Re: [ccp4bb] Mg2+ or water

[Robbie Joosten]

You could also try the original WASP here (also for coloured Indo-dutch 
catholics): http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl or you can use 
the latest WHAT_CHECK that also has an implementation of Nayal and Di Cera's 
algorithm.
 
Cheers,
Robbie
 
> Date: Mon, 20 Dec 2010 22:59:53 +
> From: paul.ems...@bioch.ox.ac.uk
> Subject: Re: [ccp4bb] Mg2+ or water
> To: CCP4BB@JISCMAIL.AC.UK
> 
> On 20/12/10 21:48, Robbie Joosten wrote:
> >
> >
> > Also note that Mg2+ is significantly smaller than water. It fits in 
> > places where water cannot go. This doesn't look like a magnesium site 
> > on first glance.
> 
> I tend to agree with Robbie. I wonder what WASP would say... (if you 
> use Coot, you can try the "Highly Coordinated Waters" validation test - 
> a symmetry-enhanced implementation of the Nayal & Di Cera (1996) algorithm).
> 
> Paul.
  

Re: [ccp4bb] Mg2+ or water

[Weiergräber , Oliver H .]

Hello,

as to the WASP server: We found it to be very useful in the past, but the link 
is no longer functional!
Is there any other address for accessing this server?

Oliver


 PD Dr. Oliver H. Weiergräber
 Institut für Strukturbiologie und Biophysik
 ISB-3: Strukturbiochemie
 Tel.: +49 2461 61-2028
 Fax: +49 2461 61-1448






From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Robbie Joosten 
[robbie_joos...@hotmail.com]
Sent: Tuesday, December 21, 2010 9:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Mg2+ or water

You could also try the original WASP here (also for coloured Indo-dutch 
catholics): http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl or you can use 
the latest WHAT_CHECK that also has an implementation of Nayal and Di Cera's 
algorithm.

Cheers,
Robbie

> Date: Mon, 20 Dec 2010 22:59:53 +
> From: paul.ems...@bioch.ox.ac.uk
> Subject: Re: [ccp4bb] Mg2+ or water
> To: CCP4BB@JISCMAIL.AC.UK
>
> On 20/12/10 21:48, Robbie Joosten wrote:
> >
> >
> > Also note that Mg2+ is significantly smaller than water. It fits in
> > places where water cannot go. This doesn't look like a magnesium site
> > on first glance.
>
> I tend to agree with Robbie. I wonder what WASP would say... (if you
> use Coot, you can try the "Highly Coordinated Waters" validation test -
> a symmetry-enhanced implementation of the Nayal & Di Cera (1996) algorithm).
>
> Paul.



Forschungszentrum Juelich GmbH
52425 Juelich
Sitz der Gesellschaft: Juelich
Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498
Vorsitzender des Aufsichtsrats: MinDirig Dr. Karl Eugen Huthmacher
Geschaeftsfuehrung: Prof. Dr. Achim Bachem (Vorsitzender),
Dr. Ulrich Krafft (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt,
Prof. Dr. Sebastian M. Schmidt

Re: [ccp4bb] Mg2+ or water

[James Stroud]

On Dec 20, 2010, at 1:53 PM, Jacob Keller wrote:
> what is the .odp file extension?

http://tinyurl.com/mjokqs

A .odp file is an "open document presentation". It is the open version of a 
power point presentation.

   http://en.wikipedia.org/wiki/OpenDocument

An .odp file is an ISO standard--like the country code you dial when you call 
your favorite Aunt.

You can open a .odp file with the free office suit called OpenOffice. Just 
download it from http://www.openoffice.org/ and start double-clicking to open 
the file just like you would if you were using some other presentation software.

Also, Jlliu Liu set a good example by sending the document in an open format so 
anyone can open it (even though some may not have heard of an odp file before). 
By using an open format, Jlliu Liu has catered to convenience rather than 
catering to ignorance, and has increased the range of people who can provide 
him with help.

