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

[MARTYN SYMMONS]

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.

[ccp4bb] Postdoc Position in Molecular Neuroscience

[Quyen Hoang]

A postdoctoral position is available to study the mechanisms of  
Parkinson disease - please see attached file for detail.


Genetic studies in the past decade have identified a number of genes  
as causal factors of Parkinson disease. Our research focuses on  
understanding the biochemical functions of the disease associated  
proteins and the biological pathways in which they function. Our core  
research technique is structural biology (protein X-ray  
crystallography) and structure-based drug design, but we use a wide  
range of techniques including, computational chemistry, biophysical  
techniques, enzymology, molecular biology and cell biology.


 Our laboratory is affiliated with the Department of Biochemistry and  
Molecular Biology (http://www.medicine.iu.edu/body.cfm?id=998) as well  
as the Stark Neurosciences Research Institute (http:// 
snri.iusm.iu.edu/) at IUPUI (http://www.iupui.edu/) in Indianapolis  
Indiana (http://en.wikipedia.org/wiki/Indianapolis).


 We are looking for an enthusiastic person who holds a PhD in  
biochemistry or related field, has a strong background in molecular  
biology and protein biochemistry. An ideal candidate should have  
genuine interest in science and desire to learn new techniques to  
tackle challenging and important questions.


 Please email a cover letter, CV, and three reference letters to:

qqho...@iupui.edu

Thank you for looking and happy holidays!

Cheers,
Quyen
___
Quyen Hoang, Ph.D
Department of Biochemistry and Molecular Biology,
Stark Neurosciences Research Institute
Indiana University School of Medicine
635 Barnhill Drive, Room MS0013D
Indianapolis, Indiana 46202-5122

Phone: 317-274-4371
Fax: 317-274-4686
email: qqho...@iupui.edu

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

[David Briggs]

Hi Martyn,

this recipe tends to work for me...

Lysozyme: 50 mg/ml in 0.1 M Sodium Acetate pH 4.8
Reagent: 8% w/v Sodium Chloride, 0.1 M Sodium Acetate pH 4.8
Additional Reagents: Index Reagent 8, 22, 28, 31, 34, 40, 58, 59, 69, 86, 88

Mix equal amounts of lysozyme with reagent, incubate at 4 or 22
degrees Celsius. Batch or vapor diffusion works fine.

==> Direct copy/paste from http://hamptonresearch.com/experiments.aspx <==

HTH and Happy Christmas.

Cheers,

Dave


David C. Briggs PhD

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

Twitter: @xtaldave
Skype: DocDCB




2009/12/21 MARTYN SYMMONS :
> 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.
>

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  
 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] where I have been going wrong in crystallization?

[Jeffrey Wilson]

I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M  
NaCl method mentioned in a Hampton Research catalog and attributed to  
Enrico Stura.  I see that he has also just commented on this thread.   
I found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant  
ratio, lysozyme crystallized in about 1 hour.  Jumping that up to  
150mg/ml allowed for crystallization in minutes.  Hanging drop behaved  
similarly.  I was using lysozyme from Sigma.


Jeff

Jeffrey Wilson, Ph.D.
University of Cincinnati College of Medicine
Molecular Genetics Department
231 Albert Sabin Way
MSB 3109A
Cincinnati, OH 45267-0524
(513) 558-1360


On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS 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.

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

[Mark J. van Raaij]

- I think the original poster was only calling attention to the fact  
that some proteins want to be treated respectfully in order to  
crystallise (and the fact that Rigaku Japan realises this). I find  
that indeed the case.
Other proteins, however, prefer the attitude "I don't know why I am  
setting up these drops, this protein is too crappy to crystallise",  
i.e. a challenge.
Lysozyme, on the other hand, even crystallises under conditions of  
complete indifference. At least I find that every student in a  
practical course can get nice crystals of lysozyme, and a majority of  
these drops have been set up under conditions of complete  
indifference...maybe lysozyme is not a protein after all, but a salt:  
lysozyme-chloride / LyCl7 ?

Mark
PS the detailed protocols and experiences are useful though.


