Re: [ccp4bb] salt bridges etc..

[Eleanor Dodson]

Thank you Gert - very clear explanation..
Eleanor

On 16 July 2018 at 13:31, Gert Vriend  wrote:

> Dear Eleanor,
>
> Salt bridges are a compromise between entropy and enthalpy. If, say, an
> Asp and an Arg side chain are a bit restricted in their freedom and the
> charges are close, enthalpy wins, and if they are very exposed, and not
> close at all, entropy wins. The enthalpic gain upon protein folding from
> obtaining one salt bridge has been given many values in the literature, but
> in practice boils down to about 1 kCal/Mole. The enthalpic contribution is
> a bit higher than 1kcal/Mole when the positive and negative charges are
> very close to each other (in which case you loose entropy upon folding).
> Most salt bridges are at the surface where they continuously compromise
> between entropy and enthalpy. So, they move around, but most of the time
> the charges are close together, and that is why you can see them with Xray.
> When the two charged groups come close to each other there are always
> certain local conformations that are preferred over others. Those
> (sometimes multiple) locally preferred conformations we see in Xray if we
> have good crystals. It does not matter, however, how many local
> conformations we observe. It is just one salt bridge, and its energetic
> contribution to protein folding remains (very roughly, and this is
> practical experience for which no good theory exists) about 1kCal/Mole.
>
> Gert
>
> On 11-7-2018 16:52, Eleanor Dodson wrote:
>
> How do people decide on what is a salt bridge within a molecule and how to
> count them for those Tables?
>
> I have been looking at 2z2f - paper claims some score..-
>
> But there are several residues in alternate conformation
>
> with NZ A  to OE1Aand NZ A to OE1B  and NZ B to OE1B etc
>
> Is that one salt bridge   or 3 salt bridges
>
> PISA lists salt bridges between molecules but not within a molecule I dont
> think?
>
> Suggestions gratefully received.
> Eleanor
>
>
>
>
>
> --
>
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>
>
>



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Re: [ccp4bb] salt bridges etc..

[Gert Vriend]

Dear Eleanor,

Salt bridges are a compromise between entropy and enthalpy. If, say, an 
Asp and an Arg side chain are a bit restricted in their freedom and the 
charges are close, enthalpy wins, and if they are very exposed, and not 
close at all, entropy wins. The enthalpic gain upon protein folding from 
obtaining one salt bridge has been given many values in the literature, 
but in practice boils down to about 1 kCal/Mole. The enthalpic 
contribution is a bit higher than 1kcal/Mole when the positive and 
negative charges are very close to each other (in which case you loose 
entropy upon folding). Most salt bridges are at the surface where they 
continuously compromise between entropy and enthalpy. So, they move 
around, but most of the time the charges are close together, and that is 
why you can see them with Xray. When the two charged groups come close 
to each other there are always certain local conformations that are 
preferred over others. Those (sometimes multiple) locally preferred 
conformations we see in Xray if we have good crystals. It does not 
matter, however, how many local conformations we observe. It is just one 
salt bridge, and its energetic contribution to protein folding remains 
(very roughly, and this is practical experience for which no good theory 
exists) about 1kCal/Mole.


Gert


On 11-7-2018 16:52, Eleanor Dodson wrote:
How do people decide on what is a salt bridge within a molecule and 
how to count them for those Tables?


I have been looking at 2z2f - paper claims some score..-

But there are several residues in alternate conformation

with NZ A  to OE1A    and NZ A to OE1B  and NZ B to OE1B etc

Is that one salt bridge   or 3 salt bridges

PISA lists salt bridges between molecules but not within a molecule I 
dont think?


Suggestions gratefully received.
Eleanor







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[ccp4bb] salt bridges etc..

[Eleanor Dodson]

How do people decide on what is a salt bridge within a molecule and how to
count them for those Tables?

I have been looking at 2z2f - paper claims some score..-

But there are several residues in alternate conformation

with NZ A  to OE1Aand NZ A to OE1B  and NZ B to OE1B etc

Is that one salt bridge   or 3 salt bridges

PISA lists salt bridges between molecules but not within a molecule I dont
think?

Suggestions gratefully received.
Eleanor



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Salt or protein?

[Jan van Agthoven]

Thanks your suggestions! I tried Dials but couldn’t get any further. There just 
too few spots like Artem suggested. I’ll follow Patrick’s advice to seed a new 
screening with this crystal. This one was really tiny. I may get a bigger one.
Jan
> On Feb 17, 2018, at 2:43 PM, Harry Powell  wrote:
> 
> Hi Jan
> 
> What happens if you use a program that can easily use spots from all images 
> to index, like DIALS or XDS? My recollection (which may be wrong) is that 
> this is not straigthforward in HKL3000.
> 
> 
>> On 17 Feb 2018, at 19:32, Jan van Agthoven  wrote:
>> 
>> Dear all,
>> At first I thought this was a salt crystal, though after diffraction I 
>> obtained low resolution spots (10 to 6 Å resolution), some of them are close 
>> to each other. I indexed the pattern and obtained P1 with
>> a= 92, b=132 c=252, and alpha=103, beta=92 and gamma=94 (see pictures).
>> 
>> So I’m not sure anymore. Can anyone help to interpret?
>> 
>> Thanks,
>> Jan
>> 
>> 
> 

Re: [ccp4bb] Salt or protein?

