Re: [ccp4bb] side chain density

[Matthew Franklin]

Dear Remy and Faisal -

Just a geeky bioinformatics note:  searching my local copy of the PDB 
(somewhat out of date), shows that 22,165 out of 72,000 structures 
contain a 'REMARK 470' line, which is how the PDB notes that atoms are 
missing from a residue.  Some of these are no doubt mistakes by the 
depositors, but most probably reflect the decision to remove invisible 
side chain atoms from the coordinates (e.g. trimming back to Cbeta) 
while keeping the correct residue name.


I think we can all agree that virtually every structure in the PDB will 
have a few residues where some of the atoms are not visible in the 
density.  So the "trim the side chains" crowd is a well-represented 
minority at 30%, but 70% of depositors chose another option.


I personally like to leave all atoms on the side chains; they look wrong 
to me when beheaded.  I just try to put the invisible atoms in a 
stereochemically plausible conformation, leave the occupancy set to 1, 
and let the refinement program deal with them.


- Matt


On 11/9/12 3:47 PM, Remy Loris wrote:

Dear Faisal,

You definitely do not mutate to alanine as that would imply for the 
future "user" of your pdb file that it is a mutant.
Some people feel they have to keep the side chain but put the 
occupancies at zero. I think this is a bad practice and strongly 
oppose to it as for the future  "user" of your deposited pdb file, who 
often is not a crystallographer, you suggest a specific conformation 
for your side chain that may be interpreted in terms of biology, while 
in reality it addopts a huge, disordered ensemble of conformations.
Personally I am of the opinion that you should simply remove the side 
chain atoms (but keep the residue name). And that is the same as what 
you do with a whole loop that is disordered. I think it is lso the 
most common practice in deposited structures.


Remy Loris
Vrije Universiteit Brussel

On 09/11/12 20:22, Faisal Tarique wrote:

Dear all

i have solved a structure ( at 2A resolution) whose Rwork and Rfree 
is 22 and 25 respectively..the Ramachandran plot shows 90% of the 
residues in the most favorable region and with 6 residues in 
generously allowed and no residues in disallowed region. But in some 
areas i can see density missing for side chains ( in loop regions 
)..i have question do i need to mutate them to alanine or leave them 
as such..The density fit analysis in COOT ( traffic light) showing 
those regions with side chain as red..


thanx in advance

Regards

Faisal
School of Life Sciences
JNU







--
Matthew Franklin, Ph. D.
Senior Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(212) 939-0660 ext. 9374

Re: [ccp4bb] model protein for ligand soaking

[Dmitry Rodionov]

Hi Uli,

I think Proteinase K and PMSF should work, though I never tried.

Cheers, 
Dmitry


On 2012-11-09, at 9:12 AM, Ulrich Zander wrote:

> Hi All,
> 
> 
> For a tutorial, we would like to demonstrate the method of ligand soaking in 
> protein crystals. I am thinking about using some sort of native ligand or 
> inhibitor that can be easily identified in the electron density rather than 
> halide or heavy atom derivatization.
> 
> Does anybody have a suggestion for a protein/ligand combination that could be 
> used for that and that is commercially available?
> 
> 
> Thanks!
> 
> Uli
> 
> 
> 
> 
> 
> 
> 
> 
> -- 
> 
> Ulrich Zander
> High Troughput Crystallization Lab/Diffraction Instrumentation Team
> EMBL Grenoble Outstation
> 6 rue Jules Horowitz
> 38000 Grenoble, France
> Phone: +33 47620-9487
> 

Re: [ccp4bb] low-resolution and zinc

[Roger Rowlett]

Are you trying to have Coot display the Zn-ligand bonds? That's a 
different issue from having refmac recognize and use the metal-ligand 
restraints in refinement. (I don't bother to have Coot do this.) Looking 
at your environment distances, it looks like refmac has done a 
restrained refinement, as your Zn-ligand bond distances look right. You 
can tell in the refmac log file if you get a message describing that a 
Zn-ligand distance was found and it is not listed as "not be used." (If 
you get this latter message, you have the wrong link usage setting 
chosen in the CCP4i GUI.) I just had an undergraduate student do the 
procedure yesterday to do a restrained refinement for a ZnCys2HisAsp 
environment. Refmac picks up the Zn-Cys link in the library without any 
intervention, and the Zn-His and Zn-Asp bonds have to be taken care of 
in the refmac-generated .cif file. All the links show up in the LINKR 
records. There may be a more elegant way to do this but it works.


___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 11/9/2012 1:09 PM, SD Y wrote:

Dear All,

Thanks for all the suggestions on low resolution, SG and Zn and I 
learned a  lot. I am not done yet.


I am trying model co-ordinated ZN+2. I got lot of help from Prof. 
Roger Rowlett. Also an excellent protocol is available at 
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. Protocol 
seems to be very straight forward. I am not getting the result I wanted.


1. When ever I generate cif file,  its not writing any for ZN-SG links 
at all.


2. After refmac refinement it only draw in LINK for His-ZN. Please see 
the images

https://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.png
https://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png

3. I get this His-Zn link without using .cif file, so what stage do I 
use this .cif file?


4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not 
writing LINK in PDB. I only see
LINKR   ZNZN H   1 SG  CYS A  
83ZN-CYS


I dont know what is missing, I also attached the log file which 
generated the ZN-His coordination.


Any help is highly appreciated.

Thanks
SDY


Date: Thu, 8 Nov 2012 14:36:59 +0200
From: mbfro...@post.tau.ac.il
Subject: Re: [ccp4bb] low-resolution and zinc
To: CCP4BB@JISCMAIL.AC.UK

The experiment should be very problematic if I can't determine point 
group on the base of the symmetry merging statistics.

Watch CHI2 :-)
Dr Felix Frolow
Professor of Structural Biology and Biotechnology, Department of 
Molecular Microbiology and Biotechnology

Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

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

On Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com 
 wrote:


Then we agree. I got confused because you mentioned"space group"
and not "point group" in your phrase about PHASER and MOLREP and
was afraid others might have gotten confused as well. Also, in
case of twinning or almost crystallographic non-crystallographic
symmetry, determining the point group on the basis of processing
statistics alone can be inconclusive or even misleading. If I
recall correctly, there has recently been a thread about this in
the bulletin board.
Herman

Re: [ccp4bb] Assemble Protein-DNA complex

[David Schuller]

On 11/09/12 15:43, Tommi Kajander wrote:
It is not very surprising that the affinity gets higher with lower 
salt, right? 


Not at all. I wanted to ask if their assay could distinguish between 
specific binding and nonspecific binding, but I decided not to sidetrack 
the discussion.


--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

Re: [ccp4bb] side chain density

[Remy Loris]

Dear Faisal,

You definitely do not mutate to alanine as that would imply for the 
future "user" of your pdb file that it is a mutant.
Some people feel they have to keep the side chain but put the 
occupancies at zero. I think this is a bad practice and strongly oppose 
to it as for the future  "user" of your deposited pdb file, who often is 
not a crystallographer, you suggest a specific conformation for your 
side chain that may be interpreted in terms of biology, while in reality 
it addopts a huge, disordered ensemble of conformations.
Personally I am of the opinion that you should simply remove the side 
chain atoms (but keep the residue name). And that is the same as what 
you do with a whole loop that is disordered. I think it is lso the most 
common practice in deposited structures.


