[ccp4bb] Postdoctoral vacancy in YSBL York

[Keith Wilson]

There is a vacancy for a postdoctoral research assistant in my group
in the YSBL in York. For further details please see:


http://www.york.ac.uk/univ/mis/cfm/vacancies/vac_detail.cfm? 
vacno=BR07286&mode=standard



I am away until 15th August. Please contact Caroline Myers  
([EMAIL PROTECTED])

if you have difficulty with the web site.

Keith Wilson





*
Prof. Keith S. Wilson,
Structural Biology Laboratory,
University of York,
YORK  YO10 5YW
UK
tel 44-1904 328262
fax  44-1904 328266

Re: [ccp4bb] advice for crystallizing hydrophobic small molecules

[Li Sheng]

Anthony had given some good advice. 
In addition, Any organic solvent may be helpful. We often use methanol and 
chloroform.



= 2007-7-19 6:12, your message:  Re: [ccp4bb] advice for 
crystallizing hydrophobic small molecules=

Hi Todd

I have worked in the past with crystallization of dozens of small  
molecules, several of them steroids which are hydrophobic. What I did  
was, first get the small  molecule into the solution. The solvent can  
be dimethyl formamide, dimethyl sulfoxide, chloroform or anything  
that I can find on my shelf. Also hydrophobic solvents like benzene,  
hexane etc.

Leave the compound in the semi-open containers and hope for crystals  
to appear. Some times I also used to poison the solution by adding  
other solvents that include water.

Here, I should say that steroids crystalize really well, at least  
those I synthesized.

People, some times use crystallization techniques similar to protein  
like hanging or sitting drops for some small molecules, particularly  
those are water soluble.

Good luck

Anthony

Anthony Addlagatta, PhD
Institute of Molecular Biology
University of Oregon
Eugene, OR-97403
Phone: (541) 346-5867
Fax: (541)346-5870
Web: http://uoregon.edu/~anthony




On Jul 18, 2007, at 2:56 PM, Green, Todd wrote:

> Hello all,
>
> I am asking this question for a colleague(a chemist not a  
> crystallographer) who would like to crystallize a small molecule 
> (for clarification this is just the small molecule not a protein  
> complex). The compound is quite hydrophobic and is rather "greasy."  
> He has a free alcohol which could be a site of modification if this  
> would help. I have only worked with proteins and was hoping that  
> someone might be knowledgible and could point me in the direction  
> of some help(literature, websites, etc) that might aide as a ground  
> level tutorial on crystallization of small molecules, and if  
> possible more specifically crystallization of hydrophobic/"greasy"  
> small molecules.
>
> Thanks in advance-
> Todd Green
> University of Alabama at Birmingham
>

.





Sincerely yours,
Li Sheng
2007-07-19
__
Email:[EMAIL PROTECTED]
Institute of Biochemistry and Cell Biology
Institutes for Biological Sciences
Chinese Academy of Sciences
320 Yue-Yang Road, Shanghai 200031, China
Tel: +86-21-5492-1217
__

Re: [ccp4bb] advice for crystallizing hydrophobic small molecules

[Anthony Addlagatta]

Hi Todd

I have worked in the past with crystallization of dozens of small  
molecules, several of them steroids which are hydrophobic. What I did  
was, first get the small  molecule into the solution. The solvent can  
be dimethyl formamide, dimethyl sulfoxide, chloroform or anything  
that I can find on my shelf. Also hydrophobic solvents like benzene,  
hexane etc.


Leave the compound in the semi-open containers and hope for crystals  
to appear. Some times I also used to poison the solution by adding  
other solvents that include water.


Here, I should say that steroids crystalize really well, at least  
those I synthesized.


People, some times use crystallization techniques similar to protein  
like hanging or sitting drops for some small molecules, particularly  
those are water soluble.


Good luck

Anthony

Anthony Addlagatta, PhD
Institute of Molecular Biology
University of Oregon
Eugene, OR-97403
Phone: (541) 346-5867
Fax: (541)346-5870
Web: http://uoregon.edu/~anthony




On Jul 18, 2007, at 2:56 PM, Green, Todd wrote:


Hello all,

I am asking this question for a colleague(a chemist not a  
crystallographer) who would like to crystallize a small molecule 
(for clarification this is just the small molecule not a protein  
complex). The compound is quite hydrophobic and is rather "greasy."  
He has a free alcohol which could be a site of modification if this  
would help. I have only worked with proteins and was hoping that  
someone might be knowledgible and could point me in the direction  
of some help(literature, websites, etc) that might aide as a ground  
level tutorial on crystallization of small molecules, and if  
possible more specifically crystallization of hydrophobic/"greasy"  
small molecules.