[Harry Powell]

Hi Jan

What happens if you use a program that can easily use spots from all images to 
index, like DIALS or XDS? My recollection (which may be wrong) is that this is 
not straigthforward in HKL3000.


> On 17 Feb 2018, at 19:32, Jan van Agthoven  wrote:
> 
> Dear all,
> At first I thought this was a salt crystal, though after diffraction I 
> obtained low resolution spots (10 to 6 Å resolution), some of them are close 
> to each other. I indexed the pattern and obtained P1 with
> a= 92, b=132 c=252, and alpha=103, beta=92 and gamma=94 (see pictures).
> 
> So I’m not sure anymore. Can anyone help to interpret?
> 
> Thanks,
> Jan
> 
> 

[ccp4bb] AW: [ccp4bb] Salt bridge-hydrogen bonds

[Herman . Schreuder]

Yes, Arg has 3 potential Hbond donor/acceptors and Glu 2. Also a single atom 
can have multiple interactions.
Look in coot at the structures and which atom has which interaction with which 
partner atom.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mohamed 
Noor
Gesendet: Freitag, 20. Januar 2017 11:45
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Salt bridge-hydrogen bonds

Dear all

Is it possible for two residues to form a salt bridge between them, and at the 
same time**, each of them form a hydrogen bond with another residue? In other 
words:

Arg1 - Glu10 (salt bridge)

Arg1 - Tyr600 (H bond)

Glu10 - Thr590 (H bond)


** I understand proteins are not static structures, so I am referring to an 
exact time and space here and not something formed and broken throughout a 
sampled period of time.

[ccp4bb] Salt bridge-hydrogen bonds

[Mohamed Noor]

Dear all

Is it possible for two residues to form a salt bridge between them, and at the 
same time**, each of them form a hydrogen bond with another residue? In other 
words:

Arg1 - Glu10 (salt bridge)

Arg1 - Tyr600 (H bond)

Glu10 - Thr590 (H bond)


** I understand proteins are not static structures, so I am referring to an 
exact time and space here and not something formed and broken throughout a 
sampled period of time.

[ccp4bb] Salt!

[Bishop, Catherine E.]

I have been attempting to obtain a protein crystal of my protein for just over 
2 years at this point.  We have attempted removing the tag, binding the protein 
to its ligand, removing as much salt as possible (crashes at at too low a salt 
concentration)--this lead us to try reverse vapor diffusion--seeding, additive 
trays, optimization around the conditions an ortholog of one of the domains 
crystallized well in, and a plethora of other methods. We do get crystals, but 
they all diffract as salt. Most of these salts are found in wells containing a 
cation (ie. lithium sulfate, nickel chloride, magnesium acetate, cobalt 
chloride, etc.). When crystals are too small to shoot, I do set up optimized 
trays; however, if I get larger crystals, they diffract as salt as well. Most 
of these set ups, at this point, have also been conducted in hands other than 
mine and at other facilities known for their successful crystallization of 
proteins.

Has anyone else run into this problem and seen light at the other side?? Any 
suggestions are appreciated.

Re: [ccp4bb] Salt!

[Chris Fage]

Hi Catherine,

If they are indeed salt crystals, have you tried preparing different
truncations of the gene encoding your target? I've seen a number of
successes in which, after a codon or two was added to the termini of the
gene being overexpressed, the target crystallized beautifully.

Best,
Chris


On Wed, Jul 16, 2014 at 9:55 AM, Bishop, Catherine E. cati...@ou.edu
wrote:

  I have been attempting to obtain a protein crystal of my protein for
 just over 2 years at this point.  We have attempted removing the tag,
 binding the protein to its ligand, removing as much salt as possible
 (crashes at at too low a salt concentration)--this lead us to try reverse
 vapor diffusion--seeding, additive trays, optimization around the
 conditions an ortholog of one of the domains crystallized well in, and a
 plethora of other methods. We do get crystals, but they all diffract as
 salt. Most of these salts are found in wells containing a cation (ie.
 lithium sulfate, nickel chloride, magnesium acetate, cobalt chloride,
 etc.). When crystals are too small to shoot, I do set up optimized trays;
 however, if I get larger crystals, they diffract as salt as well. Most of
 these set ups, at this point, have also been conducted in hands other than
 mine and at other facilities known for their successful crystallization of
 proteins.

 Has anyone else run into this problem and seen light at the other side??
 Any suggestions are appreciated.

Re: [ccp4bb] Salt!

[David Briggs]

Hi Catherine,

What buffer  salt do you have your protein in when you set up screens -
maybe this is leading to your salt crystal issues? How long does it take
for the salt crystals to appear?

Have you tried setting up trays at different temperatures? Microbatch
crystallisation under oil?

Failing that - try altering the construct - add / remove a few amino acids
at each end, truncate any predicted loops...