James




> 
> 
> 
> JPK
> 
> On Mon, Dec 20, 2010 at 3:48 PM, Robbie Joosten
>  wrote:
>> Dear jlliu liu,
>> 
>> Also note that Mg2+ is significantly smaller than water. It fits in places
>> where water cannot go. This doesn't look like a magnesium site on first
>> glance. If you can (privately) give the PDBid of the previous publication, I
>> can have a look in 3D.
>> 
>> Cheers,
>> Robbie
>> 
>>> Date: Mon, 20 Dec 2010 21:31:58 +
>>> From: p...@mrc-lmb.cam.ac.uk
>>> Subject: Re: [ccp4bb] Mg2+ or water
>>> To: CCP4BB@JISCMAIL.AC.UK
>>> 
>>> Mg2+ is (almost) aways octahedrally coordinated, usually by oxygen atoms,
>>> with distances of ~2A.
>>> Phil
>>> 
>>> On 20 Dec 2010, at 21:16, jlliu liu wrote:
>>> 
 Hi All,
 
 I am refining a structure and encountered a problem of modeling a
 difference density as water or Mg2+, and would like to hear opinions from
 the community. It has the following coordinations (attached): the 
 water/Mg2+
 forms salt bridge/H-bonding interaction with a carboxylate group from the
 ligand, it also forms salt bridge/H-bonding interaction with a Glu residue
 from the protein, it is also within hydrogen bonding distance to the main
 chain N of another protein residue. In provious publication, it was 
 modelled
 as a Mg2+ and the author reasoned the dual salt-bridge stabilizes the
 liganding binding, also the Mg2+ is present in the protein solution for
 crystallization. For my case, I have no Mg2+ present in the protein buffer,
 also modelling it with water refines perfectly with no indication of
 positive difference density even at 2.0 sigma cut off. Should I modelled
 this density as water or as Mg2+. Your opinions are appreciated.
 
 JL
 
 
 
>> 
> 
> 
> 
> -- 
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***

Re: [ccp4bb] Mg2+ or water

[Vellieux Frederic]

James Stroud wrote:

On Dec 20, 2010, at 1:53 PM, Jacob Keller wrote:

what is the .odp file extension?


http://tinyurl.com/mjokqs

A .odp file is an "open document presentation". It is the open version 
of a power point presentation.


   http://en.wikipedia.org/wiki/OpenDocument

An .odp file is an ISO standard--like the country code you dial when 
you call your favorite Aunt.


You can open a .odp file with the free office suit called OpenOffice. 
Just download it from http://www.openoffice.org/ and start 
double-clicking to open the file just like you would if you were using 
some other presentation software.


Also, Jlliu Liu set a good example by sending the document in an open 
format so anyone can open it (even though some may not have heard of 
an odp file before). *By using an open format, Jlliu Liu has catered 
to convenience rather than catering to ignorance,* and has increased 
the range of people who can provide him with help.


James


But there was a double extension in the name of the file provided 
initially (.png.odp if I remember well).


Programs that check viruses in incoming emails remove all files that 
carry double extensions because this is a way to "hide" the real nature 
of the file. They also remove .exe files and others (like having too 
many spaces in a file name).


Fred.

Re: [ccp4bb] Mg2+ or water

[Tim Gruene]

[christmas flame]
The 'hiding' only applies to Windows, and that's your own fault then...
and your responsibility to look after it.  And since attachments are deprecated
on this list it further imposes no real problem at all.
[/christmas flame]