Remy Loris
Vrije Universiteit Brussel

On 09/11/12 20:22, Faisal Tarique wrote:

Dear all

i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 
22 and 25 respectively..the Ramachandran plot shows 90% of the 
residues in the most favorable region and with 6 residues in 
generously allowed and no residues in disallowed region. But in some 
areas i can see density missing for side chains ( in loop regions )..i 
have question do i need to mutate them to alanine or leave them as 
such..The density fit analysis in COOT ( traffic light) showing those 
regions with side chain as red..


thanx in advance

Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] usefulness of cacodylate?

[Bryan Lepore]

On Fri, Nov 9, 2012 at 7:26 AM, Frank von Delft  wrote

> Anybody know
> a) how hazardous is cacodylate?
> b) does it really matter for crystallization screens?

[...]

>

(We're being subjected to a safety review.)


I know you are in the UK but this wouldn't have anything to do with this:

http://bostonglobe.com/lifestyle/health-wellness/2012/11/05/what-telling-patients-about-arsenic-and-rice/8VkGh3K3TtXEaucIzPEHvJ/story.html

would it...?

-Bryan

Re: [ccp4bb] Assemble Protein-DNA complex

[Tommi Kajander]

It is not very surprising that the affinity gets higher with lower salt, right? 
Why dont you measure it under _physiological_ salt concentration? (or i assume 
maybe you did?)
and of course its not as high affinity due to screening (but physiological 
conditions)
of the electrostatic interactions. 

If the complex doesnt stay together in ca. 150 mM salt (e.g in TBS) in gel 
filtration (which you
dont say if you tried) then why not just try mixing it in the drop and screen, 
this is what
you would do if you cant purify the complex. and try different rations. 

There are texts on preparation of DNA complexes in case you dont have advice 
for it.

Tommi

On Nov 9, 2012, at 6:50 PM, Tim Gruene wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Dear Wei Huang,
> 
> if you are lucky you can form the complex as crystal in the drop (I
> assume you want to crystallise the protein-DNA complex):
> 
> set up drops at high salt concentration (as low as possible to keep
> the protein in solution at reasonable concentration) in the presence
> of DNA.
> 
> Prepare the buffer the same way as the protein buffer, but with
> reduced salt concentration (btw, you can also try divalent ions, eg.
> MgCl2).
> This way water evaporates into the drop, diluting the salt concentration.
> 
> - From your discription the solubility of the protein drops faster than
> its concentration, hence it should precipitate. And, as I said, if you
> are lucky, it does so as a crystal in complex with the DNA.
> 
> Best,
> Tim
> 
> On 11/09/2012 05:14 PM, Wei Huang wrote:
>> Dear CCP4BBers,
>> 
>> I have a problem in purifying protein-DNA complex for a protein
>> that I am interested in.
>> 
>> The purification of protein only has been optimized and I've get
>> enough yield for what I need (10 mg/2.4 L growth). And I've
>> measured DNA binding using Fluorescence Anisotropy. The results
>> show that my protein has the tightest binding (Kd=8 nM) to the DNA
>> at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5).
>> 
>> However, I came across several problems when I assemble protein-DNA
>> complex in large scale.
>> 
>> First, my protein is unstable at low salt condition. When I
>> dialyzes my protein into low salt buffer (tried 30 mM and 100 mM
>> KCl) for binding DNA, the protein precipitates. What I don't quite
>> understand is that the DNA binding assay performed at low salt
>> condition doesn't seem to be affected by this instability of
>> protein. I guess it may be due to the assay was performed at very
>> diluted protein concentration (in nM).
>> 
>> Second, I can not purify protein-DNA complex at high salt condition
>> with gel filtration column. Because of the first problem, I tried
>> to assemble the complex at high salt condition (150 mM KCl, 150 mM
>> NaCl). However, the elution profile shows no binding of DNA to my
>> protein (no increase in the observation of protein peak and a large
>> peak around expected position for DNA). This may be due to weaker
>> binding at high salt as my DNA binding assay shows that the Kd
>> under this buffer condition is ~1100 nM.
>> 
>> Third, a lot of protein is lost during dialysis of protein-DNA
>> complex into low salt condition. I tried add DNA directly into
>> protein in high salt buffer, then dialyze very slowly against low
>> salt buffer. However, I still lost quite a lot of protein due to
>> precipitation. I was able to load some sample onto the gel
>> filtration column with low salt running buffer. And I saw the shift
>> of protein peak in the elution profile, also protein concentration
>> measured by Bradford assay shows that the protein concentration is
>> much less than that expected from uv trace, suggesting the 
>> contribution to the absorbance from DNA. But the yield is very low,
>> less than 0.2 mg of protein is left and the complex seems to be
>> unhappy when I concentrate it. So I can not get protein sample
>> concentrated enough for my study.
>> 
>> My previous experience with another DNA binding protein is much
>> better. I purified it in high salt, dialyzed into low salt to
>> binding DNA and finally purify with gel filtration column. However,
>> the one I am currently working on seems to be very picky. If you
>> have any suggestion regarding to my problems, I will be thankful.
>> 
>> Best regards,
>> 
> 
> - -- 
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
> 
> GPG Key ID = A46BEE1A
> 
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.12 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
> 
> iD8DBQFQnTRFUxlJ7aRr7hoRAvscAJ9Bm/3ix2ln69wZz74LWwaPMdBhFQCfS9C6
> nvrZMfMmeekAauJ26jy6jbE=
> =nbV+
> -END PGP SIGNATURE-

Tommi Kajander, Ph.D., Docent
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1
(P.O. Box 65)
00014 Helsinki
Finland
p. +358-9-19158903
tommi.kajan...@helsinki.fi
http://www.biocenter.helsinki.fi/bi/kajander/

Re: [ccp4bb] side chain density

[Ethan Merritt]

On Friday, 09 November 2012, Faisal Tarique wrote:
> Dear all
> 
> i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22
> and 25 respectively..the Ramachandran plot shows 90% of the residues in the
> most favorable region and with 6 residues in generously allowed and no
> residues in disallowed region. But in some areas i can see density missing
> for side chains ( in loop regions )..i have question do i need to mutate
> them to alanine or leave them as such.

Mutating to alanine is not an option.
They are not alanine.
If nothing else, when you get to the point of depositing your
structure in the PDB it will fail validation checks because
the sequence is not correct at those points.

But if you mean should you delete sidechain atoms beyond
CB, that is another question.  That is a legitimate option.
I suggest trying that and then looking in difference density
maps to see if any density shows up to guide placement of
the sidechain.