HTH,

Dave


[image: David Briggs on about.me]

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


On 16 July 2014 15:55, Bishop, Catherine E. cati...@ou.edu wrote:

  I have been attempting to obtain a protein crystal of my protein for
 just over 2 years at this point.  We have attempted removing the tag,
 binding the protein to its ligand, removing as much salt as possible
 (crashes at at too low a salt concentration)--this lead us to try reverse
 vapor diffusion--seeding, additive trays, optimization around the
 conditions an ortholog of one of the domains crystallized well in, and a
 plethora of other methods. We do get crystals, but they all diffract as
 salt. Most of these salts are found in wells containing a cation (ie.
 lithium sulfate, nickel chloride, magnesium acetate, cobalt chloride,
 etc.). When crystals are too small to shoot, I do set up optimized trays;
 however, if I get larger crystals, they diffract as salt as well. Most of
 these set ups, at this point, have also been conducted in hands other than
 mine and at other facilities known for their successful crystallization of
 proteins.

 Has anyone else run into this problem and seen light at the other side??
 Any suggestions are appreciated.

Re: [ccp4bb] Salt!

[Jon Agirre]

Hi Catherine,

first of all, I'm sorry that you're feeling so frustrated.

In addition to the many sensible suggestions that you're surely going to
read here, I would like to point you to a paper I read a while ago in
Nature: In situ proteolysis for protein crystallization and structure
determination, http://www.ncbi.nlm.nih.gov/pubmed/17982461

Sometimes proteases can remove from the target protein just those external
bits that hinder crystallisation. I would try everybody else's suggestions
before attempting this, but here it goes just in case everything else fails.

Good luck,

Jon


On 16 July 2014 16:13, Chris Fage cdf...@gmail.com wrote:

 Hi Catherine,

 If they are indeed salt crystals, have you tried preparing different
 truncations of the gene encoding your target? I've seen a number of
 successes in which, after a codon or two was added to the termini of the
 gene being overexpressed, the target crystallized beautifully.

 Best,
 Chris


 On Wed, Jul 16, 2014 at 9:55 AM, Bishop, Catherine E. cati...@ou.edu
 wrote:

  I have been attempting to obtain a protein crystal of my protein for
 just over 2 years at this point.  We have attempted removing the tag,
 binding the protein to its ligand, removing as much salt as possible
 (crashes at at too low a salt concentration)--this lead us to try reverse
 vapor diffusion--seeding, additive trays, optimization around the
 conditions an ortholog of one of the domains crystallized well in, and a
 plethora of other methods. We do get crystals, but they all diffract as
 salt. Most of these salts are found in wells containing a cation (ie.
 lithium sulfate, nickel chloride, magnesium acetate, cobalt chloride,
 etc.). When crystals are too small to shoot, I do set up optimized trays;
 however, if I get larger crystals, they diffract as salt as well. Most of
 these set ups, at this point, have also been conducted in hands other than
 mine and at other facilities known for their successful crystallization of
 proteins.

 Has anyone else run into this problem and seen light at the other side??
 Any suggestions are appreciated.





-- 
Dr Jon Agirre
York Structural Biology Laboratory / Department of Chemistry
University of York, Heslington, YO10 5DD, York, England
http://www.york.ac.uk/chemistry/research/ysbl/people/research/jagirre/
+44 (0) 1904 32 8253

Re: [ccp4bb] Salt!

[Edward Snell]

Hi Catherine,

At the Hauptman-Woodward Medical Research Institute High Throughput 
Crystallization Screening laboratory we've just introduced SONICC and UV two 
photon fluorescence into the imaging process. I don't think it's been announced 
on the website 
(http://www.hwi.buffalo.edu/faculty_research/crystallization.html) but the 
images are now being sent to users routinely and they are proving extremely 
useful in identifying (a) salt from protein and (b) protein crystals that were 
not otherwise noticed due to obscuration by precipitate or other gunk. I highly 
recommend the application of these two techniques in your case. If you don't 
have it available locally, while trying not to advertise, it is available to 
the community as a service - just don't ask me!  The lab can be contacted 
directly at hts...@hwi.buffalo.edumailto:hts...@hwi.buffalo.edu.

Cheers,

Eddie

Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Senior Scientist, Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: esn...@hwi.buffalo.edu
Telepathy: 42.2 GHz


Heisenberg was probably here!

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bishop, 
Catherine E.
Sent: Wednesday, July 16, 2014 10:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Salt!

I have been attempting to obtain a protein crystal of my protein for just over 
2 years at this point.  We have attempted removing the tag, binding the protein 
to its ligand, removing as much salt as possible (crashes at at too low a salt 
concentration)--this lead us to try reverse vapor diffusion--seeding, additive 
trays, optimization around the conditions an ortholog of one of the domains 
crystallized well in, and a plethora of other methods. We do get crystals, but 
they all diffract as salt. Most of these salts are found in wells containing a 
cation (ie. lithium sulfate, nickel chloride, magnesium acetate, cobalt 
chloride, etc.). When crystals are too small to shoot, I do set up optimized 
trays; however, if I get larger crystals, they diffract as salt as well. Most 
of these set ups, at this point, have also been conducted in hands other than 
mine and at other facilities known for their successful crystallization of 
proteins.

Has anyone else run into this problem and seen light at the other side?? Any 
suggestions are appreciated.

Re: [ccp4bb] salt or not?

