Re: [ccp4bb] salt sensitive complex

[David Briggs]

Hi Jerry,

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

Ok.

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

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

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

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

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

Hope this (rather lengthy reply) helps!

Dave


On 23/01/2008, Jerry McCully <[EMAIL PROTECTED]> wrote:
>
>  Dear All:
>
>  Recently I am pursuing the crystallziation of a complex formd by
> two individual proteins and I met several interesting problems though  they
> are kind of off-topic.
>
>  Any suggestions for these problems will be highly appreciated.
>
>  BIAcore showed about submicromolar affinity(both Kinetic and
> steady-state fitting) for these two proteins in the complex. However,
> precipitates immediately appeared when these two proteins were mixed
> together even at 10uM(<0.3mg/ml) concentration in the condition of low
> salt(less than 20mM NaCl).
>  By the way, these two proteins completely precipitated when the molar ratio
> is 1:1 in this condition.
>
>   THerefore, I increased the salt concentraion step by step and finally I
> can keep both of them soluble in the solution with 25mM Tris(pH8) and 60mM
> NaCl(the minimum of salt concentration).   Wierd thing happened when ITC
> experiments were carried out to confirm the binding affinity.  20uM in the
> sample cell and 200uM in the syringe could not give enough heat for a good
> curve fitting. The optimistic estimation of the affinity is lower than 5uM,
> which is much lower than the affinity given by BIAcore in the same
> buffer(25mM Tris plus 150mM NaCl).
>
>Now I am suspecting the capability of the interaction between these
> two proteins. However, I can not explain why these two guys precipitated
> stoichiometrically if they do not interact with each other.
>
>Is the complex salt-sensitive therefore there was just minor binding
> in the high-salt condition revealed by ITC?
>
> I am planning to do the ITC again in the condition of 25mMTris and
> 60mM NaCl.
>
> What if the affinity given by ITC is still much lower than that by
> BIAcore. Which one should I choose to believe?
>
>Are there some better ways that  I can validate the binding affinity?
>
>
>   Thanks again for your great ideas.
>
>  Jerry McCully
>
>
>
>
>
>
> 
> Need to know the score, the latest news, or you need your Hotmail(R)-get your
> "fix". Check it out.


-- 

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

Re: [ccp4bb] Characterization of common salt crystal forms?

[David Briggs]

Hi Joe,

I've known most salt crystals in Phosphate - and I think most people
are weary of phosphate.

Also, Calcium Sulphate is a fairly common one, esp if your buffers are
titrated with sulphuric acid. Fluoride Ions are also prone to form
salt crystals with transition metal ions.

HTH,

Dave


On 22/01/2008, Joe Krahn <[EMAIL PROTECTED]> wrote:
> Salt crystals are common in macromolecular crystallography. Has anyone
> tried to tabulate salt crystal forms that commonly occur?
>
> I just identified a salt crystal as Mirabilite, made of Na2SO4·10H2O.
> The high water content makes them rather soft, and may not be recognized
> as salt right away. In this case, it probably happened because the
> buffer was made with Na·Citrate + HCl instead of citric acid, while
> trying to optimize conditions. So, characterization of salt crystals can
> help to avoid the conditions that cause them.
>
> There is probably a reasonably small number of salt crystal forms that
> are very common in crystallization trials. Maybe it would be useful to
> tabulate common salt crystals to help guide optimization experiments.
> Has anyone else tried to use salt crystal information beyond ensuring
> that it is not protein?
>
> Joe Krahn
>


-- 

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

Re: [ccp4bb] microsoft 3-button wheel mouse with OS X 10.5

[mb1pja]

Dear Bill

William Scott wrote


Aqua simply behaves by slightly different rules.  Although I am a
slobbering OS X fan, this lack of customizability to me, as well as  
a lack

of focus-follows-mouse, it a negative.



To get focus-follows-mouse in Aqua, type the following in your  
Terminal window:


defaults write com.apple.Terminal FocusFollowsMouse -string YES

and then logout and log in again or quit and restart Terminal.