Ethan

> .The density fit analysis in COOT (
> traffic light) showing those regions with side chain as red..
> 
> thanx in advance
> 
> Regards
> 
> Faisal
> School of Life Sciences
> JNU
> 

Re: [ccp4bb] side chain density

[Lari Lehtiö]

Hi,

There has been quite a lot of discussion about this and I think  
different opinions exist. I would try to keep the side chains, if  
there is some evidence where they are. Otherwise I would just delete  
atoms and I would not mutate them to alanine.


http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg20437.html

Best regards,

Lari

__
Lari Lehtiö, PhD, Adjunct Professor
Biocenter Oulu
Department of Biochemistry
P.O.Box 3000
FIN-90014 University of Oulu
Finland
http://cc.oulu.fi/~llehtio/
_


Quoting Faisal Tarique :


Dear all

i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22
and 25 respectively..the Ramachandran plot shows 90% of the residues in the
most favorable region and with 6 residues in generously allowed and no
residues in disallowed region. But in some areas i can see density missing
for side chains ( in loop regions )..i have question do i need to mutate
them to alanine or leave them as such..The density fit analysis in COOT (
traffic light) showing those regions with side chain as red..

thanx in advance

Regards

Faisal
School of Life Sciences
JNU

[ccp4bb] side chain density

[Faisal Tarique]

Dear all

i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22
and 25 respectively..the Ramachandran plot shows 90% of the residues in the
most favorable region and with 6 residues in generously allowed and no
residues in disallowed region. But in some areas i can see density missing
for side chains ( in loop regions )..i have question do i need to mutate
them to alanine or leave them as such..The density fit analysis in COOT (
traffic light) showing those regions with side chain as red..

thanx in advance

Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] low-resolution and zinc

[SD Y]

I am using  Refmac_5.7.0029 in CCP4ThanksSDY Date: Fri, 9 Nov 2012 20:35:48 
+0200
From: mbfro...@post.tau.ac.il
Subject: Re: [ccp4bb] low-resolution and zinc
To: CCP4BB@JISCMAIL.AC.UK

What program do you use for refinement?FF

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

Acta Crystallographica F, co-editor

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


On Nov 9, 2012, at 20:09 , SD Y  wrote:Dear All,
Thanks for all the suggestions on low resolution, SG and Zn and I learned a  
lot. I am not done yet.
I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger 
Rowlett. Also an excellent protocol is available at 
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. 
Protocol seems to be very straight forward. I am not getting the result I 
wanted.
1. When ever I generate cif file,  its not writing any for ZN-SG links at all.
2. After refmac refinement it only draw in LINK for His-ZN. Please see the 
imageshttps://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.pnghttps://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png
3. I get this His-Zn link without using .cif file, so what stage do I use this 
.cif file?
4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing 
LINK in PDB. I only see LINKR   ZNZN H   1 SG  CYS A  
83ZN-CYS
I dont know what is missing, I also attached the log file which generated the 
ZN-His coordination.
Any help is highly appreciated.
ThanksSDY
Date: Thu, 8 Nov 2012 14:36:59 +0200
From: mbfro...@post.tau.ac.il
Subject: Re: [ccp4bb] low-resolution and zinc
To: CCP4BB@JISCMAIL.AC.UK

The experiment should be very problematic if I can't determine point group on 
the base of the symmetry merging statistics.Watch CHI2 :-)
Dr Felix Frolow   
Professor of Structural Biology and Biotechnology, Department of Molecular 
Microbiology and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608
On Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com wrote:Then we agree. I 
got confused because you mentioned"space group" and not "point group" in your 
phrase about PHASER and MOLREP and was afraid others might have gotten confused 
as well. Also, in case of twinning or almost crystallographic 
non-crystallographic symmetry, determining the point group on the basis of 
processing statistics alone can be inconclusive or even misleading. If I recall 
correctly, there has recently been a thread about this in the bulletin 
board.Herman



<74_refmac5_1.log>
  

Re: [ccp4bb] low-resolution and zinc

[Felix Frolow]

What program do you use for refinement?
FF
Dr Felix Frolow   
Professor of Structural Biology and Biotechnology, Department of Molecular 
Microbiology and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

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

On Nov 9, 2012, at 20:09 , SD Y  wrote:

> Dear All,
> 
> Thanks for all the suggestions on low resolution, SG and Zn and I learned a  
> lot. I am not done yet.
> 
> I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger 
> Rowlett. Also an excellent protocol is available at 
> http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. 
> Protocol seems to be very straight forward. I am not getting the result I 
> wanted.
> 
> 1. When ever I generate cif file,  its not writing any for ZN-SG links at all.
> 
> 2. After refmac refinement it only draw in LINK for His-ZN. Please see the 
> images
> https://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.png
> https://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png
> 
> 3. I get this His-Zn link without using .cif file, so what stage do I use 
> this .cif file?
> 
> 4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing 
> LINK in PDB. I only see 
> LINKR   ZNZN H   1 SG  CYS A  83ZN-CYS
> 
> I dont know what is missing, I also attached the log file which generated the 
> ZN-His coordination.
> 
> Any help is highly appreciated.
> 
> Thanks
> SDY
> 
> Date: Thu, 8 Nov 2012 14:36:59 +0200
> From: mbfro...@post.tau.ac.il
> Subject: Re: [ccp4bb] low-resolution and zinc
> To: CCP4BB@JISCMAIL.AC.UK
> 
> The experiment should be very problematic if I can't determine point group on 
> the base of the symmetry merging statistics.
> Watch CHI2 :-)
> Dr Felix Frolow   
> Professor of Structural Biology and Biotechnology, Department of Molecular 
> Microbiology and Biotechnology
> Tel Aviv University 69978, Israel
> 
> Acta Crystallographica F, co-editor
> 
> e-mail: mbfro...@post.tau.ac.il
> Tel:  ++972-3640-8723
> Fax: ++972-3640-9407
> Cellular: 0547 459 608
> 
> On Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com wrote:
> 
> Then we agree. I got confused because you mentioned"space group" and not 
> "point group" in your phrase about PHASER and MOLREP and was afraid others 
> might have gotten confused as well. Also, in case of twinning or almost 
> crystallographic non-crystallographic symmetry, determining the point group 
> on the basis of processing statistics alone can be inconclusive or even 
> misleading. If I recall correctly, there has recently been a thread about 
> this in the bulletin board.
> Herman
> 
> 
> 
> 
> <74_refmac5_1.log>

Re: [ccp4bb] Assemble Protein-DNA complex

[James Stroud]

I meant "where the drops have the concentration of ammonium acetate needed".

James


On Nov 9, 2012, at 10:06 AM, James Stroud wrote:

> This sounds like a job for ammonium acetate. Use it as your salt. Purify your 
> complex in it and then set up drops where they wells have the amount of 
> ammonium acetate needed to keep your protein stable and the wells have none, 
> or a range of concentrations. The ammonium acetate will equilibrate by vapor 
> diffusion, lowering the concentration in the drop and causing your complex to 
> come out of solution.
> 
> James
> 
> On Nov 9, 2012, at 9:14 AM, Wei Huang wrote:
> 
>> Dear CCP4BBers,
>> 
>> I have a problem in purifying protein-DNA complex for a protein that I am 
>> interested in.
>> 
>> The purification of protein only has been optimized and I've get enough 
>> yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding 
>> using Fluorescence Anisotropy. The results show that my protein has the 
>> tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 
>> 20 mM HEPES (pH 7.5).
>> 
>> However, I came across several problems when I assemble protein-DNA complex 
>> in large scale. 
>> 
>> First, my protein is unstable at low salt condition. When I dialyzes my 
>> protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, 
>> the protein precipitates. What I don't quite understand is that the DNA 
>> binding assay performed at low salt condition doesn't seem to be affected by 
>> this instability of protein. I guess it may be due to the assay was 
>> performed at very diluted protein concentration (in nM). 
>> 
>> Second, I can not purify protein-DNA complex at high salt condition with gel 
>> filtration column. Because of the first problem, I tried to assemble the 
>> complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the 
>> elution profile shows no binding of DNA to my protein (no increase in the 
>> observation of protein peak and a large peak around expected position for 
>> DNA). This may be due to weaker binding at high salt as my DNA binding assay 
>> shows that the Kd under this buffer condition is ~1100 nM.
>> 
>> Third, a lot of protein is lost during dialysis of protein-DNA complex into 
>> low salt condition. I tried add DNA directly into protein in high salt 
>> buffer, then dialyze very slowly against low salt buffer. However, I still 
>> lost quite a lot of protein due to precipitation. I was able to load some 
>> sample onto the gel filtration column with low salt running buffer. And I 
>> saw the shift of protein peak in the elution profile, also protein 
>> concentration measured by Bradford assay shows that the protein 
>> concentration is much less than that expected from uv trace, suggesting the 
>> contribution to the absorbance from DNA. But the yield is very low, less 
>> than 0.2 mg of protein is left and the complex seems to be unhappy when I 
>> concentrate it. So I can not get protein sample concentrated enough for my 
>> study.
>> 
>> My previous experience with another DNA binding protein is much better. I 
>> purified it in high salt, dialyzed into low salt to binding DNA and finally 
>> purify with gel filtration column. However, the one I am currently working 
>> on seems to be very picky. If you have any suggestion regarding to my 
>> problems, I will be thankful.
>> 
>> Best regards,
>> 
>> -- 
>> Wei Huang, PhD
>> Postdoctoral Associate
>> Center for Proteomics and Bioinformatics
>> Case Western Reserve University
>> Cleveland, OH 44106
>> 
> 

Re: [ccp4bb] Assemble Protein-DNA complex

[James Stroud]

This sounds like a job for ammonium acetate. Use it as your salt. Purify your 
complex in it and then set up drops where they wells have the amount of 
ammonium acetate needed to keep your protein stable and the wells have none, or 
a range of concentrations. The ammonium acetate will equilibrate by vapor 
diffusion, lowering the concentration in the drop and causing your complex to 
come out of solution.

James

On Nov 9, 2012, at 9:14 AM, Wei Huang wrote:

> Dear CCP4BBers,
> 
> I have a problem in purifying protein-DNA complex for a protein that I am 
> interested in.
> 
> The purification of protein only has been optimized and I've get enough yield 
> for what I need (10 mg/2.4 L growth). And I've measured DNA binding using 
> Fluorescence Anisotropy. The results show that my protein has the tightest 
> binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES 
> (pH 7.5).
> 
> However, I came across several problems when I assemble protein-DNA complex 
> in large scale. 
> 
> First, my protein is unstable at low salt condition. When I dialyzes my 
> protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, 
> the protein precipitates. What I don't quite understand is that the DNA 
> binding assay performed at low salt condition doesn't seem to be affected by 
> this instability of protein. I guess it may be due to the assay was performed 
> at very diluted protein concentration (in nM). 
> 
> Second, I can not purify protein-DNA complex at high salt condition with gel 
> filtration column. Because of the first problem, I tried to assemble the 
> complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the 
> elution profile shows no binding of DNA to my protein (no increase in the 
> observation of protein peak and a large peak around expected position for 
> DNA). This may be due to weaker binding at high salt as my DNA binding assay 
> shows that the Kd under this buffer condition is ~1100 nM.
> 
> Third, a lot of protein is lost during dialysis of protein-DNA complex into 
> low salt condition. I tried add DNA directly into protein in high salt 
> buffer, then dialyze very slowly against low salt buffer. However, I still 
> lost quite a lot of protein due to precipitation. I was able to load some 
> sample onto the gel filtration column with low salt running buffer. And I saw 
> the shift of protein peak in the elution profile, also protein concentration 
> measured by Bradford assay shows that the protein concentration is much less 
> than that expected from uv trace, suggesting the contribution to the 
> absorbance from DNA. But the yield is very low, less than 0.2 mg of protein 
> is left and the complex seems to be unhappy when I concentrate it. So I can 
> not get protein sample concentrated enough for my study.
> 
> My previous experience with another DNA binding protein is much better. I 
> purified it in high salt, dialyzed into low salt to binding DNA and finally 
> purify with gel filtration column. However, the one I am currently working on 
> seems to be very picky. If you have any suggestion regarding to my problems, 
> I will be thankful.
> 
> Best regards,
> 
> -- 
> Wei Huang, PhD
> Postdoctoral Associate
> Center for Proteomics and Bioinformatics
> Case Western Reserve University
> Cleveland, OH 44106
> 

Re: [ccp4bb] usefulness of cacodylate?

[Bernhard Rupp (Hofkristallrat a.D.)]

Math may be frightening but cacodylate seems not...

With a MW of 214  for the trihydrate

a 70 kg clone needs at the 0.5 g/kg LD50 to consume about 35 g of it, which
is 0.16M.

Of a 0.1M solution you'd therefore have to drink  1.6 L or almost 4 pints.

So, prost, cheers, gsuffa, bescheid, slantje, na strovje etc!

BR

PS: it is really not 0.5mg/g for the LD50 - it is indeed 0.25 to 0.5 g/L - I
checked. For waterflea it is lower though...

PPS: I remember with horror from my inorganic Chemistry Lab taking
place in Boltzmann's Labs (and with the same curriculum as I suspect now), 
a smell test (as in sniffing into the test tube) for As, called the
Cacodylprobe 
(aka Krokodilprobe). As I am still around (although the As sniffing may
explain a few things)
panic appears unwarranted.

-
Bernhard Rupp
001 (925) 209-7429
+43 (676) 571-0536
b...@ruppweb.org
hofkristall...@gmail.com
http://www.ruppweb.org/
---
The road to scientific serfdom is paved with Nature papers
---


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank
von Delft
Sent: Friday, November 09, 2012 4:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] usefulness of cacodylate?

Hi all -

Anybody know
 a) how hazardous is cacodylate?
 b) does it really matter for crystallization screens?

It seems by far the most hazardous component of the standard screens; this
2011 paper seems to think so (bizarrely, I can't access it from
Oxford):
http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstra
ct

and this is site says lethal dose is 0.5-5g/kg:
http://cameochemicals.noaa.gov/chemical/4468
meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone
should check my maths...)  [Coarse screens come mixed 2ml per condition.]


Has anybody done careful experiments that showed it really mattered for 
a given crystal -- or even an entire screen?

So I'm inclined to toss it out entirely rather than make crystallization 
screening a "hazardous activity".  (We're being subjected to a safety 
review.)


Thoughts welcome.
phx

Re: [ccp4bb] Assemble Protein-DNA complex

[Nicolas Foos]

Dear Wei,

If i understand your different experiment, you try to obtain your 
protein DNA complex at different salt concentration with different 
method to reach the final concentration.


I read that you try 150 mM KCl + 150 mM NaCl as high concentration salt, 
it result in 300 mM cations and 300 mM Cl. To my mind, and according 
your experiment it's too high. But when you try low concentration you 
dialized the protein alone in 30 and 100 mM salt concentration. In my 
opinion the best way, is to dialize the protein mixed with the DNA. 
Because the protein will probably be stablize by DNA. If the protein 
meet slowly DNA at the same time the salt concentration decrease, you 
minimize the precipitation (it's your third experiment in fact). You 
have not precised the final salt concentration with this method. If you 
try 30 mM, maybe it's too low, i suggest 100mM Salt. I remember paper 
about nucleosome where author explain solution with multiples steps 
dializis. It's more gradual and it could help if you want reach very low 
salt concentration.


One other idea, is to play with the pH, because of the protein's pI. In 
function of the pI you have to try different pH for the complex 
preraration. You can optimise the protein DNA interaction through the 
protein charge.


HtH

Nicolas


Le 09/11/12 17:14, Wei Huang a écrit :

Dear CCP4BBers,

I have a problem in purifying protein-DNA complex for a protein that I 
am interested in.


The purification of protein only has been optimized and I've get 
enough yield for what I need (10 mg/2.4 L growth). And I've measured 
DNA binding using Fluorescence Anisotropy. The results show that my 
protein has the tightest binding (Kd=8 nM) to the DNA at low salt 
condition (30 mM KCl) in 20 mM HEPES (pH 7.5).


However, I came across several problems when I assemble protein-DNA 
complex in large scale.


First, my protein is unstable at low salt condition. When I dialyzes 
my protein into low salt buffer (tried 30 mM and 100 mM KCl) for 
binding DNA, the protein precipitates. What I don't quite understand 
is that the DNA binding assay performed at low salt condition doesn't 
seem to be affected by this instability of protein. I guess it may be 
due to the assay was performed at very diluted protein concentration 
(in nM).


Second, I can not purify protein-DNA complex at high salt condition 
with gel filtration column. Because of the first problem, I tried to 
assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). 
However, the elution profile shows no binding of DNA to my protein (no 
increase in the observation of protein peak and a large peak around 
expected position for DNA). This may be due to weaker binding at high 
salt as my DNA binding assay shows that the Kd under this buffer 
condition is ~1100 nM.


Third, a lot of protein is lost during dialysis of protein-DNA complex 
into low salt condition. I tried add DNA directly into protein in high 
salt buffer, then dialyze very slowly against low salt buffer. 
However, I still lost quite a lot of protein due to precipitation. I 
was able to load some sample onto the gel filtration column with low 
salt running buffer. And I saw the shift of protein peak in the 
elution profile, also protein concentration measured by Bradford assay 
shows that the protein concentration is much less than that expected 
from uv trace, suggesting the contribution to the absorbance from DNA. 
But the yield is very low, less than 0.2 mg of protein is left and the 
complex seems to be unhappy when I concentrate it. So I can not get 
protein sample concentrated enough for my study.


My previous experience with another DNA binding protein is much 
better. I purified it in high salt, dialyzed into low salt to binding 
DNA and finally purify with gel filtration column. However, the one I 
am currently working on seems to be very picky. If you have any 
suggestion regarding to my problems, I will be thankful.


Best regards,

--
Wei Huang, PhD
Postdoctoral Associate
Center for Proteomics and Bioinformatics
Case Western Reserve University
Cleveland, OH 44106

Re: [ccp4bb] Assemble Protein-DNA complex

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Wei Huang,

if you are lucky you can form the complex as crystal in the drop (I
assume you want to crystallise the protein-DNA complex):

set up drops at high salt concentration (as low as possible to keep
the protein in solution at reasonable concentration) in the presence
of DNA.

Prepare the buffer the same way as the protein buffer, but with
reduced salt concentration (btw, you can also try divalent ions, eg.
MgCl2).
This way water evaporates into the drop, diluting the salt concentration.

- From your discription the solubility of the protein drops faster than
its concentration, hence it should precipitate. And, as I said, if you
are lucky, it does so as a crystal in complex with the DNA.

Best,
Tim

On 11/09/2012 05:14 PM, Wei Huang wrote:
> Dear CCP4BBers,
> 
> I have a problem in purifying protein-DNA complex for a protein
> that I am interested in.
> 
> The purification of protein only has been optimized and I've get
> enough yield for what I need (10 mg/2.4 L growth). And I've
> measured DNA binding using Fluorescence Anisotropy. The results
> show that my protein has the tightest binding (Kd=8 nM) to the DNA
> at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5).
> 
> However, I came across several problems when I assemble protein-DNA
> complex in large scale.
> 
> First, my protein is unstable at low salt condition. When I
> dialyzes my protein into low salt buffer (tried 30 mM and 100 mM
> KCl) for binding DNA, the protein precipitates. What I don't quite
> understand is that the DNA binding assay performed at low salt
> condition doesn't seem to be affected by this instability of
> protein. I guess it may be due to the assay was performed at very
> diluted protein concentration (in nM).
> 
> Second, I can not purify protein-DNA complex at high salt condition
> with gel filtration column. Because of the first problem, I tried
> to assemble the complex at high salt condition (150 mM KCl, 150 mM
> NaCl). However, the elution profile shows no binding of DNA to my
> protein (no increase in the observation of protein peak and a large
> peak around expected position for DNA). This may be due to weaker
> binding at high salt as my DNA binding assay shows that the Kd
> under this buffer condition is ~1100 nM.
> 
> Third, a lot of protein is lost during dialysis of protein-DNA
> complex into low salt condition. I tried add DNA directly into
> protein in high salt buffer, then dialyze very slowly against low
> salt buffer. However, I still lost quite a lot of protein due to
> precipitation. I was able to load some sample onto the gel
> filtration column with low salt running buffer. And I saw the shift
> of protein peak in the elution profile, also protein concentration
> measured by Bradford assay shows that the protein concentration is
> much less than that expected from uv trace, suggesting the 
> contribution to the absorbance from DNA. But the yield is very low,
> less than 0.2 mg of protein is left and the complex seems to be
> unhappy when I concentrate it. So I can not get protein sample
> concentrated enough for my study.
> 
> My previous experience with another DNA binding protein is much
> better. I purified it in high salt, dialyzed into low salt to
> binding DNA and finally purify with gel filtration column. However,
> the one I am currently working on seems to be very picky. If you
> have any suggestion regarding to my problems, I will be thankful.
> 
> Best regards,
> 

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

GPG Key ID = A46BEE1A

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Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFQnTRFUxlJ7aRr7hoRAvscAJ9Bm/3ix2ln69wZz74LWwaPMdBhFQCfS9C6
nvrZMfMmeekAauJ26jy6jbE=
=nbV+
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[ccp4bb] Assemble Protein-DNA complex

[Wei Huang]

Dear CCP4BBers,

I have a problem in purifying protein-DNA complex for a protein that I am
interested in.

The purification of protein only has been optimized and I've get enough
yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding
using Fluorescence Anisotropy. The results show that my protein has the
tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in
20 mM HEPES (pH 7.5).

However, I came across several problems when I assemble protein-DNA complex
in large scale.

First, my protein is unstable at low salt condition. When I dialyzes my
protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA,
the protein precipitates. What I don't quite understand is that the DNA
binding assay performed at low salt condition doesn't seem to be affected
by this instability of protein. I guess it may be due to the assay was
performed at very diluted protein concentration (in nM).

Second, I can not purify protein-DNA complex at high salt condition with
gel filtration column. Because of the first problem, I tried to assemble
the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the
elution profile shows no binding of DNA to my protein (no increase in the
observation of protein peak and a large peak around expected position for
DNA). This may be due to weaker binding at high salt as my DNA binding
assay shows that the Kd under this buffer condition is ~1100 nM.

Third, a lot of protein is lost during dialysis of protein-DNA complex into
low salt condition. I tried add DNA directly into protein in high salt
buffer, then dialyze very slowly against low salt buffer. However, I still
lost quite a lot of protein due to precipitation. I was able to load some
sample onto the gel filtration column with low salt running buffer. And I
saw the shift of protein peak in the elution profile, also protein
concentration measured by Bradford assay shows that the protein
concentration is much less than that expected from uv trace, suggesting the
contribution to the absorbance from DNA. But the yield is very low, less
than 0.2 mg of protein is left and the complex seems to be unhappy when I
concentrate it. So I can not get protein sample concentrated enough for my
study.

My previous experience with another DNA binding protein is much better. I
purified it in high salt, dialyzed into low salt to binding DNA and finally
purify with gel filtration column. However, the one I am currently working
on seems to be very picky. If you have any suggestion regarding to my
problems, I will be thankful.

Best regards,

-- 
Wei Huang, PhD
Postdoctoral Associate
Center for Proteomics and Bioinformatics
Case Western Reserve University
Cleveland, OH 44106

Re: [ccp4bb] usefulness of cacodylate?

[Roger Rowlett]

Cacodylate, being an arsenic compound, is moderately toxic. You do have 
to ingest it, however, for it to be toxic. Normal lab protection should 
be sufficient. It is any more concerning to me than lithium salts, 
mercury, acrylamide, ethidium bromide, etc.


Having said that, we usually abandon it as a buffer when optimizing 
conditions that include it. Typically, substituting 0.1 M MES pH 6.5 for 
cacodylate, pH 6.5, is inconsequential for crystallization for most 
proteins, but of course there are always going to be exceptions. 
Cacodylate will react with thiols, and this can complicate 
crystallization from solutions that contain high concentrations of DTT 
or beta-ME. Cacodylate can also bind to thiol groups on proteins. (This 
may be a plus or a minus, depending on your point of view.)


Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 11/9/2012 7:26 AM, Frank von Delft wrote:

Hi all -

Anybody know
a) how hazardous is cacodylate?
b) does it really matter for crystallization screens?

It seems by far the most hazardous component of the standard screens;  
this 2011 paper seems to think so (bizarrely, I can't access it from 
Oxford):
http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract 



and this is site says lethal dose is 0.5-5g/kg:
http://cameochemicals.noaa.gov/chemical/4468
meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? 
(Someone should check my maths...)  [Coarse screens come mixed 2ml per 
condition.]



Has anybody done careful experiments that showed it really mattered 
for a given crystal -- or even an entire screen?


So I'm inclined to toss it out entirely rather than make 
crystallization screening a "hazardous activity".  (We're being 
subjected to a safety review.)



Thoughts welcome.
phx

Re: [ccp4bb] Script for removing/gathering select rows of data

[Jacob Keller]

Dear all, thanks for your help--I made a very simple little script using
awk, and it works excellently (anyone is welcome to it, of course). Thanks
so much for taking the time.

Jacob





On Wed, Nov 7, 2012 at 9:49 PM, Jacob Keller  wrote:

> Dear Crystallographers,
>
> does anyone have a script on hand which can:
>
> -read in a tab-delimited dataset
> -determine whether each row in column X is above or below some input
> cutoff value Y,
> -outputs all such rows to a new file?
>
> if someone already has this on hand, I would appreciate it--seems like it
> should be a very generally useful script to have around, and I need such
> right now...
>
> Thanks very much,
>
> Jacob Keller
>



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

[ccp4bb] Opportunity for postdocs (LCLS, XFEL)

[Bischler, Ulrike]

PETER PAUL EWALD FELLOWSHIPS
BY THE VOLKSWAGEN FOUNDATION

The recent launch of X-ray free electron lasers creates unprecedented research 
opportunities. Aiming at an early progress of this field, the Volkswagen 
Foundation awards the so called "Peter Paul Ewald Fellowships" for projects 
carried out at the LCLS in Stanford, USA, in affiliation with an institute in 
Germany.

Early phase postdoctoral researchers are invited to apply. In 2013 up to five 
fellowships for three years will be awarded.

Next application deadline: January 25, 2013.

For more information please visit our website
www.volkswagenstiftung.de/ewald-fellowships
or contact

Dr. Ulrike Bischler
VolkswagenStiftung
Kastanienallee 35
30519 Hannover
GERMANY
Phone: +49 (0) 511 8381 350
Fax: +49 (0) 511 8381 4350
bisch...@volkswagenstiftung.de
www.volkswagenstiftung.de

Re: [ccp4bb] model protein for ligand soaking

[Ed Pozharski]

On Fri, 2012-11-09 at 15:12 +0100, Ulrich Zander wrote:
> Does anybody have a suggestion for a protein/ligand combination that 
> could be used for that and that is commercially available? 

Perhaps lysozyme complexed with some sugar?

-- 
Bullseye!  Excellent shot, Maurice.
  Julian, King of Lemurs.

Re: [ccp4bb] model protein for ligand soaking

[Roger Rowlett]

Human carbonic anhydrase II and any sulfonamide. We use a variety of 
alkylsulfonamides (everyone can have a different ligand and they are 
easy to model) or you can use commercially available inhibitors like 
benzenesulfonamide, sulfanilamide, acetazolamide, etc. A 30 second soak 
is sufficient to populate the active site. Kd values are in the 
nanomolar to picomolar range for most inhibitors. 1 mM or 
superstoichiometric, whichever is smaller, is sufficient.


Doug Juers at Whitman does a nice teaching lab with bovine trypsin and 
benzamidine. I am gong to plagiarize that one (with Doug's permission) 
for my teaching lab when I get tired of human CA-II.


Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 11/9/2012 9:12 AM, Ulrich Zander wrote:

Hi All,


For a tutorial, we would like to demonstrate the method of ligand 
soaking in protein crystals. I am thinking about using some sort of 
native ligand or inhibitor that can be easily identified in the 
electron density rather than halide or heavy atom derivatization.


Does anybody have a suggestion for a protein/ligand combination that 
could be used for that and that is commercially available?



Thanks!

Uli

Re: [ccp4bb] model protein for ligand soaking

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Uli,

the geometric ligands (I3C, B3C, B4C) by Tobias Beck
(http://www.protein.ethz.ch/people/tobias/) are both commercially
available and easily identified. I3C is suitable for inhouse data
collection and provide a strong anomalous signal.

Of course they are not 'native' but seem to suit your needs.

Cheers,
Tim

On 11/09/2012 03:12 PM, Ulrich Zander wrote:
> Hi All,
> 
> 
> For a tutorial, we would like to demonstrate the method of ligand 
> soaking in protein crystals. I am thinking about using some sort
> of native ligand or inhibitor that can be easily identified in the
> electron density rather than halide or heavy atom derivatization.
> 
> Does anybody have a suggestion for a protein/ligand combination
> that could be used for that and that is commercially available?
> 
> 
> Thanks!
> 
> Uli
> 
> 
> 
> 
> 
> 
> 
> 

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

GPG Key ID = A46BEE1A

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Re: [ccp4bb] usefulness of cacodylate?

[Mooers, Blaine H.M. (HSC)]

Hi Frank,

In our hands, some RNAs only crystallize out of cacodylate buffers. We would
otherwise stop using it out of health and safety concerns.

Blaine


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

Letter address:  Shipping address:
P.O. Box 26901, BRC 466  975 NE 10th Street, BRC 466
Oklahoma City, OK 73190 Oklahoma City, OK 73104-5419

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

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


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft 
[frank.vonde...@sgc.ox.ac.uk]
Sent: Friday, November 09, 2012 6:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] usefulness of cacodylate?

Hi all -

Anybody know
 a) how hazardous is cacodylate?
 b) does it really matter for crystallization screens?

