[ccp4bb] co-crystallization

[Yvonne TAN Yih Wan]

Hi ,

I am co-crystallizing a protein with compound and would like to know how much 
of compound to add to protein solution to start with. I know that the protein 
binds compound in a 1 to 1 ratio but also noticed that the compound 
precipitates out of solution when DMSO is diluted off. Where should I start of? 
A 1 protein :2 compound ratio or more? And what is the best method to determine 
if the binding is homogeneous (that all protein has got a compound in it)?

Any suggestions would help. Thanks

TY

[ccp4bb] Creating CIF Files and Renaming Ligands

[Ai Fen Chai]

Hi all,

I want to add 2 new ligands (that do not exist in PDB) to my structure. I
generated the ligands from PRODRG. The problem is that both of the ligands
have the same name DRG. After renaming the ligands in both PDB and CIF
files, I always get this error message "CIF dictionary does not contain
restraints" whenever I try to do refinement. Prior to renaming, both ligands
were recognized by COOT when they were named DRG. So, the CIF files
themselves are ok and it must be something to do with the renaming that is
causing the error. I am wondering whether renaming the ligands has changed
the CIF file subtly and causing COOT and other refinement programs to not
read it right?

Cheers,
Ai Fen

Re: [ccp4bb] spherulites and PEG3350

[Tom Peat]

You might to consider that PEG 3350 has phosphate contamination, so playing 
around with small amounts of phosphate (or removing it) might be worthwhile.
Cheers, tom

From: Regina Kettering [mailto:reginaketter...@yahoo.com]
Sent: Thursday, August 25, 2011 04:46 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] spherulites and PEG3350

Something to consider is the quality of the PEG 3350.  We have found that 
different qualities of PEG 3350 can give different results, depending on the 
type and amount of contaminants.  What used to be the Fluka PEG 3350 is now the 
pharm grade of PEG 3350 (aka Miralax).  We use high quality PEG 3350 for normal 
screening, but switch to the highest quality grade we can get for optimizing.

Regina


From: Jan van Agthoven 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, August 24, 2011 2:05 PM
Subject: [ccp4bb] spherulites and PEG3350

Dear all,

I recently obtained some spherulites while trying to crystallize my protein. 
The spherulites are manually reproducible, but changing pH, protein 
concentration, and salt concentration does not result in crystal formation. 
Microseeding with crushed spherulites isn't a solution either as it only yields 
new spherulites. Next stepp is the use of an optimization kit but I have a 
limited amount of material, and I start doubting that these are protein 
spherulites, as the spherulites are not particularly soft. The condition 
contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms 
easily spherulites around that concentration?


Thanks,

Re: [ccp4bb] Aging PEGs

[Van Den Berg, Bert]

Is there anything that consistently matters in crystallography? ;-)

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft 
[frank.vonde...@sgc.ox.ac.uk]
Sent: Wednesday, August 24, 2011 5:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Aging PEGs

And now,   does anybody know of systematic data indicating how consistently 
all this matters?
phx

On 24/08/2011 21:45, Prince, D Bryan wrote:

For those of us truly controlling types :), I used to make the PEG
solutions and filter them over a Bio-Rad resin that filtered out all the
junk added to stabilize the PEG solution. Then, of course I had to
freeze all my PEG solutions in aliquots, or wrap them in foil and store
at 4C in the dark. This would take several days, depending on the FW of
the PEG. If you are really sensitive about what is in your PEG
solutions, try GC-grade PEG's. The FW profile is much more restricted
around the reported value (i.e. PEG 3350 molecular biology grade has a
broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter
peak profile.) Back before you could buy Crystal Screen I, II or HT, you
had to make the stock solutions, then make the screen. But at least when
you did that, you had all the stocks. Now, I just buy pre-made
solutions, and keep them in a drawer with a date opened written on the
bottle. Isn't progress grand? :)

Bryan


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Confidentiality Notice: This message is private and may contain confidential 
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notify us and remove it from your system and note that you must not copy, 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Jacob Keller
Sent: Wednesday, August 24, 2011 3:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Aging PEGs

A while ago I measured the pH's of old and new PEGs and found them
very different, and internally attributed all "old vs new PEG issues"
to pH. Upon reflection, this seems too simplistic. Are there other
known mechanisms of crystallization capacities of PEGs of various
ages?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Aging PEGs

[Frank von Delft]

And now,   does anybody know of /systematic/ data indicating how 
consistently all this matters?

phx

On 24/08/2011 21:45, Prince, D Bryan wrote:

For those of us truly controlling types :), I used to make the PEG
solutions and filter them over a Bio-Rad resin that filtered out all the
junk added to stabilize the PEG solution. Then, of course I had to
freeze all my PEG solutions in aliquots, or wrap them in foil and store
at 4C in the dark. This would take several days, depending on the FW of
the PEG. If you are really sensitive about what is in your PEG
solutions, try GC-grade PEG's. The FW profile is much more restricted
around the reported value (i.e. PEG 3350 molecular biology grade has a
broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter
peak profile.) Back before you could buy Crystal Screen I, II or HT, you
had to make the stock solutions, then make the screen. But at least when
you did that, you had all the stocks. Now, I just buy pre-made
solutions, and keep them in a drawer with a date opened written on the
bottle. Isn't progress grand? :)

Bryan


--
Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.
  
-Original Message-

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Jacob Keller
Sent: Wednesday, August 24, 2011 3:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Aging PEGs

A while ago I measured the pH's of old and new PEGs and found them
very different, and internally attributed all "old vs new PEG issues"
to pH. Upon reflection, this seems too simplistic. Are there other
known mechanisms of crystallization capacities of PEGs of various
ages?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Aging PEGs

[Prince, D Bryan]

For those of us truly controlling types :), I used to make the PEG
solutions and filter them over a Bio-Rad resin that filtered out all the
junk added to stabilize the PEG solution. Then, of course I had to
freeze all my PEG solutions in aliquots, or wrap them in foil and store
at 4C in the dark. This would take several days, depending on the FW of
the PEG. If you are really sensitive about what is in your PEG
solutions, try GC-grade PEG's. The FW profile is much more restricted
around the reported value (i.e. PEG 3350 molecular biology grade has a
broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter
peak profile.) Back before you could buy Crystal Screen I, II or HT, you
had to make the stock solutions, then make the screen. But at least when
you did that, you had all the stocks. Now, I just buy pre-made
solutions, and keep them in a drawer with a date opened written on the
bottle. Isn't progress grand? :)

Bryan


--
Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.
 
