49:50 +0200, Toby Longwood  
toby.longw...@gmail.com wrote:



Dear all,

My question is related to a sample preparation.

I’m working with a complex that can be stabilized with glycerol (at least
10%) during purification. The use of detergents does not help. After
purification, the sample is homogeneous (EM) and can be concentrated
(3-4mg.mL-1) . I already set up many drops, changing several conditions
(pH, salt...) but nothing conclusive appeared.

I know that crystallogenesis in presence of glycerol works (Sousa, Acta
Cryst (1995), ...) however, because of the aspect of the drops
(precipitates that seem close to the nucleation phase), I suspect that  
the

glycerol can be one of the limiting factors of the protocol.

Has anybody else been already confronted to the same problem? Does  
someone

know if there is an alternate additive to glycerol?

Thanks in advance for suggestions/help

With best wishes


Toby



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] microseeding

[Enrico Stura]

Patrick,

This thing all things devours:
 Birds, beasts, trees, flowers;
 Gnaws iron, bites steel;
 Grinds hard stones to meal;
 Slays king, ruins town
 And beats high mountains down.

 Answer: TIME

When scaling up in seeding  the most important factor is: TIME
While the environment may be the same, the rate at which it changes
is not. Since crystal growth takes TIME, the change in volume will
affect supersaturation in a time-dependent manner.

Using salt or any other parameter to compensate for TIME will
introduce an extra variable which you may not know what its effect
will be.

Sorry to mess up your solution.

Enrico.



On Mon, 19 Mar 2012 23:39:00 +0100, Patrick Shaw Stewart  
patr...@douglas.co.uk wrote:


Sorry, I said that last part wrong.  I meant it is sometimes helpful  
to*increase

* the salt by 50% when scaling up.


On 19 March 2012 22:29, Patrick Shaw Stewart patr...@douglas.co.uk  
wrote:




Rajesh

If you set up the volumes you suggest you will probably get
precipitation.  This is counterintuitive until you realize that (as Ed
says) you will be losing a lot of protein with those small drops.  When  
you
scale up the surface area to volume ratio is lower, so a smaller  
proportion

of the protein is lost.  Therefore you go *up* on the phase diagram and
get precipitant or very small crystals.

Normally halving the amount of protein for the hits from 200 nl drops
works (suggesting that half the protein is lost from such small drops).
Try say 500+1000+500 (don't reduce the volume of seed stock because the
solution that you suspended the crystals in may be important).  Or  
dilute

the protein and use 1000+1000+500.

For the hits from the 450 nl drops you could reduce or dilute the  
protein

by say 25.%.

Or make plenty of seed-stock and try seeding into a random screen again
with larger drops, say 1.5+1+0.5 ul

Those tiny crystals should be good for seeding, don't worry about that
(provided they are protein of course).

Streak seeding may work but bear in mind that roughly a third of the
precipitant comes from the seed stock in your 250 nl drops.

You can add the seed stock with a syringe and needle if you don't have
suitable robot ;)

Experience and data-mining suggests that reducing the salt precipitant  
(in

high-salt drops) or salt additive (in PEG drops) by around 50% may be
helpful too when scaling up - I'm not sure why this works.

Good luck

Patrick






For the hits in the 250 nl drops you are probably losing

On 19 March 2012 20:31, Rajesh kumar ccp4...@hotmail.com wrote:


 Dear All,

I have few papers in hand which  explain me about microseeding, matrix
microseeding, and cross seeding.
I have also read few earlier threads and some more literature in  
google.

Using Phoenix robot, I did a matrix micro-seeding and matrix cross
seeding. I have few hits with this.
In 96 well I used 100+100+50 nL and 200+200+50 nl (protein+screen+seed)
in separate expts.
I have hard time to plan to translate this 96 sitting drop well plate  
to

24 well plate to refine the conditions to get better crystals. only 1-2
hits are small crystals and they are tiny.

 I wonder in 24 well plate, if I should do-
1)  for Example 500+500+50nl (I am sure I cant add less that 500
nL precisely)
2) to a drop of 500+500 nL do microseeding/streaking with a hair

I appreciate if you could advise and share some practical ways to  
further

my experiment.

Thanks in advance
Regards,
Rajesh





--
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 http://www.douglas.co.uk
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--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Directories Projects Spring Cleaning

[Enrico Stura]

Mark,

I am faced with the same problem. I work under Linux.
Since everything depends from the .CCP4 directory,
 this directory  can be copied to
a storage location. Example: /oldproj/dotCCP4_2011:
/home/user % cp -r .CCP4   /oldproj/dotCCP4_2011

Once this directory has been effectively backed up
all old projects can be deleted in the ccp4i interface.
The Spring cleaned .CCP4 can be moved
to a new location for example: /projects/dotCCP4_2012
/home/user % mv .CCP4  /projects/dotCCP4_2012
A logical link is established with:
/home/user % ln -s /projects/dotCCP4_2012 .CCP4
The link will point to the new directory. This new directory
will be filled up during 2012 and at the end of the year
backed up in the same way.

To re-use old environments:
/home/user % ln -s /oldproj/dotCCP4_2011 .CCP4
Will allow to return to the 2011 status.

As long as nothing is deleted, nothing is lost.
Caution: Transfered directories may need logical links to ensure that
connectivity to the directories is maintained.

This is not very elegant and I do this by hand since I am not aware of any
script that does it automatically.
Probably in a future version of ccp4i there will be an archive function.
I am sure that many other users would wlecome it too.


Enrico.

On Wed, 07 Mar 2012 19:55:18 +0100, mark Mayer may...@mail.nih.gov wrote:

I'd appreciate suggestions about how to reorganize my My  
DirectoriesProjectDir listing.
Its currently got several years worth of work and is getting hard to  
work with.
I'd like to archive old projects (say by year) but keep all the CCP4i  
files for each project intact, so that I can easily go back to them in  
the future without having to wade through the current multi year list.


Thanks



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Fwd: HR3699, Research Works Act

[Enrico Stura]

I am strongly in favour of Open Acess, but Open Access is not always helped
by lack of money for editing etc.

For example:
Acta Crystallographica is not Open Acess.
In one manner or another publishing must be financed.
Libraries pay fees for the journals. The fees help the International Union  
of Crystallography.
The money is used for sponsoring meetings, and some scientists that come  
from less rich

institutions benefit from it.

Open Acess to NIH sponsored scientific work will be for all world tax  
payers and tax doggers as well.
OR May be you would suggest that NIH sponsored work should be accessed  
only by US tax payers with a valid social security number?
The journal server will verify that Tax for the current year has been  
filed with the IRS server. A dangerous invasion of privacy!

The more legislation we add the worse off we are.

If the authors of a paper wants their work to be available to the general  
public there is Wikipedia.
I strongly support an effort by all members of ccp4bb to contribute a  
general public summary of their work on Wikipedia.

There are Open Source journals as well.

I would urge everybody NOT to sign the petition. Elsevier will not last  
for ever, and the less
accessible the work that they publish, the worse for them in terms of  
impact factor.
In the old days, if your institution did not have the journal, most likely  
you would not reference the work

and the journal was worth nothing.
We are the ones that will decide the future of Elsevier.  Elsevier will  
need to strike a balance between excellent
publishing with resonable fees or not getting referenced. A law that  
enforces a copyright will not help them.

They are wasting their money on lobbing.

The argument that NIH scientist need to publish in High Impact Factor  
Journals by Elsevier does not hold up:
1) We should consider the use of impact factor as a NEGATIVE contribution  
to science.
2) Each article can now have its own impact factor on Google Scholar,  
independent on the journal it is published in.

3) Even for journals not indexed on PubMed,  Google Scholar finds them.

I hold the same opinion for the OsX debate.
Don't buy Apple! Use linux instead. When enough people protest where it  
really hurts the
company, they will no longer have the money to lobby the American  
Congressmen.
If they make an excellent product, then they deserve the money and quite  
rightly they
can try to build a monopoly around their technology. I fight that, I use  
LINUX.


By signing petitions we acknowledge the power of the legislators. This is  
another form
of lobbing. If we disapprove of lobbing we should not engage in the  
practice even if we give

no money.
We have more powerful means of protest. The 24 Hour shutdown of Wikipedia  
meets my approval.


There is also patenting. How do we feel about it?
Some of the work I have done has also been patented. I do not feel right  
about it.


There is MONEY everywhere. This ruins our ability to acqure knowledge that  
should be free for everybody.

But since it costs to acquire it, it cannot be free.
LAWS should be for the benefit of the nation. But legislators have the  
problem of money to be re-elected.

Can we trust them?
Can we trust their laws?

Companies also play very useful roles. Some companies less so.
But at least they work for a profit and thus they must provide a worth  
while service.

This is not true for politicians.


Enrico.


--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

On Thu, 16 Feb 2012 16:47:26 +0100, Paula Salgado  
p.salg...@imperial.ac.uk wrote:



May I also suggest reading these:

https://intechweb.wordpress.com/2012/01/25/selected-reading-on-research-works-act-why-you-should-care/

https://intechweb.wordpress.com/2012/02/16/open-access-on-a-string-cut-it-and-it-will-grow-back/

Can non-US based scientists sign the petition, btw?