[Patrick Shaw Stewart]

Careina,

One thing to try if other ideas don't work or are too difficult, is
covalently (therefore unambiguously) labelling a little of your protein
with a fluorescent dye.  If you add 20 nL of this to the drop *after the
crystals have grown*, protein crystals will light up, but salt crystals
will not.  Thermo make some very easy-to-use kits for labelling.  See
methods section of our paper *Cryst. Growth Des.*, 2011, *11* (8), pp
3432–3441.

Could you also label the DNA   . . .   ?

Hope it helps, best wishes, Patrick


On 15 April 2013 11:18, Careina Edgooms careinaedgo...@yahoo.com wrote:

 Dear ccp4

 I have been performing trials on a protein DNA complex for a while now and
 have not seen any crystals form. Today I checked an old plate (over a month
 old) and I see 4 large crystals. *excitement* Three of them look tetragonal
 in shape (like a pyramid) and one of them looks hexagonal. I do not know if
 they are salt or protein. There is calcium chloride in the buffer. They
 feel quite soft to touch. They do not cause much birefringence. One of them
 does not seem to absorb much izit. It did go a bit blue but not entirely.

 How can I tell if this crystal is protein or not? Do you think its worth
 trying to see how it diffracts?

 Also, does Izit affect diffraction/ protein structures at all? Could I use
 a crystal with Izit in a diffraction experiment and ultimately to get the
 structure?

 Best
 Careina




-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36

[ccp4bb] salt or not?

[Careina Edgooms]

Dear ccp4

I have been performing trials on a protein DNA complex for a while now and have 
not seen any crystals form. Today I checked an old plate (over a month old) and 
I see 4 large crystals. *excitement* Three of them look tetragonal in shape 
(like a pyramid) and one of them looks hexagonal. I do not know if they are 
salt or protein. There is calcium chloride in the buffer. They feel quite soft 
to touch. They do not cause much birefringence. One of them does not seem to 
absorb much izit. It did go a bit blue but not entirely.

How can I tell if this crystal is protein or not? Do you think its worth trying 
to see how it diffracts?

Also, does Izit affect diffraction/ protein structures at all? Could I use a 
crystal with Izit in a diffraction experiment and ultimately to get the 
structure?

Best
Careina

Re: [ccp4bb] salt or not?

[Hargreaves, David]

Dear Careina,



I would be cautious of using dyes. Much better to 1) try in-situ
diffraction if possible as this is least invasive or 2) pick a sensible
cryo and just freeze the crystal(s). I would try to same something from
the experiment for seed stock  possibly sequencing.



Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

Discovery Sciences, Structure  Biophysics

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



Please consider the environment before printing this e-mail



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Careina Edgooms
Sent: 15 April 2013 11:18
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] salt or not?



Dear ccp4



I have been performing trials on a protein DNA complex for a while now
and have not seen any crystals form. Today I checked an old plate (over
a month old) and I see 4 large crystals. *excitement* Three of them look
tetragonal in shape (like a pyramid) and one of them looks hexagonal. I
do not know if they are salt or protein. There is calcium chloride in
the buffer. They feel quite soft to touch. They do not cause much
birefringence. One of them does not seem to absorb much izit. It did go
a bit blue but not entirely.



How can I tell if this crystal is protein or not? Do you think its worth
trying to see how it diffracts?



Also, does Izit affect diffraction/ protein structures at all? Could I
use a crystal with Izit in a diffraction experiment and ultimately to
get the structure?



Best

Careina


--
AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number: 03674842 and a registered office at 2 Kingdom Street, 
London, W2 6BD.
Confidentiality Notice: This message is private and may contain confidential, 
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Re: [ccp4bb] salt or not?

[RHYS GRINTER]

What else in in the conditions? Calcium Sulphate/Phosphate is poorly soluble, 
so if there is any sulphate or phosphate in your condition I would be 
suspicious.
The age of the plate is also a bad sign, as evaporation over an extended time 
can lead to salt crystals. Check the well solution for crystals, if there are 
any then it's almost certainly salt.
Softness and lack of birefringence are cautiously good signs, however the only 
way to know for sure is to stick them in an x-ray beam, which is always worth 
while for a crystal.

Good luck

Rhys



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Careina Edgooms 
[careinaedgo...@yahoo.com]
Sent: 15 April 2013 11:18
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] salt or not?

Dear ccp4

I have been performing trials on a protein DNA complex for a while now and have 
not seen any crystals form. Today I checked an old plate (over a month old) and 
I see 4 large crystals. *excitement* Three of them look tetragonal in shape 
(like a pyramid) and one of them looks hexagonal. I do not know if they are 
salt or protein. There is calcium chloride in the buffer. They feel quite soft 
to touch. They do not cause much birefringence. One of them does not seem to 
absorb much izit. It did go a bit blue but not entirely.

How can I tell if this crystal is protein or not? Do you think its worth trying 
to see how it diffracts?

Also, does Izit affect diffraction/ protein structures at all? Could I use a 
crystal with Izit in a diffraction experiment and ultimately to get the 
structure?

Best
Careina

Re: [ccp4bb] salt or not?

[Ulrike Demmer]

Dear Careina,

altough your crystals does't take up the Izit dye it sounds promising. The 
uptake of Izit depends on the solvent channels of the protein molecule - 
sometimes the dye just can't enter the molecule.
Concerning the Calciumchloride - if the concentration is not too high and 
without other ingredients which could cause less solube salt there is the 
possiblity that you have got protein crystals. I once had a condition whith 34 
% MPD + 0.1M buffer + 0.1 M Calciumchloride which produced nicely diffracting  
crystals
To speak from my experience I think 1 month after setting up the trays the 
drops should not be dried out. If the wells are sealed properly new crytals can 
appear even after 1 year.