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Jacob Keller
Sent: Wednesday, August 24, 2011 3:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Aging PEGs

A while ago I measured the pH's of old and new PEGs and found them
very different, and internally attributed all "old vs new PEG issues"
to pH. Upon reflection, this seems too simplistic. Are there other
known mechanisms of crystallization capacities of PEGs of various
ages?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Finding obsolete pdb entries

[Gerard DVD Kleywegt]

!Hola!

About 6 years ago I noted a couple of structures I was interested in were 
removed from the pdb. I saw in a recent email discussion that it is possible 
to access obsolete entries, however unfortunately I do not have the pdb code 
of the structure I am interested in - and neither does the original 
publication list the pdb code. Is there a way of searching 
withdrawn/obsolete entries for author name, macromolecule etc. or are 
structures that were withdrawn over 5 years ago lost for good?


Actually, you can. Tom Oldfield has written a web interface to query the PDBe 
search database directly using SQL - it lives here:


 http://www.ebi.ac.uk/pdbe-as/pdbedatabase/

If you want to look for obsoleted entries by molecule name, paste the 
following into the SQL box:


Select * from pdb_status WHERE status_code in ('OBS')  and
upper(TITLE) like '%ISOMERASE%';

Hit the "Submit SQL" button and you will get a list of hits with the word 
"ISOMERASE" in their title. Hit the "Get" (Text) button to save the complete 
list of hits.


If you know one of the authors, try the following query instead:

Select * from pdb_status WHERE status_code in ('OBS')  and
upper(AUTHOR_LIST) like '%JONES%';

!No hay de que!

--Gerard

Re: [ccp4bb] Aging PEGs

[mjvdwoerd]

 PEG is a polymer and it can be made by anionic or cationic polymerization. 
Whichever you use, you go "the other way" to terminate the reaction at an 
appropriate time (so you have the molecular weight you want). So when you start 
with an acid, you terminate with a base and vice versa. If you terminate with a 
base, your final pH is going to be high (presumably > 7) and if you terminate 
with an acid, the pH is going to low.

It is therefore important to keep track of your lot number (because as long as 
the lot number is the same, the treatment was the same). For crystallization 
recipes that do not involve buffers (there are some!) this is essential, 
because PEG and PEG are not the same thing (and you should always pH the 
solution before you use it, so you have a reference point - remember we do not 
know what we have in our crystallization drops, but we do know what we put into 
them to make them). Even if the PEG was made by the same process, the 
manufacturer is concerned with stopping the polymerization at the right time, 
but not "how hard" they stop it. In other words, the solution might be pH 8 or 
pH 11 when it is done.  

So when you say that you measured different PEGs and found the pH to be 
different, that might be accounted for by the way the PEG was made and they may 
always have been different, irrespective of age.

It is probably not known if "acid PEG" vs "basic PEG" ages at a different speed 
and with a different mechanism. As you know, the first thing to observe is 
whether PEG solutions are clear ("water") or slightly colored ("dilute 
lemonade" :-) because this is a sign of aging. Remember that PEGMME is much 
more sensitive to aging than PEG itself (you should store MME solutions in the 
dark). And if the PEG is stored in dry form, it is not very easy to age it by 
chemical reaction, because nothing is "swimming", but "not easy" is not the 
same as "impossible".

My 2 cents worth.

Mark



 


 

 

-Original Message-
From: Jacob Keller 
To: CCP4BB 
Sent: Wed, Aug 24, 2011 1:18 pm
Subject: [ccp4bb] Aging PEGs


A while ago I measured the pH's of old and new PEGs and found them
very different, and internally attributed all "old vs new PEG issues"
to pH. Upon reflection, this seems too simplistic. Are there other
known mechanisms of crystallization capacities of PEGs of various
ages?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

 

Re: [ccp4bb] Aging PEGs

[Craig A. Bingman]

Commercial polyethylene glycol is contaminated with polymers that have aldehyde 
groups at the ends.  Other impurities include some amount of internal epoxide 
linkages.  The aldehyde groups can be additionally oxidized to carboxylic 
acids.  I would assume that oxidation to terminal carboxylic acids explains the 
change in PEG pH vs. time.  The pH will also change as the PEG solution comes 
to equilibrium with atmospheric carbon dioxide.  The aldehyde content increases 
with time after the PEG is dissolved in water and stored under normal 
atmospheric gas.  These PEGs can react with protein amine groups.  

On Aug 24, 2011, at 2:18 PM, Jacob Keller wrote:

> A while ago I measured the pH's of old and new PEGs and found them
> very different, and internally attributed all "old vs new PEG issues"
> to pH. Upon reflection, this seems too simplistic. Are there other
> known mechanisms of crystallization capacities of PEGs of various
> ages?
> 
> Jacob Keller
> 
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***

Re: [ccp4bb] Aging PEGs

[Jim Pflugrath]

PEGs have small amounts of anti-oxidants added to them by the manufacturer.
I think different manufacturers may use different compounds.

And you might imagine that PEGs themselves with their -OH groups go from
alcohol to adelhyde to carboxylate.