Thanks in advance-
Todd Green
University of Alabama at Birmingham

[ccp4bb] advice for crystallizing hydrophobic small molecules

[Green, Todd]

Hello all,

I am asking this question for a colleague(a chemist not a crystallographer) who 
would like to crystallize a small molecule(for clarification this is just the 
small molecule not a protein complex). The compound is quite hydrophobic and is 
rather "greasy." He has a free alcohol which could be a site of modification if 
this would help. I have only worked with proteins and was hoping that someone 
might be knowledgible and could point me in the direction of some 
help(literature, websites, etc) that might aide as a ground level tutorial on 
crystallization of small molecules, and if possible more specifically 
crystallization of hydrophobic/"greasy" small molecules.

Thanks in advance-
Todd Green
University of Alabama at Birmingham

Re: [ccp4bb] questions about CNS

[Raji Edayathumangalam]

Well, I guess you can mess with the "overall B-factor correction" in the 
anneal.inp and rigid.inp 
files and say, turn them off. NOT a good way to proceed. That is just to let 
you know where to find 
it.

If you haven't already done so, do the following after the simulated annealing 
step. After 
annealing, run minimization (minimize.inp) and bgroup refinement (bgroup.inp). 
The grouped B 
refinement will refine the B factor.

Make sure that you keep an eye on how many cycles of refinement you run for 
each refinement 
protocol by correlating it with the drop in Rfactor and Rfree. YOu might have 
to cut back on the 
no. of refinement cycles in the later stages.

Hope that helps.
Raji


-Included Message--
>Hi everyone,
>I have some basic questions about CNS. First, I am wondering if there is 
>anywhere I can set my 
initial B-factor during my early refinement. When I generate my initial model 
in generate.inp, I 
can set the B-factore. However, after I did the rigid.inp and anneal.inp, the 
B-factor just go up 
to more than 100 by itself. I look through the input files, and couldn't find 
where to set the B-
factor. Do I have to set the B-factor by hand each time? Or will the crazy 
B-factor affect my 
refinement? Second, I am refining a new protein/DNA complex. I started with the 
DNA alone. After 
rigid body refinement I got ~1% decrease of Rf. But when I tried anneal after 
that, the Rf just 
went up much higher than where I started? If you work on a protein/DNA complex, 
what is your 
stategy? Will the anneal help refine the DNA alone? How much decrease of Rf 
usually you can get 
from refining just DNA? Third, I was wondering if I want to extend my 
resolution, do I have to make 
sure my cv file was made with the highest resolution? Thank you for your 
suggestion!
>Cortney
>_
>Don't get caught with egg on your face. Play Chicktionary!  
>http://club.live.com/chicktionary.aspx?icid=chick_wlmailtextlink
>
-End of Included Message--

[ccp4bb] questions about CNS

[m zhang]

Hi everyone,
I have some basic questions about CNS. First, I am wondering if there is 
anywhere I can set my initial B-factor during my early refinement. When I 
generate my initial model in generate.inp, I can set the B-factore. However, 
after I did the rigid.inp and anneal.inp, the B-factor just go up to more than 
100 by itself. I look through the input files, and couldn't find where to set 
the B-factor. Do I have to set the B-factor by hand each time? Or will the 
crazy B-factor affect my refinement? Second, I am refining a new protein/DNA 
complex. I started with the DNA alone. After rigid body refinement I got ~1% 
decrease of Rf. But when I tried anneal after that, the Rf just went up much 
higher than where I started? If you work on a protein/DNA complex, what is your 
stategy? Will the anneal help refine the DNA alone? How much decrease of Rf 
usually you can get from refining just DNA? Third, I was wondering if I want to 
extend my resolution, do I have to make sure my cv file was made with the 
highest resolution? Thank you for your suggestion!
Cortney
_
Don't get caught with egg on your face. Play Chicktionary!  
http://club.live.com/chicktionary.aspx?icid=chick_wlmailtextlink

Re: [ccp4bb] sleeping refmac

[Ronan Keegan]

Hi Harry,

Might not be the problem but does this machine have its ccp4 scratch 
space ($CCP4_SCR) mounted from another machine by nfs across a network? 
Refmac writes a lot of temporary files to CCP4_SCR and if this is across 
a busy network it can really slow Refmac's progress.