There are also several blogposts and discussions around regarding RWA
and subsequent calls for boycotts of publishers that support it. A few
examples (mostly from the UK):

http://cameronneylon.net/blog/the-research-works-act-and-the-breakdown-of-mutual-incomprehension/

http://gowers.wordpress.com/2012/01/21/elsevier-my-part-in-its-downfall/

http//www.elsevier.com/wps/find/intro.cws_home/elsevieropenletter

http//occamstypewriter.org/scurry/2012/02/12/an-open-letter-to-elsevier/

And my (very) personal views on the matter:
http://www.paulasalgado.org/archives/423

Best wishes
Paula

On 16 February 2012 15:24, Herbert J. Bernstein
y...@bernstein-plus-sons.com wrote:

The bill summary says:

Research Works Act - Prohibits a federal agency from adopting,  
maintaining,
continuing, or otherwise engaging in any policy, 

Re: [ccp4bb] Fwd: HR3699, Research Works Act

[Enrico Stura]

Charlie,

A much more balanced view than others have posted.

NIH Open Access requirement is a vast  overreach.

I agree.

HR 3699 appears to be as deeply flawed.

It could be made better with amendments?

Enrico.

On Thu, 16 Feb 2012 17:06:24 +0100, Charles W. Carter, Jr  
car...@med.unc.edu wrote:


For what it's worth, my own experience with the issue of scholarly  
publication and open access is nuanced enough that perhaps my two-bits  
worth can add to this discussion. In short, I agree both with Ian's  
previous message and with Herbert, and feel that the incompatibility  
between them goes to the root of a problem for which the answer is  
certainly not quite there.


I have been much influenced by the work done on this issue by Fred  
Dylla, Executive Director of the American Institute of Physics. Here is  
a link to recent information concerning his four-year effort to reach  
consensus on this issue:


http://www.aip.org/aip/aipmatters/archive/2011/1_24_11.html

I personally think that the NIH Open Access requirement is a vast  
overreach. PubMed Central is very difficult to use and ultimately has  
never satisfied me:  I always go to the UNC library holdings. There are  
several reasons why. The most immediate is that PubMed Central almost  
never gives a satisfactory copy of a paper I want to read, and the most  
serious reason is that I am convinced that the overhead exacted on  
authors and PIs by the NIH means that few, if any authors give much more  
than a glance in the direction of updating deposited manuscripts from  
journals that do not automatically deposit the version of record. For  
this reason, many PubMed Central entries are likely to have more than  
minor errors corrected in proof only in the version of record. I don't  
personally see any way around the problem that there is only one version  
of record and that version is the one for which copyright is retained by  
the publisher.


On the other hand, I am deeply sympathetic to the argument that  
publicly-funded research must be freely accessible. After talking  
intensely with the library administrators at UNC, I also believe deeply  
that university library subscriptions satisfy the need for open access.  
Casting aside for the moment the issue of Open Access journals, whose  
only real difference lies in who pays the costs of publication, I have  
long believed that careful validation through peer review constitutes  
serious added value and that journals are entitled to being paid for  
that added value. What makes this issue more difficult for me is that I  
share with many the deep suspicions of corporate (as opposed to Member  
Society) publishing organizations. Several years ago I withdrew my  
expertise from the Nature group in protest over what I felt (after,  
again, long discussions with our UNC librarians) was a power play  
designed only to weaken the library systems. I have similar views about  
Elsevier.


Finally, I am inclined to sign this petition for other reasons,  
including the fact that HR 3699 appears to be as deeply flawed in the  
other direction as the original enabling legislation that vested such  
power in the NIH and, in the same act, all but eliminated any opposition  
by diluting responsibility for compliance to the fullest possible  
extent, by penalizing PIs for non-compliance. When I first read of this  
petition, I was deeply incensed that the wing nuts in Congress would  
craft a bill so obviously designed to reward the 1%, so to speak.


In closing, I earnestly recommend that as many of you as possible look  
into Fred Dylla's work on this issue. The AIP is a publisher whose only  
revenue other than philanthropy comes from the intellectual property and  
added value of its journals, some of which represent the finest in  
physical chemistry relevant to our community. Dylla deserves kudos for  
his effort to find consensus, something that seems to have gone way out  
of fashion in recent years.


Charlie



On Feb 16, 2012, at 10:37 AM, Ian Tickle wrote:


Dear Herbert

Thanks for your detailed explanation.  I had missed the important
point that it's the requirement on the authors to assent to open
access after a year, which the proposed Bill seeks to abolish, that's
critical here.

I will go and sign the petition right now!

Best wishes

-- Ian

On 16 February 2012 15:24, Herbert J. Bernstein
y...@bernstein-plus-sons.com wrote:

The bill summary says:

Research Works Act - Prohibits a federal agency from adopting,  
maintaining,
continuing, or otherwise engaging in any policy, program, or other  
activity

that: (1) causes, permits, or authorizes network dissemination of any
private-sector research work without the prior consent of the  
publisher; or

*(2) requires that any actual or prospective author, or the author's
employer, assent to such network dissemination. *

Defines private-sector research work as an article intended to be
published in a scholarly or scientific 

Re: [ccp4bb] surface residue mutation

[Enrico Stura]

Dear All,

One of the most efficient methods to change space group and packing  
without having to change

the sequence is to change the length of N and/or C terminal tags.

An example that I am familiar with is given by the following PDB codes.

1JIZ, 1RMZ, 1JK3,  1UTT, 1UTZ, 2WOA, 2W0D, 1ROS, 1OS9, 3BA0

It includes 1 surface residue mutation, but the rest are small variations  
in length.


Complexation with any ligand that may protrude is also likely to work.

Enrico.

On Wed, 15 Feb 2012 01:35:36 +0100, Bernhard Rupp (Hofkristallrat a.D.)  
hofkristall...@gmail.com wrote:




http://services.mbi.ucla.edu/SER/


but no space group predictions are possible. BR


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of  
Prem

Kaushal
Sent: Tuesday, February 14, 2012 3:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] surface residue mutation





Hi

We have a protein that crystallized in P21212 space group. We are looking
for some different crystal forms. We tried few things did not work. Now  
we
are thinking to mutate surface residues. Anybody aware of any software  
which

can predict the mutations that might help in crystallizing protein in
different space group, please inform me.

Thanks in advance

Prem





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Why don't small crystals dissolve

[Enrico Stura]

Pius,

The situation you describe is an off-equilibrium situation. You have  
applied  a perturbation

and that may not be reversible!

Enrico.


On Wed, 08 Feb 2012 16:35:56 +0100, Pius Padayatti ppadaya...@gmail.com  
wrote:



Hi Enricho,
The scenario of streak seeding follows Ostwald ripening but will
this happen in other situations as follows

But in a special case where you have some crystals that appear as large
rods which dissolved when taken out of the incubator (or) during the
observation( these were antibody-complex crystals which were grown in
bicelles(DMPC:CHAPSO
and detergent mixtures and cholestrol, conditions citric acid pH 4.5,
with 2.4 M ammonium sulfate).
The crystals re-apparered in a day over noght incuabtion as heavy
showers of needles with heavy precipitate around.

Very hard to reproduce the conditions.
Still trying to work around these conditions.

Would like to know your thoughts if this is against the laws small
crystals to large crystals (energetically
favoured) conditions.

Also any suggestions welcome for improvements.

Pius



The answer to your question is very simple.  Small crystals will  
dissolve

when the degree of saturation
of the solution becomes too low to support their relatively high  
surface to

volume ratio.
The larger crystals will still continue to grow because of their higher
surface/volume ratio but will do so slowly.
I have achieved the dissolving of small crystals in favour of large ones
only once with 10 µl drops.
While it is difficult to achieve this with spontaneously nucleated  
crystals,

with seeding thing are very different.
This phenomenon is an every day observation if you use streak seeding on
drops that have been
equilibrated for different amount of time against different  
concentrations

of precipitant and you can
also add an additional variable by using different ratios of protein to
precipitant in the drop.
The goal is to seed at a low degree of superstauration. The small seeds  
will

be visible along the streak
immediately after seeding. When you look later on you will see only the
bigger crystals.
Streak seeding is great if you want to play this game.

Enrico.


On Wed, 08 Feb 2012 12:08:23 +0100, Theresa H. Hsu  
theresah...@live.com

wrote:

A little off from the original question. Why don't small crystals  
dissolve
to make a bigger crystal, especially when the small ones grow on top  
of each
other? Can the clustered 3D crystals (I think it is called macroscopic  
twin)

be used for full data collection?

Again, thank you.

Theresa




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90  
71







--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Freezing crystal

[Enrico Stura]

BIGGER is not always BETTER?

Theoretically it should be better because you have more scattering matter.  
If it is

not something has gone wrong in prior steps:
Purification: You were less selective and picked up more heterogeneous
protein.
Crystallization: The bigger crystals grew under conditions that were less
controlled because of changes in the state of protein supersaturation,  
precipitant or
temperature. These inhomogeneities contributed to give you bigger, but not  
better

crystals.
Cryo-soak: Bigger crystals are more prone to be shocked when transfered to  
a
less than optimal cryo-solution. This is a critical step, and crystals do  
not like too
much the cryo-chemicals. To test this I tried lower concentrations of  
various

cryo-compounds instead of a huge quantity of a single component and in most
cases the mixture was better tollerated than the single components.
Flash-freezing: Bigger crystals will cool more unevenly than small ones. A  
change from
liquid nitrogen to liquid ethane could achieve faster cooling because of  
the

greater heat capacity of the latter liquid.