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob
Keller
Sent: Wednesday, August 24, 2011 2:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Aging PEGs

A while ago I measured the pH's of old and new PEGs and found them very
different, and internally attributed all "old vs new PEG issues"
to pH. Upon reflection, this seems too simplistic. Are there other known
mechanisms of crystallization capacities of PEGs of various ages?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

[ccp4bb] Aging PEGs

[Jacob Keller]

A while ago I measured the pH's of old and new PEGs and found them
very different, and internally attributed all "old vs new PEG issues"
to pH. Upon reflection, this seems too simplistic. Are there other
known mechanisms of crystallization capacities of PEGs of various
ages?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] spherulites and PEG3350

[Prince, D Bryan]

Something else to try would be the Protic Ionic Liquid kit from Hampton.
I recently had crystals of a protein that would only grow as laminated
stacks of plates. Optimizing the conditions and using an additive screen
didn't improve crystal morphology. I tried the PIL kit from Hampton and
was able to get single, thick plates in several conditions. At the ACA
meeting in Hawaii a few years back, there was a poster about ageing your
PEG solutions by microwaving them and letting them cool on the bench.
This was the only way that the poster's author could get reproducible
crystals of her target protein.



Good Luck!

Bryan



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Regina Kettering
Sent: Wednesday, August 24, 2011 2:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] spherulites and PEG3350



Something to consider is the quality of the PEG 3350.  We have found
that different qualities of PEG 3350 can give different results,
depending on the type and amount of contaminants.  What used to be the
Fluka PEG 3350 is now the pharm grade of PEG 3350 (aka Miralax).  We use
high quality PEG 3350 for normal screening, but switch to the highest
quality grade we can get for optimizing.



Regina





From: Jan van Agthoven 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, August 24, 2011 2:05 PM
Subject: [ccp4bb] spherulites and PEG3350

Dear all,



I recently obtained some spherulites while trying to crystallize my
protein. The spherulites are manually reproducible, but changing pH,
protein concentration, and salt concentration does not result in crystal
formation. Microseeding with crushed spherulites isn't a solution either
as it only yields new spherulites. Next stepp is the use of an
optimization kit but I have a limited amount of material, and I start
doubting that these are protein spherulites, as the spherulites are not
particularly soft. The condition contains 15% PEG 3350 and 200 mM NaCl.
Does anyone know if PEG 3350 forms easily spherulites around that
concentration?





Thanks,




--
Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.
 

Re: [ccp4bb] spherulites and PEG3350

[Jan van Agthoven]

Thanks for the protocole and advice! I'll put my spherulites on gel. It will
make things clear.

Jan

2011/8/24 Regina Kettering 

> Something to consider is the quality of the PEG 3350.  We have found that
> different qualities of PEG 3350 can give different results, depending on the
> type and amount of contaminants.  What used to be the Fluka PEG 3350 is now
> the pharm grade of PEG 3350 (aka Miralax).  We use high quality PEG 3350 for
> normal screening, but switch to the highest quality grade we can get for
> optimizing.
>
> Regina
>
> --
> *From:* Jan van Agthoven 
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Sent:* Wednesday, August 24, 2011 2:05 PM
> *Subject:* [ccp4bb] spherulites and PEG3350
>
> Dear all,
>
> I recently obtained some spherulites while trying to crystallize my
> protein. The spherulites are manually reproducible, but changing pH, protein
> concentration, and salt concentration does not result in crystal formation.
> Microseeding with crushed spherulites isn't a solution either as it only
> yields new spherulites. Next stepp is the use of an optimization kit but I
> have a limited amount of material, and I start doubting that these are
> protein spherulites, as the spherulites are not particularly soft. The
> condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG
> 3350 forms easily spherulites around that concentration?
>
>
> Thanks,
>
>
>

Re: [ccp4bb] spherulites and PEG3350

[Regina Kettering]

Something to consider is the quality of the PEG 3350.  We have found that 
different qualities of PEG 3350 can give different results, depending on the 
type and amount of contaminants.  What used to be the Fluka PEG 3350 is now the 
pharm grade of PEG 3350 (aka Miralax).  We use high quality PEG 3350 for normal 
screening, but switch to the highest quality grade we can get for optimizing.

Regina



From: Jan van Agthoven 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, August 24, 2011 2:05 PM
Subject: [ccp4bb] spherulites and PEG3350


Dear all,

I recently obtained some spherulites while trying to crystallize my protein. 
The spherulites are manually reproducible, but changing pH, protein 
concentration, and salt concentration does not result in crystal formation. 
Microseeding with crushed spherulites isn't a solution either as it only yields 
new spherulites. Next stepp is the use of an optimization kit but I have a 
limited amount of material, and I start doubting that these are protein 
spherulites, as the spherulites are not particularly soft. The condition 
contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms 
easily spherulites around that concentration?


Thanks, 

Re: [ccp4bb] spherulites and PEG3350

[Gregory Verdon]

You should check these "spherulites" on SDS-PAGE gel to make sure that these
contain your protein. Then you can start thinking about optimization.

On Wed, Aug 24, 2011 at 2:05 PM, Jan van Agthoven  wrote:

> Dear all,
>
> I recently obtained some spherulites while trying to crystallize my
> protein. The spherulites are manually reproducible, but changing pH, protein
> concentration, and salt concentration does not result in crystal formation.
> Microseeding with crushed spherulites isn't a solution either as it only
> yields new spherulites. Next stepp is the use of an optimization kit but I
> have a limited amount of material, and I start doubting that these are
> protein spherulites, as the spherulites are not particularly soft. The
> condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG
> 3350 forms easily spherulites around that concentration?
>
>
> Thanks,
>

Re: [ccp4bb] spherulites and PEG3350

[Savvas Savvides]

Dear Jan
I would recommend running the following protocol on your spherulites. Just 
pretend that they are crystals :)
This was posted some time ago on the ccp4bb.
best regards
Savvas