Quoting Jeffrey Wilson :


I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M
NaCl method mentioned in a Hampton Research catalog and attributed to
Enrico Stura.  I see that he has also just commented on this thread.  I
found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant
ratio, lysozyme crystallized in about 1 hour.  Jumping that up to
150mg/ml allowed for crystallization in minutes.  Hanging drop behaved
similarly.  I was using lysozyme from Sigma.

Jeff

Jeffrey Wilson, Ph.D.
University of Cincinnati College of Medicine
Molecular Genetics Department
231 Albert Sabin Way
MSB 3109A
Cincinnati, OH 45267-0524
(513) 558-1360


On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS 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.

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

[mjvdwoerd]

 I was going to comment that I have learned the following: "respect" does not 
mean the same thing in all places in the world. Some time back I had a protein 
here that I thought needed extra respect and I had learned from a Rigaku 
employee how to do this - I bowed very very deeply in front of the protein 
before handling it. But it still did not crystallize. So when I complained to 
the Rigaku employee about the "recipe", he asked with appropriate hesitation in 
his voice: are you saying that you only bowed ONCE?

In defense of the original poster, I think the recipe on the Rigaku web site is 
entirely correct, but it does not specify how to pay proper respect. This was 
self-evident to the person who wrote down the recipe, but as we all know, what 
it obvious to one person, is not obvious to the next - especially in such 
difficult things like respect. Good recipes are indispensable and should be 
explicit about such important ingredients. 

Mark

 

 

-Original Message-
From: Mark J. van Raaij 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Mon, Dec 21, 2009 12:18 pm
Subject: Re: [ccp4bb] where I have been going wrong in crystallization?


- I think the original poster was only calling attention to the fact that some 
proteins want to be treated respectfully in order to crystallise (and the fact 
that Rigaku Japan realises this). I find that indeed the case. 
Other proteins, however, prefer the attitude "I don't know why I am setting up 
these drops, this protein is too crappy to crystallise", i.e. a challenge. 
Lysozyme, on the other hand, even crystallises under conditions of complete 
indifference. At least I find that every student in a practical course can get 
nice crystals of lysozyme, and a majority of these drops have been set up under 
conditions of complete indifference...maybe lysozyme is not a protein after 
all, but a salt: lysozyme-chloride / LyCl7 ? 
Mark 
PS the detailed protocols and experiences are useful though. 
 
Quoting Jeffrey Wilson : 
 
> I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M 
> NaCl method mentioned in a Hampton Research catalog and attributed to 
> Enrico Stura.  I see that he has also just commented on this thread.  I 
> found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant 
> ratio, lysozyme crystallized in about 1 hour.  Jumping that up to 
> 150mg/ml allowed for crystallization in minutes.  Hanging drop behaved 
> similarly.  I was using lysozyme from Sigma. 
> 
> Jeff 
> 
> Jeffrey Wilson, Ph.D. 
> University of Cincinnati College of Medicine 
> Molecular Genetics Department 
> 231 Albert Sabin Way 
> MSB 3109A 
> Cincinnati, OH 45267-0524 
> (513) 558-1360 
> 
> 
> On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS 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. 

 

Re: [ccp4bb] phenix refinement peptide bond poor geometry

[Salameh, Mohd A., Ph.D.]

I'd like to thank you all, the problem is resolved. Apparently, in the
pdb file, the carbonyl oxygen should be listed right after the carbonyl
carbon not after the side chain atoms! M 

 

** 
Mohd A. Salameh, Ph.D. 
Mayo Clinic Cancer Center 
Griffin Cancer Research building,Rm 331 
4500 San Pablo Rd 
Jacksonville, FL 32224 
Tel: (904) 953-0046 
Fax: (904) 953-0277 
salameh.m...@mayo.edu 
** 



From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Salameh, Mohd A., Ph.D.
Sent: Friday, December 18, 2009 5:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] phenix refinement peptide bond poor geometry

 

Dear all, 

I'm about to deposit  a structure into the protein data bank and I'm
encountering a disturbing problem, I have a protein site where the
peptide bond is not properly linked, the distance of C-N bond is 2.23
Angstrom, I tried to restrain that site by modifying my def file but the
problem persist after refinement. I wonder if anybody can help resolving
this problem. Thanks and happy holidays! Mohd

**

Mohd A. Salameh, Ph.D.