Large crystals are more difficult to handle than small ones but after  
experimenting with
small ones we can build up a good experimental protocol so that the big  
crystals will give exceptionally
good results. I compared small crystals on high intensity beamlines at the  
ESRF against
large crystals on BM30 and the big crystals were statistically better.  
Unfortunately, it takes
a lot of time and a certain amount of expertize to optimize the  
conditions. So ...  although I

strongly disagree with Jürgen, I will also advise to start with small ones.

Enrico.

On Tue, 07 Feb 2012 16:58:04 +0100, Bosch, Juergen jubo...@jhsph.edu  
wrote:


Something to add into this discussion is also go fro the tiny crystals  
versus the big ones.
BIGGER is not always BETTER - in particular if you try to freeze  
directly out of your conditions without an additional cryo-protectant.  
And with small or tiny I mean 10 micron, whatever you are capable of  
mounting. It is also important to keep the amount of liquid volume  
around the crystal low, so rather use a loop in which you scoop the  
crystal up instead of having a large loop with lots of liquid.


Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2  
leads to a quicker freeze of your material.


If you have the option to anneal your crystal after testing it in the  
beam try it out and assess the success or damage, this will be very  
different depending on what cryo-additives you have around.


Good luck,

Jürgen

On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote:

One last thing--sometimes crystals can be frozen as is, particularly
if you use mitegen mounts and get nearly all of the mother liquor off
the crystals by dabbing the loop on the dry surface next to the drop
several times. So simple it is always worth a try

JPK

On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij  
mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote:
Rationalising it completely may only be possible once you know the  
nature of the crystal contacts, i.e. when you have solved the structure.  
Until then it is mainly a matter of experimenting.


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Theresa H. Hsu
Sent: Monday, February 06, 2012 11:00 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Freezing crystal

Hi all

Thanks for all the suggestions which I will try soon.

How do the crystallization condition (PEG vs. salts like ammonium
sulfate) affect the croyprotectant condition? Do factors like presence
of low concentration of high molecular weight PEG ( 2000) mean PEG is
better? Do buffers and salts in protein also important?

Trying to rationalize it :)

Theresa



--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu
***

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://web.mac.com/bosch_lab/







--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 

Re: [ccp4bb] Freezing crystal

[Enrico Stura]

Theresa,

Several suggestions that have been given are excellent advice:
Li salts suggested by  Tommi Kajander is what I would use
in particular:
80% saturated lithium sulfate.
This should work. I would be very surprised if it does not.

Malonate as suggested by Sean Seaver is another great idea, but difficult  
to prepare

see ccp4bb by Doug Ohlendorf :
Malonic acid is dissolved in water and then pH adjusted to the desired  
value with NaOH. Caution: dissolving malonic acid is highly exothermic.  
Do it slowly, in a hood.


In general terms what needs to be considered is that
when one introduces a cryoprotectant it is important to
match the precipitating power of the precipitant.
If you just add glycerol, as suggested by some, you will tend to dissolve  
your

crystals with respect to 2M ammonium sulfate.

Ideally in a balanced cryoprotectant the mild solubility
enhancing effect of the glycols should be counteracted by
the addition of MPD or DMSO that can act as precipitants.
Therefore, an approach using a complex mixture of
glycols and cryoprecipitants should yield improved
results.

I have assembled a kit initially for my own personal use
that creates mixtures respecting these principles.
CEA Saclay has licenced it to Molecular Dimensions.
The details of the components, the mixtures and their use
are freely available:
http://www.moleculardimensions.com/applications/upload/CryoProtX.pdf - for  
the product flyer and:
http://www.moleculardimensions.com/shopdisplayproducts.asp?id=201cat=CryoKits  
- to get more info and order the product.


What I prefer about Lithium sulfate compared with malonate is that I just  
transfer the crystals in the 80% saturated Li2SO4
without bothering about the pH (no buffer) and it works. You may want to  
transfer with a capillary rather than a loop to avoid

shocking the crystals, but for the rest it should give good results.

Enrico.




On Sun, 05 Feb 2012 23:49:25 +0100, Theresa H. Hsu theresah...@live.com  
wrote:



Hi all

Is there a list of conditions to be tried *first* for cryoprotectant? My  
crystals diffract at room temperature capillary but no in 30% PEG 400.  
Crystals are from 2 M ammonium sulfate.


Thank you.

Theresa



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Disappearing crystals

[Enrico Stura]

Dear Christine,

I had guessed that you had more salt in the protein solution
than in the reservoir with a relatively  low protein concentration.

The protein is in:
100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration  
of ~4.3mg/mL.

 and the reservoir is:

(0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and
30% PEG MME 550).


We can suspect that no matter what temperature the equilibrium will move
towards dilution in the drop.

Basic suggestion:
Set up a range from 20-30% PEG MME 550
with at least 150mM NaCl in the reservoir.

Fine tuning:
Add high MW PEG (10K or 20K 1-5%) it should have a stabilizing effect
Try a range of salts apart from NaCl: The clasics are: Li2SO4, KSCN, MgCl2.
Try additive amounts od MPD 0.5-3%.
Try a small tight range of various pH.

Enrico.



On Mon, 28 Nov 2011 21:43:51 +0100, Harman, Christine  
christine.har...@fda.hhs.gov wrote:



Hi,
Thank you so much for your replies.  A lot of you have mentioned  
fluctuations in temp as the major contributor.  And a few of you have  
asked for more details of my protein/buffer and set up.  To my  
knowledge, the tray has been kept at constant 20 C (in an incubator)  
with exception of course to when I remove the tray to view the drops.   
It could be possible that my inspection of the tray might have  
contributed to an increase in temp, but only temporarily.  I am very  
careful about the time the drop sees intense light, but it is possible  
the temp could have changed enough to cause this problem.  Just to give  
a few more details.  My protein (a Fab fragment/peptide complex  
(hopefully) is in buffer containing 100mM Sodium Acetate pH 5.5 with  
150mM NaCl at a protein concentration of ~4.3mg/mL.

  After setting up my
drop with reservoir solution I add NaCl to well to give ~75mM NaCl to  
match ionic strength of protein in drop which is diluted 1:1 with well  
solution.  I do hope this problem is temperature.  Although I am a  
little sad to not be able to freeze those crystals I did see, I still  
consider myself lucky to get such good result from a condition right  
from the screen so there will be some definite optimization set ups with  
this condition.  Could I safely say though that the crystals I observed  
are not salt..:)  I guess that is one good thing to take from this.  Any  
more suggestions on optimization would be very welcomed.


Thanks again to all you,

Christine



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of  
Enrico Stura

Sent: Monday, November 28, 2011 1:45 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Disappearing crystals

When advice on crystallization is needed, it is important to give details
of
the protein concentration, the buffer the protein is in as well as the
method
used to grow the crystals.

Problem: The crystallization conditions are essentially low salt: 100mM
buffer
and only 50mM CaCl2. So the buffer that the protein is in is very
important !!
Fluctuation in the reservoir/drop environment will lead to crystals
dissolving.

Solution: Balance the salt in the reservoir and in the  
protein:precipitant

drop and make sure
the temperature is kept constant.

Since I do not have all the necessary information, the diagnosis and the
solution proposed
are likely to be wrong!

Enrico.

On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin yj...@brandeis.edu wrote:


On 11/28/11 12:04 PM, Harman, Christine wrote:

Hi All,
I have just noticed a very strange thing and need some help in
understanding it.  I recently found two crystals in a condition from a
screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and
30% PEG MME 550).

  The small crystals appeared after a month and

started to grow over the next 5 days after I first saw them (see
pictures attached).  I just check the same drop today and now the
crystals are gone.  So I was wondering what happened and if anyone
experienced this before.  Any insight or advice on what to do would be
greatly appreciated.
Thanks
Christine
Small   5 days later

Hi Christine,
I had similar experience. In my case, another crystal showed again with
different size a few days later. Sometimes, it seems like it is a common
event to others as well as I heard although my case only takes about a
week to be crystallized.  I'd rather wait or just set up again or in a
slightly different way.

Wish you well.

Young-Jin







--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr

Re: [ccp4bb] Disappearing crystals

[Enrico Stura]

When advice on crystallization is needed, it is important to give details  
of
the protein concentration, the buffer the protein is in as well as the  
method

used to grow the crystals.

Problem: The crystallization conditions are essentially low salt: 100mM  
buffer
and only 50mM CaCl2. So the buffer that the protein is in is very  
important !!
Fluctuation in the reservoir/drop environment will lead to crystals  
dissolving.


Solution: Balance the salt in the reservoir and in the protein:precipitant  
drop and make sure

the temperature is kept constant.

Since I do not have all the necessary information, the diagnosis and the  
solution proposed

are likely to be wrong!

Enrico.

On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin yj...@brandeis.edu wrote:


On 11/28/11 12:04 PM, Harman, Christine wrote:

Hi All,
I have just noticed a very strange thing and need some help in
understanding it.  I recently found two crystals in a condition from a
screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and
30% PEG MME 550).  The small crystals appeared after a month and
started to grow over the next 5 days after I first saw them (see
pictures attached).  I just check the same drop today and now the
crystals are gone.  So I was wondering what happened and if anyone
experienced this before.  Any insight or advice on what to do would be
greatly appreciated.
Thanks
Christine
Small   5 days later

Hi Christine,
I had similar experience. In my case, another crystal showed again with
different size a few days later. Sometimes, it seems like it is a common
event to others as well as I heard although my case only takes about a
week to be crystallized.  I'd rather wait or just set up again or in a
slightly different way.

Wish you well.