> On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete <
> kenneth.verstra...@ugent.be> wrote:
> 
> > Hi Ivan,
> >
> > there are several tests (e.g. Izit dye, crush test) you can do discern
> > protein from salt crystals but what was always very informative to me (and
> > certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the
> > crystals using the following protocol:
> >
> > - select a drop which contains some substantial crystalline material. The
> > crystals can be many and small (crystal shower) or few and large.
> > - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother
> > liquor containing a 10% higher concentration of precipitant)
> > - transfer all the crystalline material from the drop into the PCR-tube
> > using a pipet (use stabilizing buffer from the PCR tube to collect all
> > crystals)
> > - centrifuge the PCR-tube at low speed for 30-60 sec and observe the
> > crystals under the microscope. They should be at the bottom of the PCR-tube.
> > - Remove as much as supernatant as you can (make sure not to remove your
> > crystals), add stabilizing buffer to wash the crystals, and centrifuge again
> > - repeat this washing protocol a few times
> > - after the final washing step, add Laemli-buffer to the crystals and use
> > this sample to load the SDS-PAGE gel
> > - include a positive (eg. solubilize another drop directly in
> > Laemli-buffer) and a negative (final washing buffer) control
> > - use silver staining to visualize the protein
> >
> > This always works for me. If you don't see a band at this point I would be
> > worried that it is salt. You could then choose to do a Western blot instead
> > of silver staining to increase the sensitivity. Make sure to include control
> > samples then.
> >
> > Kind regards,
> >
> > Kenneth Verstraete
> > L-PROBE
> > Ghent University
> > Belgium

On 24 Aug 2011, at 20:05, Jan van Agthoven wrote:

> Dear all,
> 
> I recently obtained some spherulites while trying to crystallize my protein. 
> The spherulites are manually reproducible, but changing pH, protein 
> concentration, and salt concentration does not result in crystal formation. 
> Microseeding with crushed spherulites isn't a solution either as it only 
> yields new spherulites. Next stepp is the use of an optimization kit but I 
> have a limited amount of material, and I start doubting that these are 
> protein spherulites, as the spherulites are not particularly soft. The 
> condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 
> forms easily spherulites around that concentration?
> 
> 
> Thanks, 

[ccp4bb] spherulites and PEG3350

[Jan van Agthoven]

Dear all,

I recently obtained some spherulites while trying to crystallize my protein.
The spherulites are manually reproducible, but changing pH, protein
concentration, and salt concentration does not result in crystal formation.
Microseeding with crushed spherulites isn't a solution either as it only
yields new spherulites. Next stepp is the use of an optimization kit but I
have a limited amount of material, and I start doubting that these are
protein spherulites, as the spherulites are not particularly soft. The
condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG
3350 forms easily spherulites around that concentration?


Thanks,

Re: [ccp4bb] Modeling ligands in binding pockets when the density is weak.

[Shya Biswas]

Hi Francis,
Once I had asked Pavel Afonine the same questions and these were his
suggestions but most of these can be implemented in phenix...


I guess there is no general/unified procedure to do this, and in most of
cases the tools and outcomes vary case by case.
Some general points:
- Removing parts of model is unlikely to improve the map simply because this
makes model even more incomplete. The main purpose of computing the omit map
is not to improve the overall map but to verify the atoms in question.
- Typically, the density appears weak because the signal is buried in noise.
For example, we do not see H atoms in 2A resolution maps not because the
information about them is not there (note, H atoms being weak scatterers
contribute the most to low resolution reflections - similarly to bulk
solvent, and their contribution to high resolution data approaches zero),
but because the model quality and therefore noise at that resolution such
that it hides hydrogens' signal. However, we do see H atoms at high
resolution (say 1A and higher) not because of presence of high resolution
reflections, but because the model quality is typically high and the noise
level is below the hydrogens' signal.
Having said this, one possible way of improving your "weak density" is to
improve the model as much as you can: make sure you modeled all alternative
conformations, all solvent, etc.
- B-factor sharpening may help, although keep in mind that it will enhance
the noise too.
- Other options: kick maps, omit kick maps, b-factor sharpened kick maps...
- You can try GrowDensity method (Acta  Cryst. (1997). D53, 540-543) which
is available as phenix.grow_density. This is still under (slow) development,
so if you decide to go this route than I can help you with the details.
Pavel.

HTH,
Shya

On Tue, Aug 23, 2011 at 2:36 PM, Francis E Reyes  wrote:

> Seems to be a quiet day on the BB, so I propose this question:
>
>
> Suppose you have a ligand in the binding pocket and some mediocre data (3 A
> or so), the 'core' of the ligand is well defined in 2Fo-Fc map  using the
> model phases of your protein, however there are 'chains/tails' of the ligand
> which are not.  Composite omit or simulated annealing omit maps do not
> produce density for these 'chains'
>
> The question here is how the chains/tails should be modeled (if at all).
>
>
> [1] Model in the core, but remove the atoms for the chains  (and conclude
> the diffraction data do not support interactions with the protein and
> subsequent experiments are needed (higher resolution data, biochemical data,
> etc)).
>
> or
>
> [2] Model in the chains/tails noting that potential hydrogen bond
> donors/acceptors on the protein are within hydrogen bonding distance to the
> chains/tails. You do this and subsequent refinement still does not produce
> the expected density for the chains.
>
>
> or
>
> [3] Your solution here.
>
>
>
>  If this situation has been discussed before, please let me know .
>
> F
>
> -
> Francis E. Reyes M.Sc.
> 215 UCB
> University of Colorado at Boulder
>

Re: [ccp4bb] LABELIT with XDS

[Nicholas Sauter]

James,

After indexing the images with LABELIT it is possible to write out an
XPARM.XDS file that defines the unit cell, orientation, and beam parameters.


For example, index an image first:
labelit.index lysozyme_001.img --index_only

This gives several possible Bravais lattice solutions...e.g. triclinic=1;
tetragonal=9.  Suppose you want XDS parameters for the tetragonal setting:

labelit.xds_xparm 9

That outputs the XPARM.XDS file.  I'd like to get any feedback from the
community...this feature was added to Labelit in March 2011.