Mayo Clinic Cancer Center

Griffin Cancer Research building,Rm 331

4500 San Pablo Rd

Jacksonville, FL 32224

Tel: (904) 953-0046

Fax: (904) 953-0277

salameh.m...@mayo.edu

**

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

[Felix Frolow]

If "respect" is concerned, try to stop a blow of a sharp samurai sword with  a 
sharp word.
I myself have no problems whatsoever with the Rigaku recipes. And yes, I have 
stopped,
at leas once, a blow of samurai sword  with my bare hands. Since than I paint 
pictures with my left foot.
BTW
In ALL my crystallization attempts I ALWAYS use double distilled (rectified) 
water. However there is, I must admit, a fume
of triple distilled Jameson around (almost always).  All this is not written in 
the Rigaku recipe but is very important.
Dr  Felix Frolow
Professor of Structural Biology and Biotechnology
Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica D, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:   ++972 3640 8723
Fax:  ++972 3640 9407
Cellular:   ++972 547 459 608

On Dec 21, 2009, at 23:02 , mjvdwo...@netscape.net wrote:

> I was going to comment that I have learned the following: "respect" does not 
> mean the same thing in all places in the world. Some time back I had a 
> protein here that I thought needed extra respect and I had learned from a 
> Rigaku employee how to do this - I bowed very very deeply in front of the 
> protein before handling it. But it still did not crystallize. So when I 
> complained to the Rigaku employee about the "recipe", he asked with 
> appropriate hesitation in his voice: are you saying that you only bowed ONCE?
> 
> In defense of the original poster, I think the recipe on the Rigaku web site 
> is entirely correct, but it does not specify how to pay proper respect. This 
> was self-evident to the person who wrote down the recipe, but as we all know, 
> what it obvious to one person, is not obvious to the next - especially in 
> such difficult things like respect. Good recipes are indispensable and should 
> be explicit about such important ingredients. 
> 
> Mark
> 
> 
> -Original Message-
> From: Mark J. van Raaij 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Mon, Dec 21, 2009 12:18 pm
> Subject: Re: [ccp4bb] where I have been going wrong in crystallization?
> 
> - I think the original poster was only calling attention to the fact that 
> some proteins want to be treated respectfully in order to crystallise (and 
> the fact that Rigaku Japan realises this). I find that indeed the case. 
> Other proteins, however, prefer the attitude "I don't know why I am setting 
> up these drops, this protein is too crappy to crystallise", i.e. a challenge. 
> Lysozyme, on the other hand, even crystallises under conditions of complete 
> indifference. At least I find that every student in a practical course can 
> get nice crystals of lysozyme, and a majority of these drops have been set up 
> under conditions of complete indifference...maybe lysozyme is not a protein 
> after all, but a salt: lysozyme-chloride / LyCl7 ? 
> Mark 
> PS the detailed protocols and experiences are useful though. 
>  
> Quoting Jeffrey Wilson : 
>  
> > I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M 
> > NaCl method mentioned in a Hampton Research catalog and attributed to 
> > Enrico Stura. I see that he has also just commented on this thread. I 
> > found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant 
> > ratio, lysozyme crystallized in about 1 hour. Jumping that up to 
> > 150mg/ml allowed for crystallization in minutes. Hanging drop behaved 
> > similarly. I was using lysozyme from Sigma. 
> > 
> > Jeff 
> > 
> > Jeffrey Wilson, Ph.D. 
> > University of Cincinnati College of Medicine 
> > Molecular Genetics Department 
> > 231 Albert Sabin Way 
> > MSB 3109A 
> > Cincinnati, OH 45267-0524 
> > (513) 558-1360 
> > 
> > 
> > On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS 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. 

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

[Karthik S]

It makes slight sense if the word 'respect' can be correlated with whether
the drop is mixed (protein+ppt). i have heard some protein may indeed
crystallize if it were not mixed or vice-versa (do not expect it with
lysozyme though). it would make sense if diffusion played a role but with
few ul drop (the tip of pipette covering the surface of the drop in
96-well), i imagine what would be the real reason/advantage for this
argument of mixing vs not mixing drops.

--
Karthik

On Mon, Dec 21, 2009 at 4:19 PM, Felix Frolow wrote:

> If "respect" is concerned, try to stop a blow of a sharp samurai sword with
>  a sharp word.
> I myself have no problems whatsoever with the Rigaku recipes. And yes, I
> have stopped,
> at leas once, a blow of samurai sword  with my bare hands. Since than I
> paint pictures with my left foot.
> BTW
> In ALL my crystallization attempts I ALWAYS use double distilled
> (rectified) water. However there is, I must admit, a fume
> of triple distilled Jameson around (almost always).  All this is not
> written in the Rigaku recipe but is very important.
>
> Dr  Felix Frolow
>
> Professor of Structural Biology and Biotechnology
>
> Department of Molecular Microbiology
>
> and Biotechnology
>
> Tel Aviv University 69978, Israel
>
>
> Acta Crystallographica D, co-editor
>
>
> e-mail: mbfro...@post.tau.ac.il
>
> Tel:   ++972 3640 8723
>
> Fax:  ++972 3640 9407
> Cellular:   ++972 547 459 608
>
> On Dec 21, 2009, at 23:02 , mjvdwo...@netscape.net wrote:
>
>  I was going to comment that I have learned the following: "respect" does
> not mean the same thing in all places in the world. Some time back I had a
> protein here that I thought needed extra respect and I had learned from a
> Rigaku employee how to do this - I bowed very very deeply in front of the
> protein before handling it. But it still did not crystallize. So when I
> complained to the Rigaku employee about the "recipe", he asked with
> appropriate hesitation in his voice: are you saying that you only bowed
> ONCE?
>
> In defense of the original poster, I think the recipe on the Rigaku web
> site is entirely correct, but it does not specify how to pay proper respect.
> This was self-evident to the person who wrote down the recipe, but as we all
> know, what it obvious to one person, is not obvious to the next - especially
> in such difficult things like respect. Good recipes are indispensable and
> should be explicit about such important ingredients.
>
> Mark
>
>
>  -Original Message-
> From: Mark J. van Raaij 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Mon, Dec 21, 2009 12:18 pm
> Subject: Re: [ccp4bb] where I have been going wrong in crystallization?
>
>  - I think the original poster was only calling attention to the fact that
> some proteins want to be treated respectfully in order to crystallise (and
> the fact that Rigaku Japan realises this). I find that indeed the case.
> Other proteins, however, prefer the attitude "I don't know why I am setting
> up these drops, this protein is too crappy to crystallise", i.e. a
> challenge.
> Lysozyme, on the other hand, even crystallises under conditions of complete
> indifference. At least I find that every student in a practical course can
> get nice crystals of lysozyme, and a majority of these drops have been set
> up under conditions of complete indifference...maybe lysozyme is not a
> protein after all, but a salt: lysozyme-chloride / LyCl7 ?
> Mark
> PS the detailed protocols and experiences are useful though.
>
> Quoting Jeffrey Wilson :
>
> > I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M
> > NaCl method mentioned in a Hampton Research catalog and attributed to
> > Enrico Stura. I see that he has also just commented on this thread. I
> > found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant
> > ratio, lysozyme crystallized in about 1 hour. Jumping that up to
> > 150mg/ml allowed for crystallization in minutes. Hanging drop behaved
> > similarly. I was using lysozyme from Sigma.
> >
> > Jeff
> >
> > Jeffrey Wilson, Ph.D.
> > University of Cincinnati College of Medicine
> > Molecular Genetics Department
> > 231 Albert Sabin Way
> > MSB 3109A
> > Cincinnati, OH 45267-0524
> > (513) 558-1360
> >
> >
> > On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS 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.
>
>
>

[ccp4bb] combining AutoMR and brute

[Francois Berenger]

Hello,

Is there any way to search in AutoMR mode but have the
translation only being brute forced?

Regards,
F.

Re: [ccp4bb] combining AutoMR and brute [Phaser]

[Francois Berenger]

Sorry, I forgot to mention my previous message concerns using Phaser.

Francois Berenger wrote:

Hello,

Is there any way to search in AutoMR mode but have the
translation only being brute forced?

Regards,
F.

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

[Damian Ekiert]

Steve,

My general strategy is to start with an "ideal" glycan (an Asn linked to 
NAG-NAG-(MAN)3 ) and superimpose the Asn on the residue from my protein. 
 Then you can move the whole glycan as a rigid body until the Asn and 
first NAG are roughly positioned.  Then you can tweak any sugars further 
out on the chain to get them to fit.  Unless you have really great 
density, usually it is best to avoid real pace refine zone.  Better to 
fit the sugars using the manual rigid body fitting tools, do the best 
you can, then REFMAC usually brings them in OK.


I have some models that I could send you if you need them.

Best,

Damian Ekiert



Soisson, Stephen M wrote:

Hi everyone-

I was searching for some information on what might be the best way to 
add N-linked sugars in coot, and Google has let me down.  Searching 
"adding sugars in coot" returns a very nice recipe for Coot Pudding.


***_Recipe for_/ Coot//__/_ Pudding - American_/ Coots/* 
 It has plenty of fat,/ 
sugar/, and starch, and probably some calcium from the milk.* ...* The/ 
coots/ will not tolerate/ adding/ eggs in any form, so this is an egg* ...*
///_www.beaky_//_*coot*.com/pudding.html_/// 
/ -/ _Similar_ 
// 




I did not know that coots had such an aversion to eggs. :)

Anyway, would anyone have any top tips on adding N-linked sugars using 
coot?  I can import the NAG monomers, but linking them up to the protein 
seems non-trivial


Many thanks in advance,

Steve


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

[David Salom]

Maybe is a mistake by a non-English speaker. Instead of "respectfully" should 
say 
"respectively"

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

[Robbie Joosten]

Dear Steve,

I would also use Damian's approach, but the sequence of the core should be
NAG-NAG-BMA-(MAN)2. This is improtant because the correct stereochemistry
restraints for beta-mannose can only be applied when you call the residue
BMA.
Building carbohydrates also comes with special validation requirements.
PDB-care and CARP are both very usefull. Unfortunately, the service is
currently down (http://www.dkfz.de/spec/glycosciences.de). Just make sure
the links between your carbs are correct and, please, remove the O1 atoms
when needed ;)

Cheers,
Robbie Joosten


> Date: Mon, 21 Dec 2009 17:48:31 -0800
> From: dceki...@scripps.edu
> Subject: Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition)
> To: CCP4BB@JISCMAIL.AC.UK
>
> Steve,
>
> My general strategy is to start with an "ideal" glycan (an Asn linked to
> NAG-NAG-(MAN)3 ) and superimpose the Asn on the residue from my protein.
> Then you can move the whole glycan as a rigid body until the Asn and
> first NAG are roughly positioned. Then you can tweak any sugars further
> out on the chain to get them to fit. Unless you have really great
> density, usually it is best to avoid real pace refine zone. Better to
> fit the sugars using the manual rigid body fitting tools, do the best
> you can, then REFMAC usually brings them in OK.
>
> I have some models that I could send you if you need them.
>
> Best,
>
> Damian Ekiert
>
>
>
> Soisson, Stephen M wrote:
> > Hi everyone-
> >
> > I was searching for some information on what might be the best way to
> > add N-linked sugars in coot, and Google has let me down. Searching
> > "adding sugars in coot" returns a very nice recipe for Coot Pudding.
> >
> > ***_Recipe for_/ Coot//__/_ Pudding - American_/ Coots/*
> >  It has plenty of fat,/
> > sugar/, and starch, and probably some calcium from the milk.* ...* The/
> > coots/ will not tolerate/ adding/ eggs in any form, so this is an egg*
...*
> > ///_www.beaky_//_*coot*.com/pudding.html_///
> > / -/ _Similar_
> > //
> >
> >
> >
> > I did not know that coots had such an aversion to eggs. :)
> >
> > Anyway, would anyone have any top tips on adding N-linked sugars using
> > coot? I can import the NAG monomers, but linking them up to the protein
> > seems non-trivial
> >
> > Many thanks in advance,
> >
> > Steve
> >
> >
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