Young-Jin




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Crystalization in low PH

[Enrico Stura]

I have crystallized in PEG with citrate at pH 3. If you want to go lower
I would suggest maleate:

effective pH range   pKa 25°Cbuffer
1.2-2.6   1.97   maleate (pK1)
2.2-6.53.13  citrate (pK1)

Enrico.


On Mon, 07 Nov 2011 14:15:02 +0100, Craig A. Bingman  
cbing...@biochem.wisc.edu wrote:


I'm not convinced that you need a conventional buffer at pH 2 or 3.  At  
pH 2, the hydrogen ion concentration is 10 mM.  If you want to use  
something else, the second pKa for sulfuric acid is around 2.  The first  
pKa for phosphoric acid is slightly higher than 2.  Lactic acid has a  
pKa close to 3.  Formic acid has a pKa just under 4.  Most of these  
numbers were in an appendix in the first chemistry text you ever used.   
wink  These numbers imply pretty strongly that most crystallization  
screens emphasizing common salts will require determined modification to  
hit these low pH values, because many stabilizing anions in the  
Hoffmeister series will be partially or completely protonated at these  
pH values.  PEG and organic screens will require a smaller hammer to  
retrofit.


On Nov 6, 2011, at 11:19 PM, Sam Arnosti wrote:


Hi everyone

I have a protein that is extraordinarily stable at PH=3.0 or even 2.0.

I want to crystallize it in the  low PH and compare the differences  
between the crystals in regular PH and low PH.


I was wondering how people set up the boxes in low PH, as usual buffers  
are mostly less acidic.


Regards

Sam



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] IUCr committees, depositing images

[Enrico Stura]

With improving techniques, we should always be making progress!
If we are trying to answer a biological question that is really important,  
we would be better off
improving the purification, the crystallization, the cryo-conditions  
instead of having to rely on

processing old images with new software.

I have 10 years  worth of images. I have reprocessed very few of them and  
never made any
sensational progress using the new software. Poor diffraction is poor  
diffraction.

Money can be better spent buying a wine cellar, storage works for wine.

Enrico.

On Tue, 18 Oct 2011 14:51:27 +0200, Boaz Shaanan  
bshaa...@exchange.bgu.ac.il wrote:



Hi Felix,

Excuse my question, but what have you discovered about lysozyme that we  
haven't already known before which justifies all these efforts?
After all, we're mostly after finding solutions to biological problems,  
aren't we?


  Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Felix  
Frolow [mbfro...@post.tau.ac.il]

Sent: Tuesday, October 18, 2011 1:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] IUCr committees, depositing images

I could not agree less. There is constant development of the software  
for refinement that allow to do things that were not
possible or were not necessary  in the past such as intelligent  
refinement of occupancies of mutually exclusive sites, entities and  
conformations.
I frequently remeasure lysozyme crystals. I use them as a test system  
for the beam lines, new detectors, novel software developments,  
refinement improvement etc. Sometimes I am collecting data in quite  
different wavelength than of existing structures. And what about  
diffraction  data from a chemically modified lysozyme molecule?
They are good data that show evolution of the beam line stations if they  
are keeper in historical order.

To store them all, or not to store at all…
Storage of the diffraction data is not a drinking club with muscle-bound  
selectors outside :-)

Felix Frolow
Dr Felix Frolow
Professor of Structural Biology and Biotechnology
Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On Oct 18, 2011, at 12:52 , Chris Morris wrote:

Some crystals are hard to make, so storing all the data the best way to  
get reproducibility. On the other hand, no one needs more images of  
lysozyme. So using the same standard for every deposition doesn't sound  
right.


The discussion should be held on the basis of overall cost to the  
research budget - not on the assumption that some costs can be  
externalised. It is too easy to say you should store the images, in  
case I want to reprocess them sometime. IT isn't free, nor is it  
always cheaper than the associated experimental work. The key  
comparison is:


  Cost of growing new crystals + cost of beam line time

With:

  Cost of storing images * probability of processing them again

At present, detectors are improving more quickly than processing  
software. Sample preparation methods are also improving. These forces  
both press downward the probability that a particular image will ever  
be reprocessed.


regards,
Chris

Chris Morris
chris.mor...@stfc.ac.uk
Tel: +44 (0)1925 603689  Fax: +44 (0)1925 603634
Mobile: 07921-717915
Skype: chrishgmorris
http://pims.structuralbiology.eu/
http://www.citeulike.org/blog/chrishmorris
Daresbury Lab,  Daresbury,  Warrington,  UK,  WA4 4AD



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Precipitating Crystallization Condition

[Enrico Stura]

When you give the composition of the screen condition, you must also give  
the conditions of

the protein buffer, since you get crystals when the two are mixed.

If you analyse many small salt crystals by SDS-PAGE you may still get  
staining since protein

will precipitate on the salt crystals.

Enrico.


On Tue, 27 Sep 2011 18:02:18 +0200, Mark J van Raaij  
mjvanra...@cnb.csic.es wrote:



Hi Christopher,
you'll have to try to find out how they mix the components at MD and if  
they pH the solution afterwards or before (or not at all...) and to  
which pH. You can ask them; or try different mixing/pHing protocols on a  
small scale and see if one works in keeping all in solution.

Mark

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/content/research/macromolecular/mvraaij





On 27 Sep 2011, at 17:55, Browning Christopher wrote:


Dear All,

My question might be a bit out of place, but perhaps someone can help.  
I've screened my protein in the Nuc-Pro screen from Molecular  
Dimensions and found some crystals in condition B10. They seem to be  
protein crystals as they are not highly optically active and look  
different to salt crystals. So condition B10 consist of 10% PEG 4K,  
50mM Imidazole pH 7.2, 20mM Zinc sulfate. In the screen, this condition  
is perfectly clear, but when I try and make my own screen, the whole  
solution turns white. Apparently, Zinc sulfate/Imidazole can be used  
for staining SDS-gels, but that does not really help my a lot. Does  
anybody have an idea how I might be able to keep everything in  
solution, seeing that the MD guys managed somehow.. I'm a bit  
desperate as I don't have many hits for this protein.


Cheers,

Chris B

--
Dr. Christopher Browning
Post-Doctor to Prof. Petr Leiman
EPFL
BSP-416
1015 Lausanne
Tel: 0041 (0) 02 16 93 04 40



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Precipitating Crystallization Condition

[Enrico Stura]

The PEG could also be the problem, so you can mix your stock solutions by  
the method described

below and end up with the same result as soon as you add the PEG.
See:
Frances Jurnak: Effect of chemical impurities in polyethylene glycol on  
macromolecular crystallization

Journal of Crystal Growth
Volume 76, Issue 3, 2 August 1986, Pages 577-582

Enrico.



On Tue, 27 Sep 2011 18:43:09 +0200, Roger Rowlett rrowl...@colgate.edu  
wrote:


Imidazole is basic. If you mix it directly with zinc salts you will get  
zinc
hydroxide. Most screens are prepared by mixing stock solutions. In your  
case

I would make up the screen from 1 M imidazole, pH 7.2, 1 M ZnSO4, and 50%
PEG-4000. Add the PEG last. This should work. You may or may not need  
this
specific salt or buffer to get crystals, and could swap them out or  
change

concentration as required. 50 mM buffer may not be sufficient to control
condition pH depending on the protein storage buffer composition.

Roger Rowlett
On Sep 27, 2011 11:56 AM, Browning Christopher 
christopher.brown...@epfl.ch wrote:

Dear All,

My question might be a bit out of place, but perhaps someone can help.
I've screened my protein in the Nuc-Pro screen from Molecular Dimensions  
and

found some crystals in condition B10. They seem to be protein crystals as
they are not highly optically active and look different to salt  
crystals. So

condition B10 consist of 10% PEG 4K, 50mM Imidazole pH 7.2, 20mM Zinc
sulfate. In the screen, this condition is perfectly clear, but when I try
and make my own screen, the whole solution turns white. Apparently, Zinc
sulfate/Imidazole can be used for staining SDS-gels, but that does not
really help my a lot. Does anybody have an idea how I might be able to  
keep
everything in solution, seeing that the MD guys managed somehow..  
I'm a

bit desperate as I don't have many hits for this protein.


Cheers,

Chris B

--
Dr. Christopher Browning
Post-Doctor to Prof. Petr Leiman
EPFL
BSP-416
1015 Lausanne
Tel: 0041 (0) 02 16 93 04 40



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Complex seeding

[Enrico Stura]

The idea is not at all crazy. In a sense it is quite similar to  
Stoichiometric variation screening* if you consider that the lattice  of  
the crystallized subunit may contain planes

that might be conserved in the crystal of your hope for 3 protein complex.

*Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G.,  
Silverman, G.J., Charbonnier, J.-B. (2001)
Crystallization of macromolecular complexes: Stoichiometric variation  
screening. J. Cryst. Growth 232:580-590.


Yet if the size discrepancy is quite large, the chances that will work  
will be quite slim.

Good luck,

Enrico.

On Wed, 21 Sep 2011 19:04:43 +0200, Peter Hsu hsuu...@u.washington.edu  
wrote:



Hi all,

I've been trying to crystallize a 3 protein complex recently with little  
success. However, crystals of each subunit have previously been  
crystallized. I was wondering if any one knows of any  
literature/experiences where people have used seeds from an individual  
subunit to seed for a complex and succeeded? Or is this just a crazy/bad  
idea?


Thanks in advance for any input.

Peter



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] crystallization of complex and ...

[Enrico Stura]

Dear Min,

Regarding the stoichiometry that you should use in crystallizing two  
proteins

that form a complex. I have looked at this question before. See:
Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G.,  
Silverman, G.J., Charbonnier, J.-B. (2001)
Crystallization of macromolecular complexes: Stoichiometric variation  
screening. J. Cryst. Growth 232:580-590.

http://www.sciencedirect.com/science/article/pii/S0022024801011721

Briefly: The stoichiometry can, and should, be varied in your screening.

The relative protein solubility of the two individual proteins and the  
solubility of the complex should be
analysed under various potential crystallization conditions. Conditions  
where the complex is less soluble
than the individual ptoteins should be chosen if the complex has a  
tendency to dissociate.


Since both  proteins have been crystallized before you may also use
crystals of both the free forms of the two proteins to stimulate  
nucleation of the complex

The final  composition of the asymmetric unit may include free poteins as
well as the complex.

The paper will give you a lot more methodology to use to obtain crystals  
of the complex, if

such complex really exists, is homegeneous and relatively stable.

Enrico.


On Fri, 16 Sep 2011 04:20:26 +0200, m zhang mzhang...@hotmail.com wrote:



Dear all,
I have two questions:
First, I was trying to crystallize a complex of two proteins. Both  
proteins has been crystallized before. The two proteins bind to each  
other based on Biacore study, but they didn't form a single peak on gel  
filtration. When I mixed them at 1:1 ratio, the crystals I got contain  
only one of the two proteins. I was suggested to increase the ratio, for  
example 1.5:1, to increase the probability of co-crystallization which I  
will try. But I do want to hear if there are other possible ways to try.  
What would you try if you were in my situation?
Second is about reusing of Ni-NTA resin. According to Qiagen's  
instruction, after using fresh Ni-NTA resin, one only needs to wash the  
used Ni resin first with 0.5M NaOH, then with your own buffer. After  
that the resin is ready to be reused until it needs being recharged. But  
my question is: Once immidazole competes with His-tagged protein and  
binds to Ni-resin, how can immidazole be rinsed off with the same  
buffer(usually pH is above 7) one uses to purify the protein?

Thank you for any suggestion or comment.
Min




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] drops swelling

[Enrico Stura]

Anita,

see the message I posted yesterday on the CCP4bb:
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg22638.html

or google stura glycerol
Re: [ccp4bb] How to help crystal grow bigger

In your case the sentence you need is:
You need to have a higher (double) glycerol concentration in the  
reservoir else you will risk finding that your drops will get biggger and  
not smaller. This note of caution applies to vapour diffusion set ups as  
equilibration can be tricky in such context: Vera,L., Czarny, B.,  
Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in  
Protein Crystallization. Cryst. Growth  Des. 11 :2755–2762. 

http://pubs.acs.org/doi/abs/10.1021/cg101364m

Enrico.


On Fri, 09 Sep 2011 13:22:53 +0200, anita p crystals...@gmail.com wrote:


Dear Crystallographers,
I have set hanging drop trays with 2ul of protein and 2 ul of resorvior
solution. I have seen in some cases the drops are swelling. My protein
buffer has 15% glycerol in it.
This is happening mainly when I have peg 400 or peg MME or MPD or  
Jeffamine

in the buffer condition.
Could any one suggest a remedy for this.

with regards
Anita



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] How to help crystal grow bigger

[Enrico Stura]

SnowDeer,

Additives are indeed a good idea, make sure you do it in an informed  
manner, each additive is different and you will
get a benefit only if you know how to do it right. I do not recommend a  
random approach.


For example, you mention glycerol. There is a lot to know on the subject  
and probably more to discover.
Glycerol will help freezing/thawing cycles and improve protein stability  
if you want to work at higher temperatures than
4°C. You should check increased stability effect out since it is easier to  
work outside a cold room.
I assume you are trying to set up your drops at the same temperature at  
which they will equilibrate. Which is a good thing to do.


Yet, plan your experiments carefully. Also look at previous messages on  
ccp4bb on the subject of glycerol
in particular regarding propanediol. Annie Hassell among others has posted  
some very good comments. This bullettin board
has been very keen on the subject in the past, so you can learn a lot from  
the archives.


Glycerol is also great to reduce nucleation. If you decide to add glycerol  
to the protein solution (for solubility, but in your case it might be for  
stability
reasons), you also need to have a higher (double) glycerol concentration  
in the reservoir else you will risk finding that your drops will get  
biggger and
not smaller. This note of caution applies to vapour diffusion set ups as  
equilibration can be tricky in such context:
Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011)  
Practical Use of Glycerol in Protein Crystallization. Cryst. Growth  Des.  
11 :2755–2762.

http://pubs.acs.org/doi/abs/10.1021/cg101364m

Good luck, but most important work with precision. To go from small to big  
crystals you need to be very
precise on how you set up your experiments and understand the rate at  
which you equilibrate your drops.



Enrico.

On Thu, 08 Sep 2011 09:52:40 +0200, SnowDeer huanxu...@gmail.com wrote:

To Boaz: I seperated the large crystals and checked its diffraction  
already.

While the diffraction is quite poor, only several dots could be seen. T_T
I washed the seeds twice with my buffer and seed the drop immediately  
after

setting it. Thanks for your advices and I will try the additives.

To Charles, Enrico, Bernie  Tiantian: Thanks for your kindly advices, I  
set

different conditions for the buffers with glycerol and different
protein/reservoir volume ratios already following your instructions. :)

To David: Hmm...my crystals are smaller than the smallest loop T_T. It's
quite hard for me to pick them up (due to my clumsy fingers...lol).  
Thanks

for your advices and the review.

I have another question: I usually stored my protein samples aliquots at  
-80
degree and thaw the small aliquots when I need to use. While my senior  
said
it will harm the protein so she suggested to keep them at 4 degree. So  
it is

possible that I got the small crystals coz the freezing and thawing alter
the proteins?

Thanks very much.
SnowDeer





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] How to help crystal grow bigger

[Enrico Stura]

dear SnowDeer,

The first step to see if bigger crystals can be grown is to use bigger  
protein drops and vary
the precipitant/protein ratio at the beginning of the vapour diffusion  
experiment.

You can work out for yourself why this
should give you all the informations that you need in subsequent  
experiments.


Enrico.


On Mon, 05 Sep 2011 10:06:22 +0200, SnowDeer huanxu...@gmail.com wrote:


Dear All:

Recently I am working on a protein which can already grow nice  
pyramid-like
crystals after the condition was optimized, while the crystals are too  
small
to be picked up. The crystals grew quite fast and densely, so I tried to  
put

100ul paraffin oil inside the 600ul reservoir solution or put the plate
under 16 degree to slow down the evaporation, while the crystals were  
still
the same. I also tried macro or micro seeding with or without the  
paraffin

oil. Macroseeding would give a larger crystal (not very nice) with many
small crystals in the drop even I washed the seeds carefully. For
microseeding, the same small crystals grew.

I don't have many experiences in crystallography, so I have no idea how  
to

make it grow bigger...

Any suggestion is most welcome.

Thanks.
SnowDeer



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo

[Enrico Stura]

Chris,

Crystals can be very tollerant of cryo-solutions that respect the degree  
of hydration

of the lattice. You can use any cryo-solution you want, including oil.
Try 10% Di-ethylene glycol, 10% 1.2-propanediol, 10% glycerol, 10% PEG  
10K, 10% buffer solution, 1M NaCl

(pH close to  the one you use now; slightly higher or lower may help).
1M NaCl should provide enough ionic strength for the duration of the  
cryo-soak

to prevent the crystals from dissolving.
Test one crystal in this solution. If the crystals crack reduce the PEG  
10K concentration

if they dissolve increase the NaCl concentration or try:
Try 8% Di-ethylene glycol, 8% 1.2-propanediol, 8% glycerol, 8% PEG 10K, 8%  
MPD  10% buffer solution,  1M NaCl.


As already suggested cryosalts may work, but  Ammonium di-hydrogen  
phosphate 0.4M is on the low side
as far as ionic strength is concerned and if the crystals are very big  
they are likely to crack. No problems

if you have needles or smallish crystals.

Enrico.


On Thu, 26 May 2011 13:01:08 +0200, herman.schreu...@sanofi-aventis.com  
wrote:



Dear Chris,
I have not used this particular condition, but as a rule of thumb I use
like with like, e.g. glycerol, ethylene glycol, PEG400 etc. with PEG
conditions, and saltlike compounds (sucrose, xylitol, salts) for salt
conditions. In your case I would try glucose or xylitol or just look
what happens if you increase the ammonium phosphate concentration to say
2M without adding glycerol.

Good luck!
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Chris Ulens
Sent: Thursday, May 26, 2011 12:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate
cryo

Hi,
I would like to get recommendations for a proper cryo solution for a
crystallization hit from the Hampton crystal screen Ammonium di-
hydrogen phosphate 0.4M. I tried increasing glycerol up to 30% with the
same ammonium phosphate concentration or increasing glycerol up to 30%
in the presence of 1.3M ammonium phosphate. Both gave iced up drops (I
only tried quick and dirty tests by dipping a cover glass in liquid
nitrogen).

Thank you.
-Chris



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Peptide Crystallization

[Enrico Stura]

I would discourage using pre-made screens on a project outside the norm.

The main problems are:

Zinc: At mM concentrations will easily crystallize and give false positives
in many screens. It will also act as a precipitant for the peptide.

The best approach would be to separate the zinc adduct from the  
non-complexed

to avoid heterogeneity.

 During crystallization to avoid loosing the zinc it might be good to
add 50-200mM imidazole with 0.5-10mM zinc. You can either
add this to each precipitant solution (best) or to the peptide solution
(dangerous as it may all precipitate).

Then  you can use precipitants except phosphate as it will give false  
positives
avoid EDTA and other strong chelators (but weak chelators like imidazole  
will be good).


If there are specific problems, many from CCP4BB will provide suggestions.

Enrico.

On Wed, 25 May 2011 14:04:23 +0200, Buz Barstow b...@mac.com wrote:


Dear all,

I am considering trying to crystallize a small peptide (around 15 amino  
acids). The peptide is soluble in neutral water or buffer (pH 7.0) until  
at least 10 mM (13 mg/mL), and adopts a turn conformation when bound to  
Zn.


What are your thoughts on attempting this?

If you think that it is worthwhile, what crystallization conditions  
would you try? I am thinking of a sparse matrix screen using the Hampton  
Crystal Screen 1 and 2 kits, using hanging drop crystallization in  
Hampton Vdx trays.


Thanks! and all the best,

---Buz



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Detergents

[Enrico Stura]

Rashmi,

Yes, you should use the needle for seeding.
The first thing to do is to seed your control drop to make sure that the  
needles
are not bicine, as this buffer can give needles at certain pH at high  
PEG400

concentrations.
Next (or at the same time) also seed other protein drops that you have  
already set up
under similar conditions. I recommend streak seeding, so that even if  
crystal growth

is slow you will see the streak line early on.

Enrico.

On Fri, 29 Apr 2011 12:43:21 +0200, anita p crystals...@gmail.com wrote:


Hi,
I had set up crystallization with a bicine  as buffer and peg 400 as
precipitant. I used the detergent DDAO/LDAO as an additive to the
crystallization drop (one of the hampton additive screen condition, it  
says

5% on the vial)
I have a clear drop and in the centre there is a shiny precipitate (looks
like granules attached to eah other).
Then I opened the drop to touch those granules with a needle, but
suprisingly its like a skin and the skin surrounded my needle, and then  
some

how I was able to but back the skin in the ppt.
orelse the drop is clear if I remove the skin from the drop.
I couldnot see the same on the control drop with no protein and just  
buffer.

Does anyone have any experience with  such kind of of shiny skins on
crystallization drops??

Is it protein? Can I go forward and use it for seeding??

Please suggest
with regards
Rashmi



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Automatic LINK generation

[Enrico Stura]

Does anybody know the pdb codes with proteins with O-linked
sugars on THR.

I support a program to help interpret sugars in poor electron density
by stabilizing links. Coot helps a bit but branching poses a problem.
Rather than based on distance like coot operates at present it should be  
best

if one could specify the type of link.

I have crystals where sugars are in poor electron density. I can recognize
sialic acid moieties at a crystal contact with symmetry related molecule,
but fitting the weak density for the sugars leading to the crystal contact  
is

almost impossible.

There are good reasons to combine knowledge of normal human glycosylation  
with
weak electron density to produce a reasonable model. With natural products  
and
heterogeneous sugars, the density in never going to be fantastic except  
for the few
sugar moieties close to the protein. Yet interpreting all existing density  
may be a step

better than outright modeling.

Enrico.


On Thu, 10 Mar 2011 09:45:57 +0100, herman.schreu...@sanofi-aventis.com  
wrote:



Dear Hailiang,

While apparently no response came, here my 2 cents:
Even if there would be an automatic utitility to generate links based on
distances, I would never use it. Either the sugars have been properly
refined and then the link cards are present in the pdb file, or the
sugars have been fitted manually and may have been real space refined in
coot or some other model-building program. In this case, the sugar
positions are likely off, since sugars are very often rather disordered
and have poor electron density. In this case distance-based link
generators very likely will make wrong connections. I just cut and paste
link cards from an other pdb file and manually edit them. I eagerly
await the moment there will be a create link option in coot, where you
just click on the two atoms to be linked and the link card gets
generated.

Best,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Hailiang Zhang
Sent: Wednesday, March 09, 2011 1:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Automatic LINK generation

Hi there,

I am trying to build the LINK information in PDB header for
sugar-containing protein, and I am wondering whether there is some
utility in CCP4 (or any others) can do it automatically (eg by measuring
inter-sugar distances). Thanks in advance!

Best Regards, Hailiang



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate

[Enrico Stura]

80% saturated lithium sulfate should have about the correct ionic strength  
to match

your crystallization conditions.
The crystals need to be transfered with as little mother liquor as  
possible to

avoid lithium phosphate crystallization.

Robert Kirchdoerfer suggestion is also excellent, but careful about  
matching the pH.


Enrico.

--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
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http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] crystal growth

[Enrico Stura]

ODD INDEED!
When you get crystals with the same morphology for three different  
proteins in same condition
you should start suspecting that they are not protein crystals but  
something to do with
a combination of the common buffer for each of the proteins or/and the  
precipitant used


Enrico.

On Sat, 16 Oct 2010 05:56:23 +0200, Seema Nath seema.n...@saha.ac.in  
wrote:


All the crystals I got for three different proteins in same condition  
looked similar. I think crystal morphology may vary with the  
crystallizing conditions.





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] database-assisted data archive

[Enrico Stura]

Knowing where all the important files are is really all that is needed.  
Sofistication can come later.

I would welcome a CCP4 database-assisted data archive system.

Here is my contribution to the discussion:

I agree with Paul Paukstelis that getting users to use any  
database-assisted data archive system
is the biggest obstacle. I have had problems with compliance with my  
system, where all that the student
has to do is to provide file and directory names each Friday to keep the  
database up to date.


It is a simple html based access system where through hyperlinks one can  
access the data anywhere
where it is stored. Users need only provide the directories names of where  
the various pieces
of data are stored within the accessible network and the data manager (any  
HTML competent individual)
can then set-up the links to the main control platform (start-up html  
page).
The advantage of such system is that it is platform independent and needs  
only a well configured browser.

It is backward compatible with any old data.

George Pelios may want to consider an automated system where mosflm, scala  
and all subsequent
programs contribute to create and update a raw data retrieval file  on the  
basis of the files
they have used. When the project is finished a backup program should be  
able to retrieve
all such files to be stored in a consolidated manner for transfer to a  
long term storage server.


A brief description of the system I use for synchrotron data collection:

Prior to the synchrotron trip, each sample taken to the synchrotron is  
entered in a table that represents its position in
the puck with hyperlinks to a file describing its position in the  
crystallization tray (this file will have hyperlinks

to crystallization and all prior preparation steps).
As data is collected a short comment (resolution and number of frames is  
included if data has been
collected) as the data is transfered in the home lab a link to the  
directory where the data is

stored is then added.
To give an idea of data quality Mosflm and gimp screen capture are used to  
create a jpg of
the first data image (with the frame filename added) which is stored in  
the same directory as

the raw data frames. This image is accessed when clicking on the comment.
Compliance with the system can be checked by clicking on comments other  
than not tested.


It is all manual but is not very time consuming once the initial html  
templates have been
set up. Still I am looking foward to a simple CCP4 designed system that  
can do something similar

automatically.

I would also recommend looking at ispyb implemented at the ESRF which is  
also web based:

www.esrf.eu/UsersAndScience/Experiments/MX/Software/ispyb

Enrico.

--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] control of nucleation

[Enrico Stura]

Dear Zq  CCP4BB readers,The precipitant is the main component that affects nucleation.In specific cases other factors can be used to modulate nucleation as mentioned before by others: protein concentration, temperature, drop size, initial protein/precipitant ratio etc. All good components of a very long list that will give a student years of work ahead.Just to take the first item: "Protein concentration"Most will think that "Protein concentration" should be reduced to reduce nucleation. Unfortunately,what will happen when the protein concentration is reduced is not so easily predictable.Let's do the opposite! Just for fun, let's increase the protein concentration instead.We will increase the protein concentration while severely reducing the precipitant concentration. The 15 mins lysozyme crystallization is a good example of this. Enrico's 15mins Lysozyme recipeLysozyme concentrations of 100-150 mg/ml are used. The high protein concentration allows crystals to grow rapidly, so each nucleus has a chance to grow before more nuclei are formed. This is because each growing nucleus ends up depleting its local environment and making the nucleation of others nearby less likely. Nucleation requires a higher degree of supersaturation than crystal growth. Getting it to work and controlling it requires accuracy, but it is great fun to do ... and lysozyme is cheap.HOT STUFF crystallization is ! In the case of lysozyme: Do it in a hot room you for better control. This ambiguity persists for other items:Thomas Edwards suggests that: "dioxane - it is supposed to reduce nucleation.""Supposed to" when it does not do the opposite:Ménétrey, J., Perderiset, M., Cicolari, J., Houdusse, A.  Stura, E.A. (2007) Improving Diffraction from 3 to 2 Å for a Complex between a Small GTPase and Its Effector by Analysis of Crystal Contacts and Use of Reverse Screening. Cryst. Growth Des. 7:2140-2146.This is the one additive that really increases nucleation in the above paper.Online access:  Improving DiffractionThe procedures in "reverse screening" are used to identify what each proposed effector really does and use it toachieve better crystals.As screening is done with smaller and smaller drops, the conditions that will emerge more often are those where the nucleation rate is very high. Nanodrops will yield "overnucleation".To conclude dear Zq, listen to all the advice, but unless you really understand your protein you willfind that you will achieve the opposite of what you are trying to do.Enrico.On Thu, 06 May 2010 10:10:37 +0200, Thomas Edwards t.a.edwa...@leeds.ac.uk wrote: Dear Zq, A few ideas: 1) Vary protein concentration, temperature, or protein : mother liquor   ratio. 2) Try dioxane - it is supposed to reduce nucleation. 3) give your protein a good hard spin before you set up drops to remove   aggregates. 4) seeded factorial screen. 5) re-purify on gel filtration? Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- Nature composes some of her loveliest poems for the microscope and   the telescope.  ~Theodore Roszak  From: zq deng dengzq1...@gmail.com Reply-To: zq deng dengzq1...@gmail.com Date: Thu, 6 May 2010 09:03:46 +0100 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] control of nucleation hello,everybody . due to excess nucleation,I often get many tiny   crystals instead of  few,large crystals.i wana optimize the condition,   does anyone have adivce about this? Best regards.-- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 OfficeRoom 19, Bat.152,   Tel: 33 (0)1 69 08 9449LabLTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE   http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-sturahttp://www.chem.gla.ac.uk/protein/mirror/stura/index2.htmle-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Protein-antibody complex

[Enrico Stura]

Jan,

Are you dealing with a whole IgG or an Fab?

Most Fab-antigen complexes will not be affected by 5mM DTT.
Fab production makes use of reducing agents that allow the
antibody hinge region to become exposed to proteases.
If you need DTT to prevent your antigen from aggregating, try
to crystallize the Fab-antigen complex without worring about the
DTT.

Enrico.

On Thu, 22 Apr 2010 10:29:18 +0200, Jan Rash jan...@googlemail.com wrote:


Hi All,



 I have a simple question about the complex formation between  
macromolecular

complex and antibody. My protein is stable in the presence of the 5mM DTT
and under these conditions the reducing environment is too strong for the
antibody to survive. I am also now trying to check the stability of the
protein  in lower molar concentration of DTT, but as DTT being a strong
reducing agent it might still pose a threat to the disintegrate  the
antibody.



Does anybody have experience in handling protein-antibody complexes using
other reducing agents? Your answers and help in this regard will be  
highly

appreciated.



Thanks,



Jan



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] how to make cholesterol solution

[Enrico Stura]

Jerry,

Steroids have a certain solubility also in ethylene glycol and PEG.
You might be able to work out a crystallization strategy around this.
The ethanol/PEG combination was used in:
http://www.nature.com/nature/journal/v437/n7055/full/nature03923.html


Enrico.


Dear ALL:

 Sorry for this kind of off-topic question.

I am going to co-crystallize one protein with cholesterol. I read  
some papers saying that
their protein can be pre-incubated with 1mM cholesterol in the presence  
of 5% (v/v) ethanol.


   TO do so, I first dissolved cholesterol in 100% ethanol at a  
concentration of 10mM. However, it is very difficult to make the  
dilution into 5% ethanol either just in water or some buffers.


   Does anyone have such experience to make cholesterol solution in  
normal buffers plus some ethanol?


   Thanks a lot,



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Need isolated crystals

[Enrico Stura]

Amit Sharma,

I second Tim's suggestions.

The first experiment is the following.
Set up several experiments at constantly decreasing precipitant  
concentrations.
Streak seed all of them. If in any of those you can get non-clustered  
crystals

non matter what size, seeding can solve your problem.
To restore crystal size follow the rest of Tim's list.

Enrico.


On Mon, 12 Apr 2010 23:01:10 +0200, Tim Gruene t...@shelx.uni-ac.gwdg.de  
wrote:



Have you tried anything, yet? The list of answers can be quite long.
A couple of keywords:
- micro-seeding
- macro-seeding
- cross-seeding
- additive screens
- extend to purification protocol
- change temperature, pH, etc.
- have you searched around the current conditions
 ...

if you describe a little what you have done so far we can also get an  
idea of

your status of experience and adapt the answers accordingly.

Kind regards, Tim


--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] question about the zinc binding protein

[Enrico Stura]

What is 1mM BTW? I am not familiar with this abbreviation.

PBS phoshate buffered saline (Phosphate + NaCl) not suitable for zinc  
binding proteins

BBS borate buffered saline (Borate + NaCl) wrong pH for zinc binding.
BTW ? (? ? ?) No idea what this is. Charles, do you know if it includes  
HEPES or Phosphate?


Enrico.

On Thu, 01 Apr 2010 11:06:10 +0200, Charles Allerston  
charles.allers...@sgc.ox.ac.uk wrote:



Hi,

are you sure it is your protein precipitating?  You will get a cloudy  
precipitate appearing in HEPES and Phosphate buffers on addition of  
ZnCl, without protein.



cheers

charlie


dengzq1987 dengzq1...@gmail.com 3/31/2010 5:08 pm 
hello everyone, recently i purify  a protein conteining zinc binding  
domain,and i want to determine its structure.i get the crystal,but poor  
diffraction.so i try to adding zinc into the protein to optimize the  
crystal,but the protein precipitate immidiately  even the znic is 1  
mM.BTW,we use the protein to do zinc scan,we don't find the zinc. does  
anyone have some advice?


2010-03-31



dengzq1987



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] question about zinc binding protein

[Enrico Stura]

Not strange at all:

Reading from:

http://www.chem.gla.ac.uk/research/groups/protein/mirror/stura/cryst/add.html

Zinc acetate or sulfate   0.2-5mM It inevitably reduces protein solubility.
It can act as an inhibitor. Essential for activity of various enzymes

Zinc reduces protein solubility and often aggregates proteins. In a case
of a zinc binding protein you may need to add 100-200 mM imidazole to
control even 0.2mM concentrations of this ion.
Without good control, you will end up by depleating the zinc within the  
precipitate

and end up without zinc in your binding site.

This is what you got.

Enrico.

On Wed, 31 Mar 2010 18:11:46 +0200, zq deng dengzq1...@gmail.com wrote:


hello everyone, recently i purify  a protein conteining zinc binding
domain,and i want to determine its structure.i get the crystal,but poor
diffraction.so i try to adding zinc into the protein to optimize the
crystal,but the protein precipitate immidiately  even the znic is 1
mM.BTW,we use the protein to do zinc scan,we don't find the zinc. does
anyone have some advice?hello everyone, recently i purify  a protein
conteining zinc binding domain,and i want to determine its structure.i  
get
the crystal,but poor diffraction.so i try to adding zinc into the  
protein to
optimize the crystal,but the protein precipitate immidiately  even the  
znic
is 1 mM.BTW,we use the protein to do zinc scan,we don't find the zinc.  
does

anyone have some advice?



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] crystallization of a macromolecular complex

[Enrico Stura]

Jan,

Salting in/salting out.
Have you considered having your protein in very low salt
and crystallizing it in high concentration of high MW PEG, again at very  
low salt.


Enrico.


On Fri, 05 Mar 2010 15:02:55 +0100, Jan Rash jan...@googlemail.com wrote:


Dear All,

This is about the crystallization of the macromolecular complex which is
highly soluble and shows no signs of the aggregation (even at high
concentration). We have tried several salts, precipitants and even high
protein concentration (around 20g/L) for its crystallization without any
genuine hit. Any suggestions for growing the crystals of this  
macromolecular

complex will be highly appreciated.

Thanks,

Jan



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] how to improve resolution

[Enrico Stura]

On Fri, 05 Feb 2010 05:39:14 +0100, rui ruis...@gmail.com wrote:


Hi, All,

We are trying to crystallize a protein and found some initial hit in the
following conditions,

pH 4.8, 0.2 M AS or some other salts ( NaCl,LiCl, MgCl2 ), 32% PEG4000 or
PEG3350 ). However the quality of the crystal is not so great,some of  
them

look like needle cluster(very long in length), some of them look like
multi-crystals or hollow inside.

Such growth problems are likely due to the quality of the protein solution.
Changes in precipitant concentrations are likely to be ineffective. Try
ion exchange purification.


We tried to optimize the pH and PEG and
tested one that diffracts at 2.9A. For the next, how to improve
resolution?Any suggestions? Even mutate the protein to get a high  
resolution

is ok, generally what kind of mutation would make proteins crystallize
better? Thanks.

It is not mutations that will improve diffraction, it is small changes
in crystal contacts.
The Discussion section from:
http://pubs.acs.org/cgi-bin/article.cgi/cgdefu/2007/7/i11/html/cg700698d.html

may help.

Enrico

--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Help with MR in P21

[Enrico Stura]

It may be a good exercise to look at the possibility of weak reflections  
in the
original images. Such reflections are not picked up by the automatic  
methods and

could be a possible  source for your problems.
Use manual spots picking in mosflm or an equivalent program. This will  
ensure that

such spots are taken into account in the indexing.

Alternatively, it is possible that the space group is wrong? And if so,  
how can I figure out the

correct one?


Thank you in advance,
Michele



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] smeared spot in diffraction

[Enrico Stura]

Fengxia,

From the images it is clear that the degree of smearing is variable. From  
this you can
deduce that you should be able to get sharp images by finding better  
cryo-conditions.


There is no magic involved. Your crystallization conditions have little  
precipitant
and a lot of water. Hence your problem. Increasing just the PEG  
concentration may
induce a cell shrinkage and worsen the problem. On the other hand  
replacing some of the
high molecular weight PEG 4K with a mixture of PEG10K + PEG200 + ethylene  
glycol (EG) might
work. The higher MW PEG10K would better stabilize the crystals and the  
PEG200/EG should provide

good cryoprotection.

Enrico.



On Thu, Jan 21, 2010 at 1:54 AM, Fengxia Liu fengxia@gmail.com  
wrote:



Dear all,
I am trying to diffract one semet-protein, it gave me some clear spots  
and

some smeared spots in one diffration, you can find maps attached.
Mother liquor condition is 0.1MTris-HCL pH8.5+10% PEG4k + 0.2M Li2SO4, I
have tried mother liquor+25%glycerol, paratone, paratone+ mineral oil,  
but

they all gave me such pattern.

Does anyone know how to solve this? I appreciate any advice.

Thank you in advance.

Best wishes,
Fengxia






--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition)

[Enrico Stura]

I am also having problems with the branching.
The fucose linked to the first NAG being linked to the second NAG of the  
carbohydrate chain.

So one has a linear chain NAG-FUC-NAG-MAN-MAN-MAN
instead of the FUC being a branch out from the first NAG. i.e. :
NAG-NAG-BMA-BMA
||
FUC MAN

Has anybody solved such problem in coot.

Enrico.


--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] where I have been going wrong in crystallization?

[Enrico Stura]

Dear Martin,

The original procedure with polyethylene glycol comes from:

 Stura, E.A. (1998) Strategy 3: Reverse Screening. In Crystallization of  
Proteins: Techniques, Strategies and Tips. A laboratory manual (Bergfors,  
T., ed) International University Line. pp. 113-124.


If you follow the procedure described within you should not have problems  
in getting crystals in 15 mins

unless there is a problem with your hen egg white lysozyme.
The method given on the Rigaku site proposes 25% (v/v) ethylene glycol  
instead of polyethylene glycol (2K-10K).
This would slow down the crystallization rate. You are better off using   
polyethylene glycol 4K and then
transfer the crystals for freezing into 15% polyethylene glycol 4K, 25%  
(v/v) ethylene glycol, 1M NaCl, pH 4.5

as your cryo-solution. Why wait 2 days when you can do it in 1 hour?

Enrico.

On Mon, 21 Dec 2009 17:12:48 +0100, MARTYN SYMMONS  
martainn_oshioma...@btinternet.com wrote:



Dear All
  checking out the Lysozyme crystallization methods on the web I  
liked the Rigaku Instructions that I found:

(http://www.rigaku.com/protein/crystallization.html)

...create a drop of 3ul lysozyme solution, and 3 ul of well solution,  
respectfully, for a total drop size of 6ul...


So perhaps sometimes I am just not respectful enough to deserve crystals  
?


good wishes to all
   regards,
 Martyn
---
Martyn Symmons
MRC-MBU Cambridge UK
'Chan fhiosrach mur feòraich.'
Gaelic proverb -
  Nothing asked, nothing learned.



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Crystallization of lysine and arginine rich proteins

[Enrico Stura]

Before deciding to change the protein sequence it is best to look at the
precipitation pattern of the protein as a function of various precipitants.
If this shows that precipitation is not within the standard range commonly
observed for other basic proteins even at high protein concentrations
then side chain mutation should be tried.
Even lysozyme would be hard to crystallize at 1 mg/ml at 38°C.

E.
--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] long needles

[Enrico Stura]

James and those that may be interested,

65% MPD at 4 degree C at pH 3.6 are conditions that stimulate the growth
of salt crystals, but one cannot exclude that the florets are indeed
protein.

1. If you are sure you are dealing with protein crystals try the  
co-precipitant

approach: Carry out a simple screeen  like 5% MPEG550 + 60% MPD ;
10% MPEG550 + 55% MPD  and so on. This can be repeated with other
co-precipitants that you may have identified for other conditions you have  
screened

previouysly.

2. Vary the protein concentration vs. the precipitant concentration.
The results should follow the theoretical phase diagram.

3. Use the results of Steps 1 and 2 do screen finely using seeding. Florets
often indicate a disordered initial nucleous, which can be caused by  
excessive protein or

precipitant concentration, but could also be due to ther factors.
Seeding of drops that do not nucleate spontaneously will in many cases  
remove

the problem.

Enrico.


On Tue, 25 Aug 2009 15:14:24 +0200, james09 pruza james09x...@gmail.com  
wrote:



Dear all,

Sorry for the non-ccp4 question. Recently I have got some long needle of  
on

35 kDa protein with 65% MPD at 4 degree C at pH 3.6 (50 mM NaOAc buffer).
The protein contains 50mM of salt. The florets are very thin. I need some
suggestions regarding the optimization of the crystallization conditions.

Thanks in advance.

James




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] precipitation of the protein in crystallisation solution

[Enrico Stura]

Given that you are able to achieve a protein concentration of 15 mg per ml
in Tris-HCl, pH 8.0, 100mM NaCl and 1% Glycerol suggests that you have no
drastic solubility problem.

Unless you have a chromatophore as an intrinsic co-factor of one of your
component proteins, the yellow slime may be due to impurities.

The standard screens in most kits are fine for standard proteins but
not for proteins that are out of the ordinary.

The fact that precipitation is absent or delayed with PEG is coherent with
your ability to concentrate the protein to 15mg/ml.

Now for the improvement steps:
The first step is to normalize your protein concentration :
## The mean crystallization condition is around 16% PEG 3350. ##
The above may not be entirely correct but is a good working hypothesis
that can be used as described below:
If 16% PEG 3350, 50mM Tris-HCl, pH 8.0, 100mM NaCl and 1% Glycerol gives
you just some precipitation and 24% PEG in the same salt and buffer  
conditions
precipitates most of the protein, then you can assume that at 15mg/ml your  
protein
concentration is just right and now you may want to screen various ions  
and different
pH to find suitable crystallization conditions. (If not ... reduce your  
protein concentration to

satisfy the test based on the hypothesis).
The next step is basic buffer component substitution to see what your  
buffer components are doing:
That implies checking how other salts can be used instead of NaCl (Li2SO4,  
KCl Na-mono,di,tri-carboxylates,  )
1% glycerol replaced by 0.01-5% organic compounds such as MPD, EtOH, PrOH,  
xylitol, etc.
Tris-HCl replaced by other basic range buffers such as imidazole, glycine,  
... pH 8.5-10.6
If the protein tollerates neutral and acidic conditions you can extend the  
pH range to be searched.


Once you understand your protein complex better you should design a screen  
of your own based on your

new knowledge.


--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Florets- How to improve it

[Enrico Stura]

Re: 65% MPD in 0.1M Acetate buffer pH 3.6
These conditions can crystallize salt impurities present in the protein  
buffer.

The first step is to ensure that the crystals are protein.
If they are protein the next step is to reduce the MPD concentration and  
seed
the drops if no crystals appear. This should allow you to pass from  
rosettes to

single crystals.



On Mon, 18 May 2009 12:42:32 +0200, james09 pruza james09x...@gmail.com  
wrote:



Deal all,

Sorry for the non-ccp4 query.

I am new to this field and need some suggestions regarding the  
improvement

in the crystallization qual
I have crystallized a protein with 65% MPD in 0.1M Acetate buffer pH 3.6  
at

4 degree. The florets appear after 2 days and covers the whole drop. What
should I do for the optimization. Its a 20kDa protein.
The needles are clusters and very long.
Please suggest how what are the various tricks one can apply.

Thanks in advance for the suggestions.

James pruza




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

[ccp4bb] POSTDOCTORAL POSITION IN X-RAY CRYSTALLOGRAPHY AT CEA SACLAY

[Enrico Stura]

POSTDOCTORAL POSITION IN X-RAY CRYSTALLOGRAPHY AT CEA SACLAY


Position Description:  The department of molecular engineering of proteins  
(SIMOPRO)
 CEA Saclay, near Paris, France (Head of the Department: Vincent DIVE) has  
an immediate career opportunity for a highly motivated and experienced  
crystallographer to work towards the identification of inhibitors of  
clostridial toxins under the direction of Enrico STURA.
The laboratory focuses on the use of X-ray crystallography for  
macromolecular structure
determination in support of research groups in the depertment, associated  
with drug development and antigen recognition.  The department is a highly  
collegial and collaborative research environment and supports a diverse  
range of investigators and research projects.

Dr. STURA will oversee the operation of the laboratory; provide expertise,
training, and/or service in crystallization, diffraction and structure
determination as required.  For further information please contact  
est...@cea.fr.


Position requirements:  Ph.D. or equivalent degree in physics, chemistry,  
biochemistry or a
closely related field. At least one year of experience in macromolecular  
X-ray structure refinement is required. This would preferably include  
experience working in collaborative and/or team-based projects and least  
two publications in leading peer-reviewed journals,

related to macromolecular X-ray structure determination.
Salary:  Competitive commensurate with experience.

This post is available immediately and is funded by the CEA under the  
program NRBC for an initial period of 9 months renewable once and it could  
potentially lead to a permanent position. The likely starting date is  
March 16, 2009 and applications will be reviewed as a a continuous process  
as applications are received

continuous process until the position is filled.

Letters of application including a CV and names and addresses of 2  
referees should be sent to Vincent DIVE vincent.d...@cea.fr or Enrico  
STURA est...@cea.fr




--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette Cedex FRANCE
http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
  e-mail: est...@cea.frFax: 33 (0)1 69 08 90 71

--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette Cedex FRANCE
http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
 e-mail: est...@cea.frFax: 33 (0)1 69 08 90 71