;-> Tim


On Tue, Dec 21, 2010 at 11:08:27AM +0100, Vellieux Frederic wrote:
> James Stroud wrote:
>> On Dec 20, 2010, at 1:53 PM, Jacob Keller wrote:
>>> what is the .odp file extension?
>>
>> http://tinyurl.com/mjokqs
>>
>> A .odp file is an "open document presentation". It is the open version  
>> of a power point presentation.
>>
>>http://en.wikipedia.org/wiki/OpenDocument
>>
>> An .odp file is an ISO standard--like the country code you dial when  
>> you call your favorite Aunt.
>>
>> You can open a .odp file with the free office suit called OpenOffice.  
>> Just download it from http://www.openoffice.org/ and start  
>> double-clicking to open the file just like you would if you were using  
>> some other presentation software.
>>
>> Also, Jlliu Liu set a good example by sending the document in an open  
>> format so anyone can open it (even though some may not have heard of  
>> an odp file before). *By using an open format, Jlliu Liu has catered  
>> to convenience rather than catering to ignorance,* and has increased  
>> the range of people who can provide him with help.
>>
>> James
>
> But there was a double extension in the name of the file provided  
> initially (.png.odp if I remember well).
>
> Programs that check viruses in incoming emails remove all files that  
> carry double extensions because this is a way to "hide" the real nature  
> of the file. They also remove .exe files and others (like having too  
> many spaces in a file name).
>
> Fred.

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

phone: +49 (0)551 39 22149

GPG Key ID = A46BEE1A



signature.asc
Description: Digital signature

Re: [ccp4bb] If it is a new structure?

[Sergii Buth]

Dear CCP4 users,

I would like to draw your attention to the topic "If it is a new 
structure?", which is one more case, when new community members were 
welcomed, and were "gently" explained about the rules of files sharing, 
as it is an evidence, that the rules about files sharing are not well 
defined and highlighted as a crucial point (as it appears to be) during 
the subscription.


I think, that proper introduction of these rules on the page of 
subscription is much more easier than to explain them to every new 
member who posts files not properly.


Best,

Sergii


On 12/21/2010 01:02 AM, CCP4BB automatic digest system wrote:

You're welcome!

Next time, irrespective of whether your structure is a new one or not,
please upload your images to picasa, flickr, your university's file
sharing server or some such thing and include the link in your email.
Don't flood several thousand inboxes with megabytes of pixels.  And
compress bitmaps, for crying out loud.  How hard can it be?

Thanks.


Andreas





On 20/12/2010 10:49,  wrote:

>  The structure of my protein is as shown as the purple one. Another one
>  ,as shown as green,is homologous .But the structure of my protein can't
>  be obtained by using molecular replacement. And both structures have
>  much different, especially in B chain. If my structure is a new one?
>  thank you for help.
-- Andreas Förster, Research Associate Paul Freemont & Xiaodong Zhang 
Labs Department of Biochemistry, Imperial College London 
http://www.msf.bio.ic.ac.uk



--
Sergii Buth
PhD student of Prof. Petr Leiman
Laboratory of Structural Biology and Biophysics
Institut de physique des systèmes biologiques
École Polytechnique Fédérale de Lausanne (EPFL)
Cubotron/BSP-416
CH-1015 Lausanne
Switzerland
Phone: +41 21 693 04 40

Re: [ccp4bb] Mg2+ or water

[Ian Tickle]

Note that the original Nayal & Di Cera algorithm uses an older and
less preferred version of the bond valence model and its associated
parameters than current implementations of BV.

The original WASP uses this formula:

bond valence = (Rij/R0)^(-N)

The most up-to-date BV parameter set
(http://www.ccp14.ac.uk/ccp/web-mirrors/i_d_brown/bond_valence_param/bvparm2006.cif
) uses this:

bond valence = exp((Ro-Rij)/B)

where Rij is the metal-ligand distance, and R0, N and B are parameters
specific to the metal-ligand pair.  Obviously these parameter sets may
give different results depending on which formula you are using.

See the IUCr monograph by I.D.Brown: "The Chemical Bond in Inorganic
Chemistry: The Bond Valence Model", chapter 3 for more info.