I had thought I originally got that useful hint from your own fabulous  
PX on OSX pages but clearly not.




best wishes

Pete Artymiuk



On 20 Jan 2008, at 15:39, William Scott wrote:


Hi David:

david lawson (JIC) wrote:

Dear All,

Sorry for the slightly off-topic subject.

We have recently bought a few iMacs for crystallography. I'm not  
keen on

the supplied "mighty mouse"


May I have them?


so I have switched to using a microsoft
3-button wheel mouse. I would like to configure it so that it  
behaves as

it would with other unix systems such as RH Linux.


You managed to use "Microsoft", "behaved" and "Linux" (albeit RH)  
all in

one sentence without a hint of irony.




i.e.
(1) double-clicking with LH button on a file name selects ALL of the
file name, not just up to the first full stop.


Although your choice of Microsoft products shows dedication to a  
company

with a firm reputation for placing the customizability needs of its
customers ahead of its own desire to make profits, the first thing to
realize is that you should never ever ever install their drivers.  
Ever. So
if you did, take them out, now, and reboot. I'll wait.  It is still  
early

Sunday morning here.



(2) clicking the scroll wheel pastes the selected text AND it can be
done multiple times without re-selecting.


When you've gotten rid of the drivers, this should now work. In  
Apple's

Terminal program (as of 10.5) and iTerm (as of 1215), you just set the
preference to do middle-button-paste and left-button select, and  
Blair's
your uncle. Unfortunately, in pretty much every other application I  
can
think of on OS X, this, sadly, does not work, and there is nothing  
Steve

Gates will let you do about it.


(2) I would like these functions to work in terminal windows, the  
ccp4i
gui and web pages (and probably a few other things I haven't  
thought of

yet!) AND be able to transfer the selected text between applications.


I'd like to be at my ideal high-school weight, be paid more than a
postdoc, and, well ...  Getting the OS X gui to play nice with X11 is
sometimes challenging.  With the exception of Terminal and iTerm,  
you have
to explicitly put stuff in the copy/paste buffer (command-C) before  
it is

in the system clipboard.  Then you can paste to X11 programs with a
middle-button click, but this only works if you uninstalled that viral
driver. Going from X11 to aqua programs requires selecting the text  
in the
usual X11 manner but explicitly issuing the paste command (command- 
p).  If

you are using KDE X11 applications, you are really in for headaches.

To get whole-string selection in iTerm or Terminal, there is a  
preference

setting that allows you to input which characters you want to have
considered parts of a "word" for click-to-select purposes.   
Unfortunately,
pretty much every other application lacks this customizability, and  
I know

of no system-wide preference setting that would enable you to do this
globally.

Aqua simply behaves by slightly different rules.  Although I am a
slobbering OS X fan, this lack of customizability to me, as well as  
a lack

of focus-follows-mouse, it a negative.

If you really need the canonical linux behavior, you can install  
gnome,

xfce4, KDE, enlightenment, or any number of other window managers via
fink. I've found KDE buggy and the XFCE4 is way out of date.  Gnome is
probably the best bet, and there is a major effort now to bring it
completely up to date in fink.



I have installed the microsoft intellipoint drivers that seem to give
more control over configuring the various buttons through "system
preferences", but I still can't get what I want.


Therein lies the problem, I am afraid. OS X will behave better using  
the
default settings.  It may be possible to tinker around with the  
driver,
including separate settings in X11, to recover canonical behavior,  
but for
purposes of sanity, uninstall them first, get everything working as  
best
as possible, verify middle-button-paste works in X11, verify X11  
coot and
pymol do the right thing, and then if you need additional  
functionality,

reinstall the drivers, verify things like coot and pymol still use the
middle button correctly, or adjust until they do, and only then try
customizing.

Best of luck!


Bill





Any help would be much appreciated.

Many thanks

Dave Lawson

---

Dr. David M. Lawson
Biological Chemistry Dept.,
John Innes Centre,
Norwich,
NR4 7UH, UK.
Tel: +44-(0)1603-450725
Fax: +44-(0)1603-450018
Email: [EMAIL PROTECTED]
Web: h

Re: [ccp4bb] Characterization of common salt crystal forms?