Nick Sauter

On Wed, Aug 24, 2011 at 5:10 AM, James Whittle
wrote:

> Dear all,
>
> I'm able to index my images in LABELIT. Is it possible to use that
> indexing solution in XDS?
>
> James
>



-- 
Nicholas K. Sauter, Ph. D.
Computer Staff Scientist/Engineer
Physical BioSciences Division
Lawrence Berkeley National Laboratory
1 Cyclotron Rd., Bldg. 64R0121
Berkeley, CA 94720-8118
(510) 486-5713

Re: [ccp4bb] number of cycles in refmac

[Garib N Murshudov]

Dear Tim

At the moment there is no option to stop refmac prematurely. I can add if it is 
necessary. I can only give my experience. 
After molecular replacement before running ARP/wARP or buccaneer I usually run 
60-100 cycles of refinement with jelly body with sigma set to 0.01.
Then automatic model building works better. I have seen this behaviour in 
several cases (if there are more than one copy I also would use local ncs 
restraints).
Using jelly body slows down convergence but shifts seem to make more sense.

regards
Garib

On 24 Aug 2011, at 17:24, Tim Gruene wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Dear all,
> 
> especially at the beginning of model building and/or at low resolution
> both Rfree and "-LL free" as reported in the refmac logfile show a
> minimum at a some cycle before rising again.
> 
> I am certainly not the only one tempted to first run refmac with a large
> number of refinement cycles, determine that minimum and rerun refmac
> with ncyc set to that minimum.
> 
> Of course I want the resulting model and phases/map to be as close to
> the what's in the crystal as possible in order to facilitate model building.
> 
> Is it therefore good practice to interrupt refmac wherever it finds a
> minimum (if so, the minimum w.r.t. which number reported in the log-file)?
> 
> Thanks for everyone's opinion and experience,
> 
> Tim
> 
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
> 
> GPG Key ID = A46BEE1A
> 
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.11 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
> 
> iD8DBQFOVReTUxlJ7aRr7hoRAqyzAKCZpMJPVSQJTDEoWGxZEymwvqfFcACeMNLL
> rvIDPlXiL5HQmoNm7yrTt6k=
> =UnKT
> -END PGP SIGNATURE-

Garib N Murshudov 
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road 
Cambridge 
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk 
Web http://www.mrc-lmb.cam.ac.uk

Re: [ccp4bb] Modeling ligands in binding pockets when the density is weak.

[Dale Tronrud]

   I agree with Herman, and would like to add to his list of
explanations why density may not be observed.  It is possible
that the compound binding to your protein simply doesn't contain
those bits without density.  I have known cases where the
compound in the vial does not match the label on the vial.  In
addition I've had cases where bits were cleaved from the compound
at some point before binding.  Sometimes you can't see it because
it simply isn't there.

Dale Tronrud

On 08/24/11 01:02, herman.schreu...@sanofi-aventis.com wrote:
> Dear Francis,
> 
> Although I am a member of the "never truncate a disordered side chain"
> camp, I think for ligands it is quite a different story. 
> 
> For me a random disordered lysine on the protein surface is completely
> uninteresting, except if one wants to examine the electrostatic surface.
> A non-expert end-user is either not aware of truncations and ends up
> with wrong results, or has to laboriously put back the side chains the
> crystallographer laboriously had removed before.
> 
> However, a bound ligand is very different. Determining its binding mode
> is usually THE goal of the study and every hydrogen bond with the
> protein is discussed in great detail. Chemists and modelers use these
> structures to design more potent ligands and theoreticians use these
> structures to improve their force fields. Also biochemists and
> biologists may be tempted to draw all kind of conclusions about
> important interactions where in fact there may be none.
> 
> Especially when the ligand has designed to make a certain interaction
> and in absence of experimental data you model the same interaction, the
> chemist and modeler will be very happy and immediately jump on it to
> design more of the same. I have seen many cases where theoretically, the
> ligand would be able to make a wonderful interaction with the protein,
> but that flexible side chains on the protein or ligand just did not want
> to give up their freedom (entropy) to become locked in such an
> interaction. Not seeing flexible parts of a ligand is not resolution
> dependent. At higher resolution you see even less of the flexible parts
> since there is less model bias possible.
> 
> So my approach: If there is weak or even very weak but real density for
> the flexible parts of the ligands (I usually scroll down to 0.6 sigma),
> I build the part, or build 2 or 3 conformations in case of discrete
> disorder. Here I think I take more liberties then most of my colleagues.
> 
> 
> However, if no convincing (weak) density is present above the noise
> level I remove the undefined parts and do not even consider to leave
> them in with occupancy zero. They will appear on the display of the end
> user and give the impression that an interaction is present where there
> is none. If the ligand would make the interaction, it would be visible
> in the electron density maps.
> 
> My choice is definitively [1], but before rushing to get more
> experimental data I would first put some brain power in it: maybe it is
> better for binding not to fix certain flexible side chains (less entropy
> loss), flexibility may endow the protein with broader substrate/ligand
> specificity, there may be crystal contacts which prevent the correct
> binding mode, components of the crystallization buffer may interfere
> with proper ligand binding etc.
> 
> Best,
> Herman
> 
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Francis E Reyes
> Sent: Tuesday, August 23, 2011 8:37 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Modeling ligands in binding pockets when the density
> is weak.
> 
> Seems to be a quiet day on the BB, so I propose this question: 
> 
> 
> Suppose you have a ligand in the binding pocket and some mediocre data
> (3 A or so), the 'core' of the ligand is well defined in 2Fo-Fc map
> using the model phases of your protein, however there are 'chains/tails'
> of the ligand which are not.  Composite omit or simulated annealing omit
> maps do not produce density for these 'chains' 
> 
> The question here is how the chains/tails should be modeled (if at all).
> 
> 
> 
> [1] Model in the core, but remove the atoms for the chains  (and
> conclude the diffraction data do not support interactions with the
> protein and subsequent experiments are needed (higher resolution data,
> biochemical data, etc)). 
> 
> or 
> 
> [2] Model in the chains/tails noting that potential hydrogen bond
> donors/acceptors on the protein are within hydrogen bonding distance to
> the chains/tails. You do this and subsequent refinement still does not
> produce the expected density for the chains. 
> 
> 
> or 
> 
> [3] Your solution here. 
> 
> 
> 
>  If this situation has been discussed before, please let me know .
> 
> F
> 
> -
> Francis E. Reyes M.Sc.
> 215 UCB
> University of Colorado at Boulder

Re: [ccp4bb] number of cycles in refmac

[Ian Tickle]

Hi Tim

The answer is a definite NO, the goal of refinement is to maximise the
likelihood from the working set and the restraints.  It is definitely not to
optimise Rfree or LLfree.  The correct value of the latter is whatever you
get at convergence of refinement, i.e. at maximum of the likelihood,
anything else is 'cheating'.