You should definately check the diffraction then you will know for sure.

Cheers,

Ulrike

Re: [ccp4bb] salt or not?

[James Holton]

I may be biased, but the only way to really be sure is to shoot them.

If you see no spots at all, be sure to do a wide oscillation (rotation 
during the exposure) shot as well.  It is not unlikely for a salt 
crystal to be oriented so that no relps are on the Ewald sphere, giving 
no spots.  But, if you sweep through 180 degrees during the exposure you 
will at least have a nice, pretty symmetric diffraction pattern to look 
at for a moment before the disappointment sets in.


-James Holton
MAD Scientist

On 4/15/2013 3:18 AM, Careina Edgooms wrote:

Dear ccp4

I have been performing trials on a protein DNA complex for a while now 
and have not seen any crystals form. Today I checked an old plate 
(over a month old) and I see 4 large crystals. *excitement* Three of 
them look tetragonal in shape (like a pyramid) and one of them looks 
hexagonal. I do not know if they are salt or protein. There is calcium 
chloride in the buffer. They feel quite soft to touch. They do not 
cause much birefringence. One of them does not seem to absorb much 
izit. It did go a bit blue but not entirely.


How can I tell if this crystal is protein or not? Do you think its 
worth trying to see how it diffracts?


Also, does Izit affect diffraction/ protein structures at all? Could I 
use a crystal with Izit in a diffraction experiment and ultimately to 
get the structure?


Best
Careina

Re: [ccp4bb] salt or not?

[Ed. Pozharski]

Protein-DNA complex crystal with channels too small for the dye is *extremely* 
unlikely, imho.

 Original message 
From: Ulrike Demmer ulrike.dem...@biophys.mpg.de 
Date: 04/15/2013  8:48 AM  (GMT-05:00) 
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] salt or not? 
 
Dear Careina,

altough your crystals does't take up the Izit dye it sounds promising. The 
uptake of Izit depends on the solvent channels of the protein molecule - 
sometimes the dye just can't enter the molecule.
Concerning the Calciumchloride - if the concentration is not too high and 
without other ingredients which could cause less solube salt there is the 
possiblity that you have got protein crystals. I once had a condition whith 34 
% MPD + 0.1M buffer + 0.1 M Calciumchloride which produced nicely diffracting  
crystals
To speak from my experience I think 1 month after setting up the trays the 
drops should not be dried out. If the wells are sealed properly new crytals can 
appear even after 1 year.

You should definately check the diffraction then you will know for sure.

Cheers,

Ulrike

Re: [ccp4bb] Salt bridge in crystallization

[Carlos Kikuti]

In think most of the salt bridges I recall in structures are either in the core 
of the protein or in interfaces (crystallin or complex interfaces with little 
or no polar solvent around). Just like charge interactions, the lower 
dielectric constant of the environment makes them stronger.

 Carlos


Em 20/10/2012, às 04:49, Ed Pozharski escreveu:

 On 10/19/2012 10:37 PM, Acoot Brett wrote:
 Will you please explain to me why the protein salt bridge can still exist in 
 the high salt concentration as used in the crystallization condition?
 
 You are saying it as if there is some fundamental law of nature that says 
 that salt bridges cannot be maintained at high salt concentration.  I know of 
 no such law.  The observation simply means that (mostly for entropic reasons) 
 the free energy of the salt bridge is still lower.  If you present your case 
 as to why the salt bridges must be disrupted, we can poke holes in it :)
 
 Cheers,
 
 Ed
 
 -- 
 Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
 Julian, King of Lemurs

[ccp4bb] Salt bridge in crystallization

[Acoot Brett]

Dear All,

A lot of 3-D crystal structures highlight the salt bridges in the structure, 
although some structures of them are got at high salt concentrations. 

Will you please explain to me why the protein salt bridge can still exist in 
the high salt concentration as used in the crystallization condition?

I am looking forward to getting your reply.

Acoot

Re: [ccp4bb] Salt bridge in crystallization

[Ed Pozharski]

On 10/19/2012 10:37 PM, Acoot Brett wrote:
Will you please explain to me why the protein salt bridge can still 
exist in the high salt concentration as used in the crystallization 
condition?




You are saying it as if there is some fundamental law of nature that 
says that salt bridges cannot be maintained at high salt concentration.  
I know of no such law.  The observation simply means that (mostly for 
entropic reasons) the free energy of the salt bridge is still lower.  If 
you present your case as to why the salt bridges must be disrupted, we 
can poke holes in it :)


Cheers,

Ed

--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] salt or protein...

[Pius Padayatti]

Sorry Jay,
They are for sure phosphate crystals.
They always looked like that.
psp

On Sun, May 29, 2011 at 5:18 PM, Jayashankar s.jayashan...@gmail.com wrote:
 Dear Friends ,

 I need to know whether phosphate can form hexagon shaped crystals.
 In one particular condition i have 4 different pattern , 3 seems to me as
 salt crystals, except for the one...
  please find the attachment for the reference..