[R.M. Garavito]

Joe,


As David said many different types of salt crystals can grow in the   
presence of phosphate, even when the concentrations of PO4 or many  
divalent cations are VERY low (10s of micromolar or less).  Struvite  
is common (NH4MgPO4) when ammonium sulfate is the precipitant, mM  
magnesium is present, and the pH is >7; struvite is a common mineral  
in kidney stones.  If we have removed phosphate just before  
crystallization, as with a spin desalting column (best way) or by  
repeated spin concentration (poorer way), we always redouble check  
"hits" in divalent cation containing conditions with a variety of  
controls (e.g., keep the filtrate fraction from the last spin  
concentration step).


Michael




R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  [EMAIL PROTECTED]



On Jan 23, 2008, at 4:06 AM, David Briggs wrote:


Hi Joe,

I've known most salt crystals in Phosphate - and I think most people
are weary of phosphate.

Also, Calcium Sulphate is a fairly common one, esp if your buffers are
titrated with sulphuric acid. Fluoride Ions are also prone to form
salt crystals with transition metal ions.

HTH,

Dave


On 22/01/2008, Joe Krahn <[EMAIL PROTECTED]> wrote:
Salt crystals are common in macromolecular crystallography. Has  
anyone

tried to tabulate salt crystal forms that commonly occur?

I just identified a salt crystal as Mirabilite, made of Na2SO4·10H2O.
The high water content makes them rather soft, and may not be  
recognized

as salt right away. In this case, it probably happened because the
buffer was made with Na·Citrate + HCl instead of citric acid, while
trying to optimize conditions. So, characterization of salt  
crystals can

help to avoid the conditions that cause them.

There is probably a reasonably small number of salt crystal forms  
that
are very common in crystallization trials. Maybe it would be  
useful to

tabulate common salt crystals to help guide optimization experiments.
Has anyone else tried to use salt crystal information beyond ensuring
that it is not protein?

Joe Krahn




--

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

Re: [ccp4bb] Why there are difference density when occupancy is 1.00

[Eleanor Dodson]

Hard to say without more information.

Have you refined the B factors for these residues? Most building 
software gives some arbitrary b value, which must then be refined. (In 
fact after any rebuilding activity you need to do a few cycles of 
refinement before looking at the maps again)


Eleanor

Sun Tang wrote:

Hello Everyone,
   
  When I refined a structure, I found strong difference density Fo-Fc at 3 sigma contour for the for five residues which already have occupancy of 1. The density is continuous and so strong as if I did not put the residues there. Why was that? Can I put greater than 1 occupancy for those residues? 
   
  Thank you very much for your opinions and suggestions!
   
  Best wishes,
   
  Sun Tang


   
-

Never miss a thing.   Make Yahoo your homepage.
  

[ccp4bb] Question about freeR tag

[Zheng Zhou]

Hi, All

Could any tell me how CCP4 handle free R flag? I know It is important to
select the same** FreeR reflections if I move to next step of refinement.
But everytime I start from fresh, the freeR Flag remains unchanged. The
Rwork and  Rfree of my models are fine (20.7% and 22.9%). I thought Rfree
was randomly generated. I asked more experienced people in the lab and
neighbor labs, and didn't get a straight answer. Did I do something wrong?

Thanks and sorry to bother others.

Zheng (Joe)

Re: [ccp4bb] microsoft 3-button wheel mouse with OS X 10.5

[William Scott]

Yes, thanks, that does it for the Terminal.app, but not for any of the
rest.  It would be great to have such a feature globally.