Cheers

-- Ian

On Wed, Aug 24, 2011 at 4:24 PM, Tim Gruene  wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
> Dear all,
>
> especially at the beginning of model building and/or at low resolution
> both Rfree and "-LL free" as reported in the refmac logfile show a
> minimum at a some cycle before rising again.
>
> I am certainly not the only one tempted to first run refmac with a large
> number of refinement cycles, determine that minimum and rerun refmac
> with ncyc set to that minimum.
>
> Of course I want the resulting model and phases/map to be as close to
> the what's in the crystal as possible in order to facilitate model
> building.
>
> Is it therefore good practice to interrupt refmac wherever it finds a
> minimum (if so, the minimum w.r.t. which number reported in the log-file)?
>
> Thanks for everyone's opinion and experience,
>
> Tim
>
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
>
> GPG Key ID = A46BEE1A
>
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.11 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
>
> iD8DBQFOVReTUxlJ7aRr7hoRAqyzAKCZpMJPVSQJTDEoWGxZEymwvqfFcACeMNLL
> rvIDPlXiL5HQmoNm7yrTt6k=
> =UnKT
> -END PGP SIGNATURE-
>

[ccp4bb] Database with protein experimental pKa values, with possibility to search for Cysteines and .pdb structure

[Thomas Juettemann]

Hello all,

this might not be the appropriate place, but I was wondering if anyone
knows of a database with protein experimental pKa values, with
possibility to search for Cysteines and .pdb structures.

"PINT: Protein-protein Interactions Thermodynamic Database" seems to be down.

The question arose on the pymol mailing list, and I  thought I give it
a shot here.

Many thanks for any pointers!

Best,
Thomas

[ccp4bb] number of cycles in refmac

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear all,

especially at the beginning of model building and/or at low resolution
both Rfree and "-LL free" as reported in the refmac logfile show a
minimum at a some cycle before rising again.

I am certainly not the only one tempted to first run refmac with a large
number of refinement cycles, determine that minimum and rerun refmac
with ncyc set to that minimum.

Of course I want the resulting model and phases/map to be as close to
the what's in the crystal as possible in order to facilitate model building.

Is it therefore good practice to interrupt refmac wherever it finds a
minimum (if so, the minimum w.r.t. which number reported in the log-file)?

Thanks for everyone's opinion and experience,

Tim

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.11 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFOVReTUxlJ7aRr7hoRAqyzAKCZpMJPVSQJTDEoWGxZEymwvqfFcACeMNLL
rvIDPlXiL5HQmoNm7yrTt6k=
=UnKT
-END PGP SIGNATURE-

[ccp4bb] Do manufacturers change their crystallogenesis screens?

[Chris Morris]

HI,

I've recently seen two examples where the description of a screen in a local 
database was different to the current one on the manufacturer's web site. This 
happened in two different labs, using different software, and with different 
screen manufacturers.

This could potentially lead to an optimisation screen that finds no hits, 
because the wrong condition is being optimised. Does anyone have experience of 
this? Am I just looking at a few one-off errors, or is there a general problem 
here?

The ideal solution is for screen manufacturers to give version numbers to their 
screens. Failing that, a good fix at the laboratory is to download the screen 
description every time a deep-well plate is received, and second best would be 
to download it every time a trial plate is set up. If there is a real concern 
here, we will implement one of these in xtalPiMS.

Regards,
Chris


Chris Morris
chris.mor...@stfc.ac.uk
Tel: +44 1925 603689  Fax: +44 1925 603825
Mobile: 07921-717915
http://pims.instruct-fp7.eu/ 
STFC, Daresbury Lab,  Daresbury,  Warrington,  UK,  WA4 4AD

Re: [ccp4bb] PISA question

[Tom Oldfield]

Tongqing

Thank you for highlighting an issue with a recent release of the 
PDBePisa service.  The  option to return the atom detail as well as the 
residue detail within the PISA service was accidentally missed from the 
production service during a major update of features.  I am sorry that 
this has effected your work.


We are have identified and fixed the problem and are currently testing 
the change and should have the  fix in production  by the end of the week.


Regards
Tom Oldfield
PDBe


Dear CCP4bb,

 

We are trying to get some detailed interface information from an 
antibody:antigen complex, PDBePISA used to have an option to select 
atomic level details, now the new web interface only gives residue 
level information. Is there a way to run PISA (in the CCP4 package) 
with a different configuration to get the detailed information?


 


Thanks,

 

 


Tongqing

 


*Tongqing Zhou, Ph.D.*

Staff Scientist

Structural Biology Section

Vaccine Research Center, NIAID/NIH

Building 40, Room 4607B

40 Convent Drive, MSC3027

Bethesda, MD 20892

(301) 594-8710 (Tel)

(301) 793-0794 (Cell)

(301) 480-2658 (Fax)

**

The information in this e-mail and any of its attachments is 
confidential and may contain sensitive information. It should not be 
used by anyone who is not the original intended recipient. If you have 
received this e-mail in error please inform the sender and delete it 
from your mailbox or any other storage devices. National Institute of 
Allergy and Infectious Diseases shall not accept liability for any 
statements made that are sender's own and not expressly made on behalf 
of the NIAID by one of its representatives.


**

 

[ccp4bb] Immediate opeining for a Post-DOC fellowship based at Diamond & the Research Complex at Harwell

[Martin Walsh]

--
RESEARCH ASSOCIATE: A Multidisciplinary Approach to Protein Function and 
Exploitation
--

Department of Physics/Strathclyde Institute of Pharmacy and Biomedical Sciences
&
DIAMOND LIGHT SOURCE

FIXED TERM (1 yr)

Salary £29,099-£35,788

===

Applications are invited for this joint post between the 
Department of Physics and the Strathclyde Institute of Pharmacy and Biomedical 
Sciences at the University of Strathclyde, and Diamond Light Source Ltd (the UK 
national synchrotron science facility situated at Harwell in Oxfordshire).  The 
post holder will be based at Diamond, with work being directed by Dr's Neil 
Hunt, Nicholas Tucker, Paul Hoskisson at Strathclyde and Martin Walsh at 
Diamond.