Cheers

-- Ian

On Tue, Dec 21, 2010 at 8:55 AM, Robbie Joosten
 wrote:
> You could also try the original WASP here (also for coloured Indo-dutch
> catholics): http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl or you can
> use the latest WHAT_CHECK that also has an implementation of Nayal and Di
> Cera's algorithm.
>
> Cheers,
> Robbie
>
>> Date: Mon, 20 Dec 2010 22:59:53 +
>> From: paul.ems...@bioch.ox.ac.uk
>> Subject: Re: [ccp4bb] Mg2+ or water
>> To: CCP4BB@JISCMAIL.AC.UK
>>
>> On 20/12/10 21:48, Robbie Joosten wrote:
>> >
>> >
>> > Also note that Mg2+ is significantly smaller than water. It fits in
>> > places where water cannot go. This doesn't look like a magnesium site
>> > on first glance.
>>
>> I tend to agree with Robbie. I wonder what WASP would say... (if you
>> use Coot, you can try the "Highly Coordinated Waters" validation test -
>> a symmetry-enhanced implementation of the Nayal & Di Cera (1996)
>> algorithm).
>>
>> Paul.
>

Re: [ccp4bb] REFMAC 5.2.0019 question

[Ian Tickle]

I think it would be a lot easier to understand what's going on if you
posted your actual scripts.

Cheers

-- Ian

On Mon, Dec 20, 2010 at 6:11 PM,   wrote:
> Thanks Ian, but I was using the output from 2a for 2b running. Results are
> still different between 2 and 1. More curious is more second question, the
> region with identical ADPs still ended up with identical ADPs (although
> different from before running) after 1, and that's why I also tried 2.
>
> Hailiiang
>
>> PS one other thought: in your run 2b you are not reading in (as TLSIN)
>> the TLSOUT file produced by run 2a.  So run 2b is not starting from
>> the same point that it would have done as in run 1.
>>
>> I.
>>
>> On Sun, Dec 19, 2010 at 11:58 PM, Hailiang Zhang  wrote:
>>> Hi,
>>>
>>> I am using REFMAC 5.2.0019 to run the following script:
>>> ***
>>> refmac5 hklin a xyzin b <>> REFI TLSC ${CTLS}
>>> REFI BREF OVERall
>>> NCYC ${CC}
>>> **
>>>
>>> I thought this script will do CTLS cycles of TLS refinement followed by
>>> CC
>>> cycles of verall B and geometry refinement. Then I did the following 2
>>> tests:
>>> 
>>> (1). CTLS=a, CC=b
>>> (2). CTLS=a, CC=0; followed by: CTLS=0, CC=b
>>> ***
>>> The results are just very different from (1) and (2), and I am not sure
>>> why.
>>>
>>> By the way, my system has a small region with identical ADPs. After
>>> doing
>>> (2), the ADPs at this region becomes different; however, after doing
>>> (1),
>>> these ADPs are still identical, although different from the original
>>> ADPs.
>>>
>>> Thanks for any clarifications!
>>>
>>> Best Regards, Hailiang
>>>
>>
>>
>
>
>

Re: [ccp4bb] Mg2+ or water

[George M. Sheldrick]

You might also like to look at the paper "Is the bond-valence method able 
to identify metal ions in protein structures", Acta Cryst. D59 (2003) 
32-37. In retrospect, the BV method is very good for identifying Mg2+ 
because it is almost always tightly octahedrally coordinated by oxygen
atoms. For other metals and low resolution the calculation can easily be 
upset by missing water ligands or inaccurate interatomic distances.
If anyone is interested I can supply a powerpoint of the lecture on the
BV method from my undergraduate solid state chemistry course, but I
should warn you that it is in German (like most of our teaching).