mb1pja wrote:
> Dear Bill
>
> William Scott wrote
>
>> Aqua simply behaves by slightly different rules.  Although I am a
>> slobbering OS X fan, this lack of customizability to me, as well as
>> a lack
>> of focus-follows-mouse, it a negative.
>
>
> To get focus-follows-mouse in Aqua, type the following in your
> Terminal window:
>
> defaults write com.apple.Terminal FocusFollowsMouse -string YES
>
> and then logout and log in again or quit and restart Terminal.
>
> I had thought I originally got that useful hint from your own fabulous
> PX on OSX pages but clearly not.
>
>
>
> best wishes
>
> Pete Artymiuk
>
>
>
> On 20 Jan 2008, at 15:39, William Scott wrote:
>
>> Hi David:
>>
>> david lawson (JIC) wrote:
>>> Dear All,
>>>
>>> Sorry for the slightly off-topic subject.
>>>
>>> We have recently bought a few iMacs for crystallography. I'm not
>>> keen on
>>> the supplied "mighty mouse"
>>
>> May I have them?
>>
>>> so I have switched to using a microsoft
>>> 3-button wheel mouse. I would like to configure it so that it
>>> behaves as
>>> it would with other unix systems such as RH Linux.
>>
>> You managed to use "Microsoft", "behaved" and "Linux" (albeit RH)
>> all in
>> one sentence without a hint of irony.
>>
>>
>>>
>>> i.e.
>>> (1) double-clicking with LH button on a file name selects ALL of the
>>> file name, not just up to the first full stop.
>>
>> Although your choice of Microsoft products shows dedication to a
>> company
>> with a firm reputation for placing the customizability needs of its
>> customers ahead of its own desire to make profits, the first thing to
>> realize is that you should never ever ever install their drivers.
>> Ever. So
>> if you did, take them out, now, and reboot. I'll wait.  It is still
>> early
>> Sunday morning here.
>>
>>
>>> (2) clicking the scroll wheel pastes the selected text AND it can be
>>> done multiple times without re-selecting.
>>
>> When you've gotten rid of the drivers, this should now work. In
>> Apple's
>> Terminal program (as of 10.5) and iTerm (as of 1215), you just set the
>> preference to do middle-button-paste and left-button select, and
>> Blair's
>> your uncle. Unfortunately, in pretty much every other application I
>> can
>> think of on OS X, this, sadly, does not work, and there is nothing
>> Steve
>> Gates will let you do about it.
>>
>>
>>> (2) I would like these functions to work in terminal windows, the
>>> ccp4i
>>> gui and web pages (and probably a few other things I haven't
>>> thought of
>>> yet!) AND be able to transfer the selected text between applications.
>>
>> I'd like to be at my ideal high-school weight, be paid more than a
>> postdoc, and, well ...  Getting the OS X gui to play nice with X11 is
>> sometimes challenging.  With the exception of Terminal and iTerm,
>> you have
>> to explicitly put stuff in the copy/paste buffer (command-C) before
>> it is
>> in the system clipboard.  Then you can paste to X11 programs with a
>> middle-button click, but this only works if you uninstalled that viral
>> driver. Going from X11 to aqua programs requires selecting the text
>> in the
>> usual X11 manner but explicitly issuing the paste command (command-
>> p).  If
>> you are using KDE X11 applications, you are really in for headaches.
>>
>> To get whole-string selection in iTerm or Terminal, there is a
>> preference
>> setting that allows you to input which characters you want to have
>> considered parts of a "word" for click-to-select purposes.
>> Unfortunately,
>> pretty much every other application lacks this customizability, and
>> I know
>> of no system-wide preference setting that would enable you to do this
>> globally.
>>
>> Aqua simply behaves by slightly different rules.  Although I am a
>> slobbering OS X fan, this lack of customizability to me, as well as
>> a lack
>> of focus-follows-mouse, it a negative.
>>
>> If you really need the canonical linux behavior, you can install
>> gnome,
>> xfce4, KDE, enlightenment, or any number of other window managers via
>> fink. I've found KDE buggy and the XFCE4 is way out of date.  Gnome is
>> probably the best bet, and there is a major effort now to bring it
>> completely up to date in fink.
>>
>>>
>>> I have installed the microsoft intellipoint drivers that seem to give
>>> more control over configuring the various buttons through "system
>>> preferences", but I still can't get what I want.
>>
>> Therein lies the problem, I am afraid. OS X will behave better using
>> the
>> default settings.  It may be possible to tinker around with the
>> driver,
>> including separate settings in X11, to recover canonical behavior,
>> but for
>> purposes of sanity, uninstall them first, get everything working as
>> best
>> as possible, verify middle-button-paste works in X11, verify X11
>> coot and
>> pymol do the right thing, and then if you need additional
>> functionality,