The appointment is initially for one year. The aim of the 
project is to combine X-ray crystallography, time resolved 2D-IR spectroscopy 
and biochemical techniques to study the structure and function of the E coli 
Hmp NO dioxygenase enzyme system and to work towards integration of ultrafast 
spectroscopy with the Diamond MX beamlines for future exploitation in 
biological research. The project will involve elements of biochemistry, 
structural biology and infrared spectroscopy, including time resolved and 
multidimensional methods. Expertise in all areas is not required though a 
strong background in structural biology, protein biochemistry, biophysics, 
infrared spectroscopy or time resolved/ultrafast spectroscopy would be 
advantageous. 

The successful candidate will join ongoing collaborative 
projects in place between Strathclyde, DLS and the Research Complex at Harwell 
(http://www.rc-harwell.ac.uk) that aim to investigate the mechanisms of protein 
systems from a multidisciplinary perspective. Regular interactions with 
Strathclyde and the Research Complex are envisaged. As such the project will 
suit an ambitious and motivated candidate with a strong commitment to 
multidisciplinary research.

For informal enquiries please contact 
martin.wa...@diamond.ac.uk, neil.h...@strath.ac.uk, nick.tuc...@strath.ac.uk or 
paul.hoskis...@strath.ac.uk.


For an application pack visit http://vacancies.strath.ac.uk or contact Human 
Resources, University of Strathclyde, Glasgow G1 1XQ. Tel. 0141 553 4133, 
quoting ref: JA/R70/2011.  

Closing date: 22 September 2011

We value diversity and welcome applications from all sections of the community
The University of Strathclyde is a Registered Scottish Charity, No SCO15263

Re: [ccp4bb] Modeling ligands in binding pockets when the density is weak.

[Ed Pozharski]

On Tue, 2011-08-23 at 15:01 -0400, Ed Pozharski wrote:
> On Tue, 2011-08-23 at 12:36 -0600, Francis E Reyes wrote:
> > Seems to be a quiet day on the BB
> 
> So you need an earthquake :)
> 
> This is similar, imho, to the issue of disordered side chains:
> 
> https://docs.google.com/spreadsheet/gform?key=0Ahe0ET6Vsx-kdHVNa3VodUtfbVQtZ2pnUFcxQkx6RHc&hl=en_US&gridId=0#chart
> 
> https://docs.google.com/spreadsheet/viewform?hl=en_US&formkey=dHVNa3VodUtfbVQtZ2pnUFcxQkx6RHc6MQ#gid=0
> 
> 
> 

The second link above happens to point at the form editing page or such,
so please disregard (my googlebox is getting flooded with requests to
share it).  If you want to cast a belated vote, use this link

https://spreadsheets.google.com/viewform?hl=en&formkey=dHVNa3VodUtfbVQtZ2pnUFcxQkx6RHc6MQ

To see the results, see this one.

https://docs.google.com/spreadsheet/gform?key=0Ahe0ET6Vsx-kdHVNa3VodUtfbVQtZ2pnUFcxQkx6RHc&hl=en_US&gridId=0#chart

Ed.

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

Re: [ccp4bb] LABELIT with XDS

[Boaz Shaanan]

Hi,

I apologise for the empty message. If you take the cell dimensions that you got 
in LABELIT into XDS it should work. I guess you couldn't get XDS to index from 
fresh, is that correct?  

  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 James Whittle 
[whit...@crystal.harvard.edu]
Sent: Wednesday, August 24, 2011 3:10 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] LABELIT with XDS

Dear all,

I'm able to index my images in LABELIT. Is it possible to use that
indexing solution in XDS?

James

Re: [ccp4bb] LABELIT with XDS

[Boaz Shaanan]

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 James Whittle 
[whit...@crystal.harvard.edu]
Sent: Wednesday, August 24, 2011 3:10 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] LABELIT with XDS

Dear all,

I'm able to index my images in LABELIT. Is it possible to use that
indexing solution in XDS?

James

[ccp4bb] LABELIT with XDS

[James Whittle]

Dear all,

I'm able to index my images in LABELIT. Is it possible to use that
indexing solution in XDS?

James

Re: [ccp4bb] PISA question

[MARTYN SYMMONS]

Dear All

I may not have understood but if I run PDBe PISA 
and go Interface>Details 
then I get a table of the form eg for H-bonds

Hydrogen bonds
 1   A:GLN 213[ NE2] 3.52A:SER   3[ O  ] 
 2   A:ASN 112[ ND2] 2.77A:SER   3[ O  ] 
 3   A:GLN 213[ NE2] 2.99A:SER   4[ O  ] 
 4   A:LYS 154[ NZ ] 2.74A:ILE   5[ O  ] 
 5   A:ARG 214[ NH2] 2.75A:GLU  16[ OE2] 
 6   A:TYR 168[ N  ] 2.86A:LYS  31[ O  ] 
 7   A:TYR 168[ OH ] 3.00A:ASN  34[ OD1] 
 8   A:LYS 175[ NZ ] 2.95A:ASP  36[ OD2] 
 9   A:ARG 129[ NH1] 3.22A:ASP 127[ O  ] 
 10  A:ARG 129[ NH2] 2.90A:ASP 127[ O  ] 
 11  A:HIS 363[ ND1] 3.47A:GLN 132[ O  ] 
 12  A:HIS 363[ ND1] 3.41A:GLN 132[ OE1] 
 13  A:TYR 350[ OH ] 2.71A:LEU 179[ O  ]

which seems to include atom details (not Hs however).

In the new pages there is no longer a pulldown for atomic detail but this now 
is the default. 