It seems by far the most hazardous component of the standard screens;
this 2011 paper seems to think so (bizarrely, I can't access it from
Oxford):
http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract

and this is site says lethal dose is 0.5-5g/kg:
http://cameochemicals.noaa.gov/chemical/4468
meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone
should check my maths...)  [Coarse screens come mixed 2ml per condition.]


Has anybody done careful experiments that showed it really mattered for
a given crystal -- or even an entire screen?

So I'm inclined to toss it out entirely rather than make crystallization
screening a "hazardous activity".  (We're being subjected to a safety
review.)


Thoughts welcome.
phx

Re: [ccp4bb] usefulness of cacodylate?

[Bosch, Juergen]

Well originally we got these in cacodylate and not much else would diffract.
http://www.pdb.org/pdb/explore/materialsAndMethods.do?structureId=2EPH
But nowadays I have another conditions, which does not require cacodylate and 
works well with Hepes.

Jürgen

On Nov 9, 2012, at 7:26 AM, Frank von Delft wrote:

> Hi all -
> 
> Anybody know
> a) how hazardous is cacodylate?
> b) does it really matter for crystallization screens?
> 
> It seems by far the most hazardous component of the standard screens;  
> this 2011 paper seems to think so (bizarrely, I can't access it from 
> Oxford):
> http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract
> 
> and this is site says lethal dose is 0.5-5g/kg:
> http://cameochemicals.noaa.gov/chemical/4468
> meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone 
> should check my maths...)  [Coarse screens come mixed 2ml per condition.]
> 
> 
> Has anybody done careful experiments that showed it really mattered for 
> a given crystal -- or even an entire screen?
> 
> So I'm inclined to toss it out entirely rather than make crystallization 
> screening a "hazardous activity".  (We're being subjected to a safety 
> review.)
> 
> 
> Thoughts welcome.
> phx

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

[ccp4bb] model protein for ligand soaking

[Ulrich Zander]

Hi All,


For a tutorial, we would like to demonstrate the method of ligand 
soaking in protein crystals. I am thinking about using some sort of 
native ligand or inhibitor that can be easily identified in the electron 
density rather than halide or heavy atom derivatization.


Does anybody have a suggestion for a protein/ligand combination that 
could be used for that and that is commercially available?



Thanks!

Uli








--

Ulrich Zander
High Troughput Crystallization Lab/Diffraction Instrumentation Team
EMBL Grenoble Outstation
6 rue Jules Horowitz
38000 Grenoble, France
Phone: +33 47620-9487

Re: [ccp4bb] usefulness of cacodylate?

[Boaz Shaanan]

Hi Frank,


As the old saying goes: it's all relative (maybe not so old actually, it's only since Einstein's relativity theory, right?). Cacodylate was used, among other things, in buffers as a substitute for phosphate, merely because it kills bugs. Nowadays, we use
 azide, which is just as hazardous (well maybe not, if you read the details on the bottle) but still not recommended. And as Raji and others have pointed out, our heavy atom compounds, acrylamide, ethidium bromide, you name them, are not nice either. We have
 to take precautions of all sorts and I guess that's what your review committee will be looking for. I'd give them the credit that they're sensible people, unlike our University administration that declare ALL chemicals as dangerous (regardless of the data
 sheet), including NaCl, glucose, Superdex columns etc. whatever we order, just so that they can charge us 1.5% extra on the price (which is already much higher than what you pay) as we order the goods,  all under the pretext  that it's for disposing of the
 material out of the lab some time in the future (of course Superdex columns are never being disposed, and so is NaCl which is used to the last grain). Meanwhile, the admins can use the money for their needs.  Clever people, right?


  Cheers,


         Boaz


 
 
Boaz Shaanan, Ph.D.

Dept. of Life Sciences  
Ben-Gurion University of the Negev  
Beer-Sheva 84105    
Israel  
    
E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan  
Fax:   972-8-647-2992 or 972-8-646-1710
 
 








From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam
 [r...@brandeis.edu]
Sent: Friday, November 09, 2012 3:26 PM
To: 
CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] usefulness of cacodylate?



Hi Frank,


I worked with protein purification buffers and crystallization buffers containing 20mM potassium cacodylate for five years or so. And yes, the only precaution I used was gloves while weighing the chemical and while making buffers etc. And not just me,
 but all of my former colleagues worked with cacodylate. Then, there's those nasty chemicals for heavy atom soaks like mercury and tantalum compounds that are equally hazardous. And... many other chemicals we used on a daily basis in the lab but we don't suspect
 as much as we should.


Nucleosome core particles stubbornly refuse to crystallize if you leave out cacodylate, not just from the crystallization buffers but also from the protein purification buffers. Yes, there are folks who accidentally left it out and never gotten crystals
 of nucleosomes. I don't rule out that someday someone may very well be able to substitute cacodylate for some other chemical and successfully crystallize nucleosomes. 


It might be hard to interpret the kind of studies you suggest and here's why, in my opinion. Even if one showed that there was no need for cacodylate for, say, a 1000 different proteins, I would definitely not exclude it from a crystallization screen for
 my favorite protein because we have not gotten to that point in crystallography where one can predict crystallization conditions for a new macromolecule with great accuracy.


In my opinion, it's all relative. There are probably more chances of me being killed by a reckless bicyclist in Boston/Cambridge than by cacodylate. ;-)


Cheerios!
Raji






On Fri, Nov 9, 2012 at 7:26 AM, Frank von Delft 
 wrote:

Hi all -

Anybody know
    a) how hazardous is cacodylate?
    b) does it really matter for crystallization screens?

It seems by far the most hazardous component of the standard screens;  this 2011 paper seems to think so (bizarrely, I can't access it from Oxford):
http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract

and this is site says lethal dose is 0.5-5g/kg:
http://cameochemicals.noaa.gov/chemical/4468
meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...)  [Coarse screens come mixed 2ml per condition.]


Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen?

So I'm inclined to toss it out entirely rather than make crystallization screening a "hazardous activity".  (We're being subjected to a safety review.)


Thoughts welcome.
phx






-- 
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University

Re: [ccp4bb] usefulness of cacodylate?

[Raji Edayathumangalam]

Hi Frank,

I worked with protein purification buffers and crystallization buffers
containing 20mM potassium cacodylate for five years or so. And yes, the
only precaution I used was gloves while weighing the chemical and while
making buffers etc. And not just me, but all of my former colleagues worked
with cacodylate. Then, there's those nasty chemicals for heavy atom soaks
like mercury and tantalum compounds that are equally hazardous. And... many
other chemicals we used on a daily basis in the lab but we don't suspect as
much as we should.

Nucleosome core particles stubbornly refuse to crystallize if you leave out
cacodylate, not just from the crystallization buffers but also from the
protein purification buffers. Yes, there are folks who accidentally left it
out and never gotten crystals of nucleosomes. I don't rule out that someday
someone may very well be able to substitute cacodylate for some other
chemical and successfully crystallize nucleosomes.

It might be hard to interpret the kind of studies you suggest and here's
why, in my opinion. Even if one showed that there was no need for
cacodylate for, say, a 1000 different proteins, I would definitely not
exclude it from a crystallization screen for my favorite protein because we
have not gotten to that point in crystallography where one can predict
crystallization conditions for a new macromolecule with great accuracy.