 Thanks in advance
 Jay
 Institute for Biophysical Chemistry
 Hannover Medical School
 Germany.




-- 
Pius S Padayatti,PhD,
Phone: 216-658-4528

[ccp4bb] salt or protein

[Ramanuj Banerjee]

Dear Jayashankar,
 I had several instances where salt crystals of 
the same component takes different morphologies. So it is very possible, as it 
appears that the hexagonal forms are also those of the salt components in the 
precipitant/buffer. You can confirm the same by fishing out the crystals and 
running it on sds-page.

Re: [ccp4bb] salt or protein crystals?

[David Briggs]

I agree - looks like small molecular diffraction.

Try increasing delta-phi to catch more of the lattice to confirm - I
often do a 5º image or two with the detector pushed as close as
possible to check for salt diffraction when screening.

The lack of low res (~15-20Å) spots around the beamstop is another
smoking gun.

HTH

Dave


David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

http://manchester.academia.edu/DavidBriggs (v.sensible)
http://xtaldave.wordpress.com/ (sensible)
http://xtaldave.posterous.com/ (less sensible)
Twitter: @xtaldave
Skype: DocDCB




On 7 December 2010 14:14, xiuwen zhang congru...@gmail.com wrote:
 Dear Colleagues,

     Currently we got several very tiny crystals. After exposuring a cluster
 of crystals one hour in home source, we could find some weak diffraction
 spots. As the spots are too few for indexing, I am not quite sure whether
 these tiny crystals are salt crystals or protein crystals. I appreciate your
 experience on similar case.

    Attached files are two diffraction images at 0 and 90 degree. Thank you
 very much for your kind help.

 Cheers,
 Xiuwen

Re: [ccp4bb] salt or protein crystals?

[Nian Huang]

Definitely small molecule crystals. You might want to push the
detector closer and use better cryo solution for further confirmation.

Nian

On Tue, Dec 7, 2010 at 8:14 AM, xiuwen zhang congru...@gmail.com wrote:
 Dear Colleagues,

     Currently we got several very tiny crystals. After exposuring a cluster
 of crystals one hour in home source, we could find some weak diffraction
 spots. As the spots are too few for indexing, I am not quite sure whether
 these tiny crystals are salt crystals or protein crystals. I appreciate your
 experience on similar case.

    Attached files are two diffraction images at 0 and 90 degree. Thank you
 very much for your kind help.

 Cheers,
 Xiuwen

Re: [ccp4bb] FW: [ccp4bb] salt sensitive complex

[Jerry McCully]

Dear All:

I am sorry that I did not know the policy.

   And thanks a lot for the kind reminder.

Jerry

CC: CCP4BB@JISCMAIL.AC.UK
From: [EMAIL PROTECTED]
Subject: Re: [ccp4bb] FW: [ccp4bb] salt sensitive complex
Date: Thu, 31 Jan 2008 09:37:01 +0100
To: [EMAIL PROTECTED]

Dear all -Sorry to intervene on a 'book keeping' issue, but indeed over the 
last few months an increasing number of people (Jerry is not the first, so 
Jerry please do not take it personally) attach pictures etc. I think in a bb 
standard practice dictates to only use text - if illustrations are needed to 
explain the problem, you can put them in eg a web site.Some text like that was 
in the 'code of conduct' off ccp4bb in the past, but I could no longer find 
it.Thus apologies if I am wrong and policies have changed, but maybe the ccp4 
crowd could tell us what is the suggested policy.And, if you really want to 
send an image please do bother to make it small. The initial posting had a 630k 
image, which it took me 1 min to make 20k and it still makes the point 
(attached so I can also violate the rules i am suggesting - I love 
inconsistency).Thanks, Tassos


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Re: [ccp4bb] salt sensitive complex

[Jerry McCully]

Dear All:

   Firstly  I would like to thank many folks here for giving me great ideas 
several days ago.

  The following are some updates for this question.

 I did ITC experiments again using 25mMTris(pH8), 60mM NaCl(low salt 
condition).

But things still turn out to be a little weird. 

I increased the concentration of both proteins(60uM in the cell and 1200uM 
in the syringe). At the end of the ITC, I saw a little of precipitation of both 
the proteins.

Fortunately I can roughly fit the curve this time. However, the heat was 
still low, around 1Kcal/mole of per injectant.  I am not sure about the fitting 
statistics.



N 1.10 ±0.17

K 1.49E5 ±1.5E5

DH   -893.5  ±213

DS   20.7Was the enthalpy was offset by the ionization of Tris buffer?Can I 
use Hepes buffer around pH8 to do ITC?
  Welcome any comments about the statistics and suggestions on how to improve 
the ITC experiments.have a nice weekend.

Jerry 










 
 Jerry McCully wrote:
  Dear All:
   
  Recently I am pursuing the crystallziation of a complex formd 
  by two individual proteins and I met several interesting problems 
  though  they are kind of off-topic.
   
  Any suggestions for these problems will be highly appreciated.
   
  BIAcore showed about submicromolar affinity(both Kinetic and 
  steady-state fitting) for these two proteins in the complex. However, 
  precipitates immediately appeared when these two proteins were mixed 
  together even at 10uM(0.3mg/ml) concentration in the condition of low 
  salt(less than 20mM NaCl).
  By the way, these two proteins completely precipitated when the molar 
  ratio is 1:1 in this condition.
   