[ccp4bb] problem on protein precipitation

[Zheng, Lei]

Hi ccp4ers,

 

Sorry for this out-topic question:

Recently we have a membrane protein expressed, after solubilized with
detergent and purified from IMAC, the protein looks beautiful in SEC.
However, it completely precipitates after the 2-3 days storage in 4
degree. We supplement 2 mM DTT in the new elute from IMAC, the protein
looks happy during weeks at 4 degree. However, it starts to form an
invisible aggregate (verified from SEC) during the protein concentration
by Centricon. I know this is not uncommon problem for both soluble and
membrane proteins and wonder if anyone has any tip and experience to
overcome this problem.

The protein pI is 8.6, buffer used is pH 7.6. Glycerol is always present
during the purification. We do have high salt (500mM) in the buffer.

 

Thank you for you input in advance,

Lei

 

Re: [ccp4bb] Question about freeR tag

[Zheng Zhou]

Sorry that I didn't explain the situation clearly. I used only one
output.sca file from HLK2000. I ran the scalepack2mtz (in CCP4i, data
reduction, import merged data) several times, on both linux Fedora and
window XP, the same computer though.

For the next step refinement, I mean add H2O, ion, ligands, double check
certain sidechains In those refmac runs, I always use the same mtz file,
which I generated from the beginning. I am as surprised as you are the Rfree
flags are identical from those different "first" runs.

Thanks

On Jan 23, 2008 9:50 AM, Zheng Zhou <[EMAIL PROTECTED]> wrote:

> Hi, All
>
> Could any tell me how CCP4 handle free R flag? I know It is important to
> select the same** FreeR reflections if I move to next step of refinement.
> But everytime I start from fresh, the freeR Flag remains unchanged. The
> Rwork and  Rfree of my models are fine ( 20.7% and 22.9%). I thought Rfree
> was randomly generated. I asked more experienced people in the lab and
> neighbor labs, and didn't get a straight answer. Did I do something wrong?
>
> Thanks and sorry to bother others.
>
> Zheng (Joe)
>
I cant understand this.
Do you mean you have multiple data sets and they all generate the same
FreeRs? If you have EXACTLY the same reflection list, and generate FreeR
flags on the same machine I guess they would be the same.. but it would
be surprising

Or if you are using the same file which already has FreeR flags then
they wont change of course - the default is always to use the
reflections flagged with FreeR = 0.  (Thats as it should be..)

By the by - your agreement between R and FreeR seems unusually low
unless you have very high resolution data or a great deal of
non-crystallographic symmetry.
Eleanor

Zheng  - I am not sure what you are doing, but as long as you work
with one dataset only, you generate the free reflections at the very
beginning when you import your data into CCP4. Then use the resulting
mtz file for all following refinement steps. There are situations
where one will have to deviate from this scheme, but those are rare.
If you feel you have such a case, then tell us more about it. Hope
that helps. Best - MM

Joe,


can you explain a bit clearer your problem?  what do you mean the 'next step
of refinement'?  are you just doing another round of refinement?  what
program are you using, REFMAC5?  also, R/Rfree of 20.7/22.9 is pretty good,
depending on resolution.

yes, you are right, Rfree flags are generated randomly and can be anywhere
from 5-10% of your reflections (your choice).  then, once the Rfree flagged
reflections are selected, they will not change (nor be refined) throughout
the rest of your structure determination.  they should have their own column
in your *.mtz file called RfreeFlag or something like this (in CCP4 that
is).  other programs handle this relatively the same, but may use various
naming conventions.  one caveat is that CCP4 flags reflections using 0 by
default (meaning 5% of your reflections are flagged with 0 and are the Rfree
set).  Other programs such as CNS and PHENIX use 1 by default, so be careful
when switching back and forth.  You can tell either program which to use for
Rfree set, but needs to be set, since it is not default.

again, not exactly sure what the problem is, but i hope some of this helps.
 feel free to email with further questions if needed.  best of luck!



cheers,
nick

Re: [ccp4bb] problem on protein precipitation

[Lisa A Nagy]

Hi Lei,

 

Try this:

50-100 mM Arginine in your buffers. Or Glutamic Acid.  Or both.

 

--

Lisa A. Nagy, Ph.D.

University of Alabama-Birmingham

[EMAIL PROTECTED]

 

 

 

From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Zheng, Lei
Sent: Wednesday, January 23, 2008 10:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] problem on protein precipitation

 

Hi ccp4ers,

 

Sorry for this out-topic question:

Recently we have a membrane protein expressed, after solubilized with
detergent and purified from IMAC, the protein looks beautiful in SEC.
However, it completely precipitates after the 2-3 days storage in 4
degree. We supplement 2 mM DTT in the new elute from IMAC, the protein
looks happy during weeks at 4 degree. However, it starts to form an
invisible aggregate (verified from SEC) during the protein concentration
by Centricon. I know this is not uncommon problem for both soluble and
membrane proteins and wonder if anyone has any tip and experience to
overcome this problem.

The protein pI is 8.6, buffer used is pH 7.6. Glycerol is always present
during the purification. We do have high salt (500mM) in the buffer.

 

Thank you for you input in advance,

Lei

 

Re: [ccp4bb] Question about freeR tag

[Roger Rowlett]

Zheng Zhou wrote:

Hi, All

Could any tell me how CCP4 handle free R flag? I know It is important 
to select the same** FreeR reflections if I move to next step of 
refinement. But everytime I start from fresh, the freeR Flag remains 
unchanged. The Rwork and  Rfree of my models are fine ( 20.7% and 
22.9%). I thought Rfree was randomly generated. I asked more 
experienced people in the lab and neighbor labs, and didn't get a 
straight answer. Did I do something wrong?


Thanks and sorry to bother others.

Zheng (Joe)
You have two options for setting the FreeR flag in CCP4i. You can do it 
at the time of scaling in SCALA by ticking the button "Ensure unique 
data & add FreeR column for x.xx fraction of data." 5% of the data is 
the default, which may be excessive depending on the number of total 
reflections you have.


If you did not set the FreeR flag during scaling, you can do it later 
using the Convert to MTZ & Standardise task under Reflection Utilities 
in the GUI. Tick "Create a full unique set of reflections and generate 
FreeR data" and select your FreeR fraction in the box near the bottom of 
the task window.


Once the FreeR flag is set it should not change, and you should not 
generate a new set of FreeR flags again during the refinement. The idea 
is to set aside some data early on that is never used to refine the 
model so that it is as unbiased as possible in evaluating your 
refinement statistics and guarding against model bias. When refining 
with Refmac5, tick the "Exclude data with freeR label FreeR_flag with 
value of 0" to ignore the FreeR data during refinement.


Cheers,

--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]

Re: [ccp4bb] Why there are difference density when occupancy is 1.00

[Sun Tang]

Hi Eleanor,

Thank you for your information. I did do the B factor refinement in CCP4i and 
at the first several rounds of refinement, there was no such difference map but 
in the almost final run of refinement (including TLS and restrained), it 
appeared.  The B-factors (about 40) are similar to nearby residues. This 
confused me.  Any ideas?

Thank you again for your suggestions.

Best,

Sun Tang

Eleanor Dodson <[EMAIL PROTECTED]> wrote: Hard to say without more information.

Have you refined the B factors for these residues? Most building 
software gives some arbitrary b value, which must then be refined. (In 
fact after any rebuilding activity you need to do a few cycles of 
refinement before looking at the maps again)

Eleanor

Sun Tang wrote:
> Hello Everyone,
>
>   When I refined a structure, I found strong difference density Fo-Fc at 3 
> sigma contour for the for five residues which already have occupancy of 1. 
> The density is continuous and so strong as if I did not put the residues 
> there. Why was that? Can I put greater than 1 occupancy for those residues? 
>
>   Thank you very much for your opinions and suggestions!
>
>   Best wishes,
>
>   Sun Tang
>
>
> -
> Never miss a thing.   Make Yahoo your homepage.
>   



   
-
Never miss a thing.   Make Yahoo your homepage.

Re: [ccp4bb] Why there are difference density when occupancy is 1.00

[Sun Tang]

Hi James,

I did check teh B-factors and they are similar to the flanking regions (about 
40). The difference density appeared at the later stage of refinement (TLS and 
restrained in CCP4i). 

What do you think and how to do it? 

Best,

Sun Tang

James Irving <[EMAIL PROTECTED]> wrote: Hi Sun,
I suggest checking that the b-factors for those residues aren't
unexpectedly high compared to those in the flankinh regions.
Cheers,
James


On 1/23/08, Sun Tang  wrote:
> Hello Everyone,
>
>   When I refined a structure, I found strong difference density Fo-Fc at 3
> sigma contour for the for five residues which already have occupancy of 1.
> The density is continuous and so strong as if I did not put the residues
> there. Why was that? Can I put greater than 1 occupancy for those residues?
>
>   Thank you very much for your opinions and suggestions!
>
>   Best wishes,
>
>   Sun Tang
>
>
> -
> Never miss a thing.   Make Yahoo your homepage.

-- 
Sent from Gmail for mobile | mobile.google.com

Dr. James Irving
NH&MRC C.J. Martin Fellow
School of Biomedical Sciences
Building 13D
Monash University
Wellington Road
Melbourne 3800
Australia


   
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Never miss a thing.   Make Yahoo your homepage.

Re: [ccp4bb] Question about freeR tag

[Edwin Pozharski]

FREEFLAG (the program which is used to generate the test set) 
description says when describing the keyword SEED:


By default, for a given job on a given machine, the random number 
generator produces the same list of "random" free-R flags each time the 
job is run. Since you would generally only produce one list of free-R 
flags for each project, this is not usually a problem. However, if you 
specify the keyword SEED, then the random number generator is seeded 
with the current time, and will produce a different list of free-R flags 
each time the job is run.


So what you see is normal.

Zheng Zhou wrote:
Sorry that I didn't explain the situation clearly. I used only one 
output.sca file from HLK2000. I ran the scalepack2mtz (in CCP4i, data 
reduction, import merged data) several times, on both linux Fedora and 
window XP, the same computer though.


For the next step refinement, I mean add H2O, ion, ligands, double 
check certain sidechains In those refmac runs, I always use the 
same mtz file, which I generated from the beginning. I am as surprised 
as you are the Rfree flags are identical from those different "first" 
runs.


Thanks

On Jan 23, 2008 9:50 AM, Zheng Zhou <[EMAIL PROTECTED] 
> wrote:


Hi, All

Could any tell me how CCP4 handle free R flag? I know It is
important to select the same FreeR reflections if I move to next
step of refinement. But everytime I start from fresh, the freeR
Flag remains unchanged. The Rwork and  Rfree of my models are fine
( 20.7% and 22.9%). I thought Rfree was randomly generated. I
asked more experienced people in the lab and neighbor labs, and
didn't get a straight answer. Did I do something wrong?

Thanks and sorry to bother others.

Zheng (Joe)

I cant understand this.
Do you mean you have multiple data sets and they all generate the same
FreeRs? If you have EXACTLY the same reflection list, and generate FreeR
flags on the same machine I guess they would be the same.. but it would
be surprising

Or if you are using the same file which already has FreeR flags then
they wont change of course - the default is always to use the
reflections flagged with FreeR = 0.  (Thats as it should be..)

By the by - your agreement between R and FreeR seems unusually low
unless you have very high resolution data or a great deal of
non-crystallographic symmetry.
Eleanor

Zheng  - I am not sure what you are doing, but as long as you work
with one dataset only, you generate the free reflections at the very
beginning when you import your data into CCP4. Then use the resulting
mtz file for all following refinement steps. There are situations
where one will have to deviate from this scheme, but those are rare.
If you feel you have such a case, then tell us more about it. Hope
that helps. Best - MM

Joe,


can you explain a bit clearer your problem?  what do you mean the 
'next step of refinement'?  are you just doing another round of 
refinement?  what program are you using, REFMAC5?  also, R/Rfree of 
20.7/22.9 is pretty good, depending on resolution.


yes, you are right, Rfree flags are generated randomly and can be 
anywhere from 5-10% of your reflections (your choice).  then, once the 
Rfree flagged reflections are selected, they will not change (nor be 
refined) throughout the rest of your structure determination.  they 
should have their own column in your *.mtz file called RfreeFlag or 
something like this (in CCP4 that is).  other programs handle this 
relatively the same, but may use various naming conventions.  one 
caveat is that CCP4 flags reflections using 0 by default (meaning 5% 
of your reflections are flagged with 0 and are the Rfree set).  Other 
programs such as CNS and PHENIX use 1 by default, so be careful when 
switching back and forth.  You can tell either program which to use 
for Rfree set, but needs to be set, since it is not default.


again, not exactly sure what the problem is, but i hope some of this 
helps.  feel free to email with further questions if needed.  best of 
luck!




cheers,
nick



--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] Question about freeR tag

[Zheng Zhou]

Thank you all for fast replying. The reason that I am trying to use a
different set of FreeR flag from the very beginning of the refinement is
that for some data set, my colleague's CNS refinement gave converged Rfree
and R work, 3% difference. However both my CNS and CCP4 refinement gave a
difference of about 7%. I was trying to use the same parameter settings as
my colleague in simulated annealing. His program is from his previous lab
and all the input files for CNS are modified as a batch file. But mine are
just individual files downloaded from CNS website.

Among those things I tried, I thought about looking at the true input data
for computing Rfree and Rwork. It appeared to me the FreeR flags in my
"First" runs are never randomized. Thanks for your reply, I guess I could
use a different flag number other than 0 (I know I am not supposed to change
this in the middle of a refinement). I even tried to use my colleague's
cross-validation file .cv for CNS. It still diverge in my runs. Anyone met
similar problem before, where CNS and CCP4 give a quite different R factors?
Thanks for your insight.

Zheng (Joe)

On Jan 23, 2008 9:50 AM, Zheng Zhou <[EMAIL PROTECTED]> wrote:

> Hi, All
>
> Could any tell me how CCP4 handle free R flag? I know It is important to
> select the same** FreeR reflections if I move to next step of refinement.
> But everytime I start from fresh, the freeR Flag remains unchanged. The
> Rwork and  Rfree of my models are fine ( 20.7% and 22.9%). I thought Rfree
> was randomly generated. I asked more experienced people in the lab and
> neighbor labs, and didn't get a straight answer. Did I do something wrong?
>
> Thanks and sorry to bother others.
>
> Zheng (Joe)
>

[ccp4bb] meeting suggestions

[Ingrid . Mechin]

Dear All,
 
 
I have a probably quite controversial question for the crystallographic
community (and there may be a strong personal bias too...).
 
Our group would like to select 4 meetings this year that would really be
focused towards our line of work (protein crystallography in
collaboration with drug design) and that are US-based. We have started
to prepare a list but may have missed some and/or need to select from
that list. So, if you were in our position, what would your top 4 be?
All suggestions welcome...
 
Thank you very much for your time and help,
 
 
Ingrid Mechin
 

 
Ingrid Mechin
Research Investigator
Chemical and Analytical Sciences
sanofi-aventis
mailstop N-103A
route 202-206
Bridgewater NJ 08807
USA
tel: + 1 908 231 3348
fax: + 1 908 231 3576
e-mail:[EMAIL PROTECTED]
 

[ccp4bb] GPCR Structural Biology Postdoctoral Position Openings

[Ray Stevens]

GPCR Structural Biology Postdoctoral Position Openings

We have several openings for postdoctoral fellows in the area of GPCR 
structural biology in the Kuhn-Stevens Laboratory at The Scripps Research 
Institute.  With the recent structure determination of the human beta2 
adrenergic receptor (Cherezov et al; Rosenbaum et al, Science 2007), we are now 
interested in studying other G-protein coupled receptors as well as focus on 
the mechanistic details of a single G-protein signal transduction system.   The 
Kuhn-Stevens laboratory is well equipped with cutting edge technologies in the 
areas of membrane protein expression, stabilization, and crystallization.  
Applicants interested in the biology, biochemistry and/or structural biology of 
this family of membrane proteins are encouraged to apply.  Interested 
applicants should send their CV, statement of research interest, and 3 letters 
of reference to [EMAIL PROTECTED]