In my opinion, it's all relative. There are probably more chances of me
being killed by a reckless bicyclist in Boston/Cambridge than by
cacodylate. ;-)

Cheerios!
Raji




On Fri, Nov 9, 2012 at 7:26 AM, Frank von Delft  wrote:

> Hi all -
>
> Anybody know
> a) how hazardous is cacodylate?
> b) does it really matter for crystallization screens?
>
> It seems by far the most hazardous component of the standard screens;
>  this 2011 paper seems to think so (bizarrely, I can't access it from
> Oxford):
> http://onlinelibrary.wiley.**com/doi/10./j.1365-2818.**
> 1977.tb01136.x/abstract
>
> and this is site says lethal dose is 0.5-5g/kg:
> http://cameochemicals.noaa.**gov/chemical/4468
> meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone
> should check my maths...)  [Coarse screens come mixed 2ml per condition.]
>
>
> Has anybody done careful experiments that showed it really mattered for a
> given crystal -- or even an entire screen?
>
> So I'm inclined to toss it out entirely rather than make crystallization
> screening a "hazardous activity".  (We're being subjected to a safety
> review.)
>
>
> Thoughts welcome.
> phx
>



-- 
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University

Re: [ccp4bb] usefulness of cacodylate?

[Ganesh Natrajan]

Hi,

I guess Cacodylate is safe as long as you don't come into direct contact 
with it, or dispose it off in a careless fashion. Aren't there 
substances in biochemical labs which are a equally if not more harmful, 
like acryamide or Ethidium Bromide, for instance?


But I agree that it should be done away with if possible, and in the 
case of Cacodylate there is an equally effective and much safer option 
in MES.



regards


Ganesh


Le 09/11/12 13:26, Frank von Delft a écrit :

Hi all -

Anybody know
a) how hazardous is cacodylate?
b) does it really matter for crystallization screens?

It seems by far the most hazardous component of the standard screens;  
this 2011 paper seems to think so (bizarrely, I can't access it from 
Oxford):
http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract 



and this is site says lethal dose is 0.5-5g/kg:
http://cameochemicals.noaa.gov/chemical/4468
meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? 
(Someone should check my maths...)  [Coarse screens come mixed 2ml per 
condition.]



Has anybody done careful experiments that showed it really mattered 
for a given crystal -- or even an entire screen?


So I'm inclined to toss it out entirely rather than make 
crystallization screening a "hazardous activity".  (We're being 
subjected to a safety review.)



Thoughts welcome.
phx

Re: [ccp4bb] usefulness of cacodylate?

[Toufic El Arnaout]

Hi Frank,
I think the lethal dose is lower than that..
It has acute toxicity (oral, dermal, inhalation) and is hazardous to the
aquatic environment.
Used as a buffer such as sodium cacodylate (less than 100-200 mM mostly).
I would read the Safety information and stick it on the screen :).. but
gloves might be enough for the tiny drops in crystal application. Apart
from that I don't know what is your policy about purchasing the powder and
storing it.. which might be a problem without even making the screen if you
are worried.
In one of the optimization screens of a condition with three components (Na
Caco is one), I couldn't get any crystals when this buffer was replaced
with another buffer (I tried a list of ten for buffer optimization covering
each a range of pHs), so yes it is sometimes critical.
It causes radiation damage when collecting data (keywords: Arsenic,
Arsenate..)... you will maybe need many crystals to collect a full
dataset.. for a small crystal you might not see the peak of Selenium in a
scan (covered because of the caco. use).
Regards


toufic el arnaout

On Fri, Nov 9, 2012 at 12:26 PM, Frank von Delft <
frank.vonde...@sgc.ox.ac.uk> wrote:

> Hi all -
>
> Anybody know
> a) how hazardous is cacodylate?
> b) does it really matter for crystallization screens?
>
> It seems by far the most hazardous component of the standard screens;
>  this 2011 paper seems to think so (bizarrely, I can't access it from
> Oxford):
> http://onlinelibrary.wiley.**com/doi/10./j.1365-2818.**
> 1977.tb01136.x/abstract
>
> and this is site says lethal dose is 0.5-5g/kg:
> http://cameochemicals.noaa.**gov/chemical/4468
> meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone
> should check my maths...)  [Coarse screens come mixed 2ml per condition.]
>
>
> Has anybody done careful experiments that showed it really mattered for a
> given crystal -- or even an entire screen?
>
> So I'm inclined to toss it out entirely rather than make crystallization
> screening a "hazardous activity".  (We're being subjected to a safety
> review.)
>
>
> Thoughts welcome.
> phx
>

Re: [ccp4bb] hkl2000 install

[Felix Frolow]

info is send to HKL.com to get cr_info if you have a legal version
FF
Dr Felix Frolow   
Professor of Structural Biology and Biotechnology, Department of Molecular 
Microbiology and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

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

On Nov 9, 2012, at 12:37 , 王瑞  wrote:

> Dear everyone:
> 
>  I'm sorry for a little off-topic! I want to install HKL2000 on
> ubuntu11.10 32bits, but it produces a file named "info" not "cr_info"
> after  run the access_prod program.And when I put "info" to
> 
> /usr/local/lib directory and typing"HKL2000" in terminal, it display:
> root@ubuntu:/usr/local/bin# HKL2000
> ERROR: Not a valid HKL-2000 license: Licence info file (cr_info) not found
> Error code: -1
> 
> So could anyone tell me how to do it ?
> 
> Rui Wang

[ccp4bb] usefulness of cacodylate?

[Frank von Delft]

Hi all -

Anybody know
a) how hazardous is cacodylate?
b) does it really matter for crystallization screens?

It seems by far the most hazardous component of the standard screens;  
this 2011 paper seems to think so (bizarrely, I can't access it from 
Oxford):

http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract

and this is site says lethal dose is 0.5-5g/kg:
http://cameochemicals.noaa.gov/chemical/4468
meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone 
should check my maths...)  [Coarse screens come mixed 2ml per condition.]



Has anybody done careful experiments that showed it really mattered for 
a given crystal -- or even an entire screen?


So I'm inclined to toss it out entirely rather than make crystallization 
screening a "hazardous activity".  (We're being subjected to a safety 
review.)



Thoughts welcome.
phx

Re: [ccp4bb] hkl2000 install

[Ian Clifton]

王瑞  writes:

>   I'm sorry for a little off-topic! I want to install HKL2000 on
> ubuntu11.10 32bits, but it produces a file named "info" not "cr_info"
> after  run the access_prod program.And when I put "info" to
>
> /usr/local/lib directory and typing"HKL2000" in terminal, it display:
> root@ubuntu:/usr/local/bin# HKL2000
> ERROR: Not a valid HKL-2000 license: Licence info file (cr_info) not found
> Error code: -1
>
> So could anyone tell me how to do it ?

Yes, you use your “info” file to obtain a “cr_info” file from HKL
Research:

http://www.hkl-xray.com/

Regards,
-- 
Ian ◎

[ccp4bb] hkl2000 install

[王瑞]

Dear everyone:

  I'm sorry for a little off-topic! I want to install HKL2000 on
ubuntu11.10 32bits, but it produces a file named "info" not "cr_info"
after  run the access_prod program.And when I put "info" to

/usr/local/lib directory and typing"HKL2000" in terminal, it display:
root@ubuntu:/usr/local/bin# HKL2000
ERROR: Not a valid HKL-2000 license: Licence info file (cr_info) not found
Error code: -1

So could anyone tell me how to do it ?

Rui Wang