Hope that helps
Martyn 








--- On Tue, 23/8/11, eugene.krissi...@stfc.ac.uk  
wrote:

> From: eugene.krissi...@stfc.ac.uk 
> Subject: Re: [ccp4bb] PISA question
> To: CCP4BB@JISCMAIL.AC.UK
> Date: Tuesday, 23 August, 2011, 17:50
> Dear Tongqing
> 
> No there is no such option in CCP4's PISA, but I put this
> on the list for further developments. Regrettably PDBe
> changed the interface after I moved out and I cannot help
> you with that. However, you may ask them to put that option
> back.
> 
> Sorry,
> 
> Eugene.
> 
> 
> On 23 Aug 2011, at 17:16, Zhou, Tongqing (NIH/VRC) [E]
> wrote:
> 
> Dear CCP4bb,
> 
> We are trying to get some detailed interface information
> from an antibody:antigen complex, PDBePISA used to have an
> option to select atomic level details, now the new web
> interface only gives residue level information. Is there a
> way to run PISA (in the CCP4 package) with a different
> configuration to get the detailed information?
> 
> Thanks,
> 
> 
> Tongqing
> 
> Tongqing Zhou, Ph.D.
> Staff Scientist
> Structural Biology Section
> Vaccine Research Center, NIAID/NIH
> Building 40, Room 4607B
> 40 Convent Drive, MSC3027
> Bethesda, MD 20892
> (301) 594-8710 (Tel)
> (301) 793-0794 (Cell)
> (301) 480-2658 (Fax)
> **
> The information in this e-mail and any of its attachments
> is confidential and may contain sensitive information. It
> should not be used by anyone who is not the original
> intended recipient. If you have received this e-mail in
> error please inform the sender and delete it from your
> mailbox or any other storage devices. National Institute of
> Allergy and Infectious Diseases shall not accept liability
> for any statements made that are sender's own and not
> expressly made on behalf of the NIAID by one of its
> representatives.
> **
>

[ccp4bb] Finding obsolete pdb entries

[Simon Kolstoe]

Dear ccp4bb,

About 6 years ago I noted a couple of structures I was interested in were 
removed from the pdb. I saw in a recent email discussion that it is possible to 
access obsolete entries, however unfortunately I do not have the pdb code of 
the structure I am interested in - and neither does the original publication 
list the pdb code. Is there a way of searching withdrawn/obsolete entries for 
author name, macromolecule etc. or are structures that were withdrawn over 5 
years ago lost for good? 

Thanks,

Simon

Re: [ccp4bb] Check PDB file for missing atoms

[Bernhard C. Lohkamp]

Coot:

Extension -> Modelling -> Residues with Missing Atoms...

Or watch out for the blue bars in the rotamer validation graph in Coot.

B


Dear all,

Does anyone know a program that will check a PDB file for missing 
atoms and output a list of the corresponding residues?


Many thanks in advance,

Tiago

***

Tiago Barros, PhD
Kuriyan lab - Molecular and Cellular Biology
University of California, Berkeley
527 Stanley Hall, QB3
Berkeley, CA 94720-3220
USA

tel:  + 1 510 643 0164
fax:  + 1 510 643 2352

***



No virus found in this message.
Checked by AVG - www.avg.com 
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Re: [ccp4bb] Modeling ligands in binding pockets when the density is weak.

[Herman . Schreuder]

Dear Francis,

Although I am a member of the "never truncate a disordered side chain"
camp, I think for ligands it is quite a different story. 

For me a random disordered lysine on the protein surface is completely
uninteresting, except if one wants to examine the electrostatic surface.
A non-expert end-user is either not aware of truncations and ends up
with wrong results, or has to laboriously put back the side chains the
crystallographer laboriously had removed before.

However, a bound ligand is very different. Determining its binding mode
is usually THE goal of the study and every hydrogen bond with the
protein is discussed in great detail. Chemists and modelers use these
structures to design more potent ligands and theoreticians use these
structures to improve their force fields. Also biochemists and
biologists may be tempted to draw all kind of conclusions about
important interactions where in fact there may be none.

Especially when the ligand has designed to make a certain interaction
and in absence of experimental data you model the same interaction, the
chemist and modeler will be very happy and immediately jump on it to
design more of the same. I have seen many cases where theoretically, the
ligand would be able to make a wonderful interaction with the protein,
but that flexible side chains on the protein or ligand just did not want
to give up their freedom (entropy) to become locked in such an
interaction. Not seeing flexible parts of a ligand is not resolution
dependent. At higher resolution you see even less of the flexible parts
since there is less model bias possible.

So my approach: If there is weak or even very weak but real density for
the flexible parts of the ligands (I usually scroll down to 0.6 sigma),
I build the part, or build 2 or 3 conformations in case of discrete
disorder. Here I think I take more liberties then most of my colleagues.


However, if no convincing (weak) density is present above the noise
level I remove the undefined parts and do not even consider to leave
them in with occupancy zero. They will appear on the display of the end
user and give the impression that an interaction is present where there
is none. If the ligand would make the interaction, it would be visible
in the electron density maps.

My choice is definitively [1], but before rushing to get more
experimental data I would first put some brain power in it: maybe it is
better for binding not to fix certain flexible side chains (less entropy
loss), flexibility may endow the protein with broader substrate/ligand
specificity, there may be crystal contacts which prevent the correct
binding mode, components of the crystallization buffer may interfere
with proper ligand binding etc.

Best,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Francis E Reyes
Sent: Tuesday, August 23, 2011 8:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Modeling ligands in binding pockets when the density
is weak.

Seems to be a quiet day on the BB, so I propose this question: 


Suppose you have a ligand in the binding pocket and some mediocre data
(3 A or so), the 'core' of the ligand is well defined in 2Fo-Fc map
using the model phases of your protein, however there are 'chains/tails'
of the ligand which are not.  Composite omit or simulated annealing omit
maps do not produce density for these 'chains' 

The question here is how the chains/tails should be modeled (if at all).



[1] Model in the core, but remove the atoms for the chains  (and
conclude the diffraction data do not support interactions with the
protein and subsequent experiments are needed (higher resolution data,
biochemical data, etc)). 

or 

[2] Model in the chains/tails noting that potential hydrogen bond
donors/acceptors on the protein are within hydrogen bonding distance to
the chains/tails. You do this and subsequent refinement still does not
produce the expected density for the chains. 


or 

[3] Your solution here. 



 If this situation has been discussed before, please let me know .

F

-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder