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
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 solubilit
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&sid=173&pid=524
Soaking will work as long as you do it correctly. A 20min soak or
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 cryst
etro
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
5 at 2:00 AM, Enrico Stura 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 micr
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 mu
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 a
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 Oc
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 w
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
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.
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
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
wrote:
Dear George, I'm not sure it's a good idea since the wet work and
coputational
work are
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/fu
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
wrote:
Dear Joern and other BBers,
While I fully agree that it is important to test a few images at
room temperature, to know t
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 menti
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 a
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
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 w
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
On Wed, 24 Jul 2013 16:36:17 +0200, Hena Dutta 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 appr
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 y
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 pro
80% saturated Li2SO4
On Thu, 23 May 2013 11:42:09 +0200, Faisal Tarique
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 adva
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
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 or
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
wrote:
Hard to say without data - but I would generate the 3 symmetry copies of
the molecule you would have in P213,
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 mor
On Mon, 07 Jan 2013 18:30:32 +0100, Joy 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 defi
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 wrote:
Dear All,
May I have your expert opini
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
wrote:
Dear all,
A very happy new year to all.
I would appreciate some expert advice on op
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,
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 no
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
glyc
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 generation
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 ob
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
Dear Marc,
I would release the PDB coordinates at the RCSB. The release will protect
you from
use of the coordinates since you have now acquired a release date prior to
referee viewing.
Assuming that an honest referee would request the coordinates only to
verify that all is well
before fina
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
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
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
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
wrote:
For what it's
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
o
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, 1O
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
wrote:
Hi Enricho,
The scenario of streak seeding follows Ostwald ripening but will
this happen
Theresa,
A little off from the original question.
You should have started a new thread.
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 larg
is almost always BETTER!
AND
Dirk Kostrewa,
Does stream-cooling beat liquid ethane as well or not?
Enrico.
On Tue, 07 Feb 2012 18:27:04 +0100, Fischmann, Thierry
wrote:
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Enrico Stura
Sent
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 conditio
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,
,
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
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 bu
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
.
Enrico.
On Tue, 18 Oct 2011 17:12:42 +0200, Peter Keller
wrote:
Dear Enrico,
Please don't get me wrong: what you are saying is not incorrect, but it
is only half the story.
On Tue, 2011-10-18 at 15:13 +0200, Enrico Stura wrote:
With improving techniques, we should always be making
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 so
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 Growt
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.
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
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 macromolecu
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 t
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
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 subsequ
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
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
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
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
i
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 c
Jerry McCully,
35% dioxane should be sufficient depending on the other components in your
buffer.
Yet, it is always advisable to try a few alternatives:
Try an extra 5-10% glycerol or ethylene glycol.
This might reduce the increase in mosaicity due to cooling shock.
If what you are cr
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.
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 arc
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
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 crystalli
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 thi
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
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
HEPE
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 an
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 wrote:
Dear All,
This is about the crystallization of the macromo
Jerry McCully and others sugars lovers,
SUGARS
==
How sweet [(it is)/(is it)] to work with glycoproteins (!/?)
Please, when asking for help, include details of the source and nature of
the glycosylation.
i.e. :
Natural (Human or Mammalian) - monoclonal (best) / polyclonal / single
dono
On Fri, 05 Feb 2010 05:39:14 +0100, rui 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 o
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
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. Hen
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
H
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 th
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
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
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
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. T
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
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