Ronan



Harry M. Greenblatt wrote:

BS"D

Dear All,

   One our our linux boxes (kernel 2.4.20) has begun recently to run 
refmac at worse than snail's pace.  This is a 3.2 GHz Xeon dual CPU 
Dell machine from 4 years ago.  All other things seem fine, including 
other CCP programs.  But refmac jobs (for various proteins) that take 
a minute or two on other machines can sit for many tens of minutes 
making very slow progress (but yes, they are getting somewhere).  
Using top, one can see that the machine is completely idle, and the 
refmac job has used up a few seconds of CPU time, even after ten 
minutes, although its priority is normal. In top, under the status 
column, it has a status of "D", which, according to the documentation, 
is "uninterruptible sleep".  Running refmac on another computer, I see 
that is does enter the "D" state a few times during the run, but never 
stays that way very long.  Any ideas?



Thanks

Harry

-

Harry M. Greenblatt

Staff Scientist

Dept of Structural Biology   [EMAIL PROTECTED] 



Weizmann Institute of SciencePhone:  972-8-934-3625

Rehovot, 76100   Facsimile:   972-8-934-4159

Israel 

[ccp4bb] sleeping refmac

[Harry M. Greenblatt]

BS"D

Dear All,

   One our our linux boxes (kernel 2.4.20) has begun recently to run  
refmac at worse than snail's pace.  This is a 3.2 GHz Xeon dual CPU  
Dell machine from 4 years ago.  All other things seem fine, including  
other CCP programs.  But refmac jobs (for various proteins) that take  
a minute or two on other machines can sit for many tens of minutes  
making very slow progress (but yes, they are getting somewhere).   
Using top, one can see that the machine is completely idle, and the  
refmac job has used up a few seconds of CPU time, even after ten  
minutes, although its priority is normal. In top, under the  
status column, it has a status of "D", which, according to the  
documentation, is "uninterruptible sleep".  Running refmac on another  
computer, I see that is does enter the "D" state a few times during  
the run, but never stays that way very long.  Any ideas?



Thanks

Harry
 
-

Harry M. Greenblatt
Staff Scientist
Dept of Structural Biology   [EMAIL PROTECTED]
Weizmann Institute of SciencePhone:  972-8-934-3625
Rehovot, 76100   Facsimile:   972-8-934-4159
Israel

Re: [ccp4bb] Combining MR and MAD phases

[Eleanor Dodson]

1) Combine your MAD 3A phases with the 1.7A native data and use DM or 
Pirate to refine and extend the phase set.

2) Make sure your MR solution is on the same origin as the MAD phases.
You can calculate PHIC and FOM from MR solution, combinre with the MAD 
phases and do an anomalous diference map, then use the sites you find to 
do the MAD phasing.


Or combine the PHIC/FOM, and the MAD phases into one mtz file and run 
the Clipper utility for phase comparison. That moves the phases so they 
are consistent .


Then you can run REFMAC using the MAD extended phases as restraints - 
you get out map coefficients with combined phase information which 
should be easy to bulid the rest of your structure into

Eleanor






Joe Batchelor wrote:

Hi,

I have a 1.7 A native dataset, a good MR solution for 2/3 of the
protein, and MAD phases to 3 A. How should I combine the MR phases
with the MAD phases?

Thanks,
Joe

Re: [ccp4bb] Problem in CNS refinement

[Sampath Natarajan]

Dear all,

I thank each and everyone who extended his/her hands to help me regarding 
problem in CNS refinement. Everyone gave valuable suggestions regarding the 
same. I could find the problem in converting to hkl file from mtz from your 
suggestions. I rectified it and now it works well.