   THerefore, I increased the salt concentraion step by step and finally 
  I can keep both of them soluble in the solution with 25mM Tris(pH8) 
  and 60mM NaCl(the minimum of salt concentration).   Wierd thing 
  happened when ITC experiments were carried out to confirm the binding 
  affinity.  20uM in the sample cell and 200uM in the syringe could not 
  give enough heat for a good curve fitting. The optimistic estimation 
  of the affinity is lower than 5uM, which is much lower than the 
  affinity given by BIAcore in the same buffer(25mM Tris plus 150mM NaCl).
   
Now I am suspecting the capability of the interaction between 
  these two proteins. However, I can not explain why these two guys 
  precipitated stoichiometrically if they do not interact with each other.
   
Is the complex salt-sensitive therefore there was just minor 
  binding in the high-salt condition revealed by ITC?
   
 I am planning to do the ITC again in the condition of 25mMTris 
  and 60mM NaCl.
   
 What if the affinity given by ITC is still much lower than that 
  by BIAcore. Which one should I choose to believe?
   
Are there some better ways that  I can validate the binding 
  affinity?
   
   
   Thanks again for your great ideas.
   
  Jerry McCully
   
   
 
   
   
 
  
  Need to know the score, the latest news, or you need your Hotmail®-get 
  your fix. Check it out. http://www.msnmobilefix.com/Default.aspx
 
 -- 
 Edwin Pozharski, PhD, Assistant Professor
 University of Maryland, Baltimore
 --
 When the Way is forgotten duty and justice appear;
 Then knowledge and wisdom are born along with hypocrisy.
 When harmonious relationships dissolve then respect and devotion arise;
 When a nation falls to chaos then loyalty and patriotism are born.
 --   / Lao Tse /
 
 

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Re: [ccp4bb] salt sensitive complex

[David Briggs]

Hi Jerry,

to summarise your problem, using (close to) physiological buffer, SPR
and ITC give you different results, you get different results in
different salt strengths and to add to your misery, the proteins
precipitate at low salt concentrations when mixed to together.

Ok.

Given the above, your interaction probably has a large electrostatic
component, and this is why the Ks are salt sensitive. This is also
possibly why you are getting precipitate. If you start with two happy
proteins, and then titrate them in together, the (presumably)
complementary electrostatic binding surfaces will interact and cancel
each other out, reducing the overall charge on the complex. If the
complex is less charged, it will be less soluble, therefore, the
complex crashes out (in 1:1 stoich).

IMHO, ITC is the more elegant experiment. Ok, so it uses *more*
material, but you aren't relying on binding surfaces to nail things
to, and you are directly(ish) measuring the heat of the interaction.

If, in an ITC cell, two proteins come together and are insoluble, they
are able to precipitate. On an SPR chip, they are already immobilised
to a surface, and so you wouldn't necessarily detect precipitation
forming, also, as you run SPR at much lower concentrations of protein
you might not induce precipitation if it is protein concentration
dependent. If I see precipitate in my sample after an ITC experiment,
I'm always weary of it - but at least I'm aware of it.

I would always choose to run an ITC experiment over an SPR (ideally
both), but sometimes, ITCs requirements for higher concentration means
that it isn't always feasible. In this case, the solubility limit of
your complex may prevent you getting good ITC data.

I would try and keep buffers consistent between your experiments
(stick to physiological salt strength - less awkward reviewer
questions), and try repeating your experiments at different pHs. I've
had complexes that precipitate at pH 7.5  9.5, but are nice and happy
at pH 5.5.

Hope this (rather lengthy reply) helps!

Dave


On 23/01/2008, Jerry McCully [EMAIL PROTECTED] wrote:

  Dear All:

  Recently I am pursuing the crystallziation of a complex formd by
 two individual proteins and I met several interesting problems though  they
 are kind of off-topic.

  Any suggestions for these problems will be highly appreciated.

  BIAcore showed about submicromolar affinity(both Kinetic and
 steady-state fitting) for these two proteins in the complex. However,
 precipitates immediately appeared when these two proteins were mixed
 together even at 10uM(0.3mg/ml) concentration in the condition of low
 salt(less than 20mM NaCl).
  By the way, these two proteins completely precipitated when the molar ratio
 is 1:1 in this condition.

   THerefore, I increased the salt concentraion step by step and finally I
 can keep both of them soluble in the solution with 25mM Tris(pH8) and 60mM
 NaCl(the minimum of salt concentration).   Wierd thing happened when ITC
 experiments were carried out to confirm the binding affinity.  20uM in the
 sample cell and 200uM in the syringe could not give enough heat for a good
 curve fitting. The optimistic estimation of the affinity is lower than 5uM,
 which is much lower than the affinity given by BIAcore in the same
 buffer(25mM Tris plus 150mM NaCl).

Now I am suspecting the capability of the interaction between these
 two proteins. However, I can not explain why these two guys precipitated
 stoichiometrically if they do not interact with each other.

Is the complex salt-sensitive therefore there was just minor binding
 in the high-salt condition revealed by ITC?

 I am planning to do the ITC again in the condition of 25mMTris and
 60mM NaCl.

 What if the affinity given by ITC is still much lower than that by
 BIAcore. Which one should I choose to believe?

Are there some better ways that  I can validate the binding affinity?


   Thanks again for your great ideas.

  Jerry McCully






 
 Need to know the score, the latest news, or you need your Hotmail(R)-get your
 fix. Check it out.


-- 

David C. Briggs PhD
Father  Crystallographer
http://www.dbriggs.talktalk.net
AIM ID: dbassophile

[ccp4bb] salt sensitive complex

[Jerry McCully]

Dear All:
 
Recently I am pursuing the crystallziation of a complex formd by two 
individual proteins and I met several interesting problems though  they are 
kind of off-topic.
 
Any suggestions for these problems will be highly appreciated.
 
BIAcore showed about submicromolar affinity(both Kinetic and 
steady-state fitting) for these two proteins in the complex. However, 
precipitates immediately appeared when these two proteins were mixed together 
even at 10uM(0.3mg/ml) concentration in the condition of low salt(less than 
20mM NaCl).
By the way, these two proteins completely precipitated when the molar ratio is 
1:1 in this condition.
 
 THerefore, I increased the salt concentraion step by step and finally I can 
keep both of them soluble in the solution with 25mM Tris(pH8) and 60mM NaCl(the 
minimum of salt concentration).   Wierd thing happened when ITC experiments 
were carried out to confirm the binding affinity.  20uM in the sample cell and 
200uM in the syringe could not give enough heat for a good curve fitting. The 
optimistic estimation of the affinity is lower than 5uM, which is much lower 
than the affinity given by BIAcore in the same buffer(25mM Tris plus 150mM 
NaCl). 
 
  Now I am suspecting the capability of the interaction between these two 
proteins. However, I can not explain why these two guys precipitated 
stoichiometrically if they do not interact with each other.
 
  Is the complex salt-sensitive therefore there was just minor binding in 
the high-salt condition revealed by ITC?
 
   I am planning to do the ITC again in the condition of 25mMTris and 60mM 
NaCl.
 
   What if the affinity given by ITC is still much lower than that by 
BIAcore. Which one should I choose to believe?
 
  Are there some better ways that  I can validate the binding affinity?
 
 
 Thanks again for your great ideas.
 
Jerry McCully
 
  

 
  
_
Need to know the score, the latest news, or you need your Hotmail®-get your 
fix.
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Re: [ccp4bb] salt or protein?

[artem]

Multiple overlapping salt lattices can sometimes look like protein
diffraction, as long as you're looking in only two dimensions. However, if
you can find the dominant rings, you should be able to discriminate since
the c-spacing of salt would nearly always be pretty small. Consider powder
patterns, and you should see what I mean.

Generally speaking, if your crystals visually appear to be single, and
give huge salt peaks - then they're probably salt. Exceptions - large,
non-diffracting protein crystals that have small salt crystals stuck to
them.

Ultimately, you can use the 'stick a fork in it' method: stick a needle in
your rod-like crystals and push. If you hear a crack, and see sharp clean
edges on the break - it's salt. If you feel the crystal 'give' and see
bending or pitting - it's probably protein.

Good luck!

Artem


 Hi All!

 I have been trying to screen for my protein crystals, from the
 crystals grown in 0.5 M Ammonium Sulphate, 1.0M Lithium Sulphate
 Monohydrate in 0.1 M TriSodium Citrate Buffer dihydrate Buffer at pH 5.6.
 Two different kinds of crystals observed: rod shaped and thin platy
 ones. Whenever I am trying to collect data from the rod shaped ones I am
 getting dominantly salt patterns but also spots at 15 A resolution bin
 repeatedly, where some of the spots very much look like from protein!
 what can be the reason for this? if anyone has come across similar
 situation please help.

 with regards,
 Sreeram Mahesh


 Research Student
 Prof S Ramakumar's lab
 PHYSICS department
 IISc Bangalore-560 012.
 ph:080-2293 2718.
 mobile: 9241145183.


 --
 This message has been scanned for viruses and
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 believed to be clean.

Re: [ccp4bb] salt or protein?

[Juergen Bosch]

Hi Sreeram,

assuming you have plenty of those crystals, why don't you loop a few 
pass them through a drop of your reservoir for washing and load them on 
a SDS gel ?


Juergen

Sreeram Mahesh wrote:


Hi All!

   I have been trying to screen for my protein crystals, from the 
crystals grown in 0.5 M Ammonium Sulphate, 1.0M Lithium Sulphate 
Monohydrate in 0.1 M TriSodium Citrate Buffer dihydrate Buffer at pH 5.6.
Two different kinds of crystals observed: rod shaped and thin platy 
ones. Whenever I am trying to collect data from the rod shaped ones I 
am getting dominantly salt patterns but also spots at 15 A resolution 
bin repeatedly, where some of the spots very much look like from protein!
what can be the reason for this? if anyone has come across similar 
situation please help.


with regards,
Sreeram Mahesh


Research Student
Prof S Ramakumar's lab
PHYSICS department
IISc Bangalore-560 012.
ph:080-2293 2718.
mobile: 9241145183.





--
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone:   +1-206-616-4510
FAX: +1-206-685-7002