Thank you once again,

Best Regards,

Sampath




 
Dr. N.SAMPATH 
Post Doctoral Fellow
  Department of Molecular & Cell Biology
Samsung Biomedical Research Institute
Sungkyunkwan Univ. School of Medicine
Suwon 440-746, S.Korea
 
tel : 82-31-299-6150 & 82-31-299-6155   




   
-
Boardwalk for $500? In 2007? Ha! 
Play Monopoly Here and Now (it's updated for today's economy) at Yahoo! Games.

Re: [ccp4bb] Twinning in C2 ?

[Demetres D. Leonidas]

Dear Eleanor,

many thanks for all your help and suggestions. We finally have solved 
the mutant structure. The trick was to use the pseudo translation (there 
was not any 2 fold axis in the self rotation) with MOLREP which found 
two molecules and then applied the pseudo translation to provide us with 
the other two molecules yielding a tetramer. I think that initially the 
programs and also ourselves  missed  the pseudo translation because it 
is very close to the  crystallographic one  (0.49 0 0.48).  Many thanks  
to  Peter  Zwart who also advised us to follow the same procedure.


Demetres


Eleanor Dodson wrote:

Oh dear - this is tricky!
You could have a dimer in the asymmetric unit, or two monomers in the 
asymm unit which generate dimers using the crystallographic 2 folds.


Things worth checking -
What does the self rotation show? Is there a clear 2 fold axis which 
is different from the crystallographic one?
If so you can use all of MOLREPs cleverness to use the self rotation 
definition and the pseudo translation.
(Under search parameters you can give the self rotation list of 
solutions - just use self rotation with 1 or 2 solutions and edit out 
the top one which will be the crystallographic 2 fold)


It will find the pseudo trans vector automatically.

If the only 2 fold is the cryst one then you need to search for 2 
molecules with the pseudo translation.


Eleanor

What is the solvent content of the native - the mutant has a smaller 
volume so less solvent - is that feasible?





I think I would generate P1 data Demetres D. Leonidas wrote:

Dear Eleanor,

Yes the native is a dimer and we did the search using the dimer as a 
model but we had similar results (i.e. all programs find one 
molecule). The graphs from TRUNCATE show rather "normal" and I am 
attaching a gif file with the plot for the cumulative intensity.


As for pseudo-translation running the "Analyse Data for MR" option in 
ccp4 in the patterson map we are getting a significant peak at 
fractional coordinates 0.4897 0.0 0.4767. How this can help ? Do we 
need to apply this pseudo translation to the solution we are getting 
from molrep ?


many thanks

Demetres

P.S. I will summarize for the members of the list all the suggestions 
I will get at the end



Eleanor Dodson wrote:
You dont say whether the molecules in the native cell form a dimer - 
if so I would search with that (you may need to turn off the packing 
search)


Or whether there is a pseudo translation vector in the mutant form..

Or what the data analysis graphs from TRUNCATE show - are they 
"normal"?


Eleanor

Demetres D. Leonidas wrote:

Dear all,

we have encountered a problem in solving one mutant structure. The 
mutant protein crystallizes in the same space group as the native 
(C2) but the unit cell dimensions are different. These for the 
native structure are 101.2 33.1 73.4 90 90.3 90 and for the mutant 
160.4 32.3 107.0 90 125.7 90. As a result the mutant structure has  
four molecules in the asymmetric unit while the native had two. 
When we run molecular replacement all programs (CNS, molrep, and 
amore) find only two molecules. Phaser finds four but when we try 
to refine the Rfree does not drop below 0.44 if we use four 
molecules and 0.53 if we only use two no matter how well we built 
the molecule and regardless of any addition of water molecules (the 
resolution of the data is 2.1). The interesting thing is that in 
the electron density map we can clearly see density for a substrate 
analog that was included in the crystallization media. Do you 
thing  that we have a case of twinning here ? We have to mention 
that Tod Yates served did not indicate any perfect merohedral 
twinning (partial merohedral twinning for this space group is not 
possible).


We would appreciate any comments

Many thanks

Demetres














--
Demetres D. Leonidas, Ph.D.
Structural Biology & Chemistry Group
Institute of Organic and Pharmaceutical Chemistry
The National Hellenic Research Foundation
48, Vassileos Constantinou Avenue
Athens 116 35, Greece
==
Tel. +30 210 7273841 (office)
+30 210 7273895 (lab) 
Fax. +30 210 7273831

E-mail: [EMAIL PROTECTED]
URL: http://athena.eie.gr
==