George

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582


On Tue, 21 Dec 2010, Ian Tickle wrote:

> Note that the original Nayal & Di Cera algorithm uses an older and
> less preferred version of the bond valence model and its associated
> parameters than current implementations of BV.
> 
> The original WASP uses this formula:
> 
> bond valence = (Rij/R0)^(-N)
> 
> The most up-to-date BV parameter set
> (http://www.ccp14.ac.uk/ccp/web-mirrors/i_d_brown/bond_valence_param/bvparm2006.cif
> ) uses this:
> 
> bond valence = exp((Ro-Rij)/B)
> 
> where Rij is the metal-ligand distance, and R0, N and B are parameters
> specific to the metal-ligand pair.  Obviously these parameter sets may
> give different results depending on which formula you are using.
> 
> See the IUCr monograph by I.D.Brown: "The Chemical Bond in Inorganic
> Chemistry: The Bond Valence Model", chapter 3 for more info.
> 
> Cheers
> 
> -- Ian
> 
> On Tue, Dec 21, 2010 at 8:55 AM, Robbie Joosten
>  wrote:
> > You could also try the original WASP here (also for coloured Indo-dutch
> > catholics): http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl or you can
> > use the latest WHAT_CHECK that also has an implementation of Nayal and Di
> > Cera's algorithm.
> >
> > Cheers,
> > Robbie
> >
> >> Date: Mon, 20 Dec 2010 22:59:53 +
> >> From: paul.ems...@bioch.ox.ac.uk
> >> Subject: Re: [ccp4bb] Mg2+ or water
> >> To: CCP4BB@JISCMAIL.AC.UK
> >>
> >> On 20/12/10 21:48, Robbie Joosten wrote:
> >> >
> >> >
> >> > Also note that Mg2+ is significantly smaller than water. It fits in
> >> > places where water cannot go. This doesn't look like a magnesium site
> >> > on first glance.
> >>
> >> I tend to agree with Robbie. I wonder what WASP would say... (if you
> >> use Coot, you can try the "Highly Coordinated Waters" validation test -
> >> a symmetry-enhanced implementation of the Nayal & Di Cera (1996)
> >> algorithm).
> >>
> >> Paul.
> >
> 
> 

Re: [ccp4bb] Mg2+ or water

[Ian Tickle]

Yes the BV method relies on summing up the 'bond valence'
contributions for each ligand co-ordinated to the metal, and comparing
the result with the formal charge on the metal ion (e.g. +2 for Mg).
This means that even if say one water that is present in the crystal
is omitted from the calculation for whatever reason (say you summed
the contributions from only 5 instead of 6 waters because you didn't
include the symmetry molecule as in the original version of WASP),
then you are likely to conclude from the result that it's not Mg even
if the distances and the octahedral co-ordination of the 5 waters that
you did include are perfect for Mg.

Obviously as George says, if the distances are inaccurate then the
computed BV contributions to the sum will be wrong and that's not good
either!  So I completely agree that it's probably wise to take the
results of applying this method with a large pinch of salt!

Cheers

-- Ian

On Tue, Dec 21, 2010 at 12:25 PM, George M. Sheldrick
 wrote:
>
> You might also like to look at the paper "Is the bond-valence method able
> to identify metal ions in protein structures", Acta Cryst. D59 (2003)
> 32-37. In retrospect, the BV method is very good for identifying Mg2+
> because it is almost always tightly octahedrally coordinated by oxygen
> atoms. For other metals and low resolution the calculation can easily be
> upset by missing water ligands or inaccurate interatomic distances.
> If anyone is interested I can supply a powerpoint of the lecture on the
> BV method from my undergraduate solid state chemistry course, but I
> should warn you that it is in German (like most of our teaching).
>
> George
>
> Prof. George M. Sheldrick FRS
> Dept. Structural Chemistry,
> University of Goettingen,
> Tammannstr. 4,
> D37077 Goettingen, Germany
> Tel. +49-551-39-3021 or -3068
> Fax. +49-551-39-22582
>
>
> On Tue, 21 Dec 2010, Ian Tickle wrote:
>
>> Note that the original Nayal & Di Cera algorithm uses an older and
>> less preferred version of the bond valence model and its associated
>> parameters than current implementations of BV.
>>
>> The original WASP uses this formula:
>>
>> bond valence = (Rij/R0)^(-N)
>>
>> The most up-to-date BV parameter set
>> (http://www.ccp14.ac.uk/ccp/web-mirrors/i_d_brown/bond_valence_param/bvparm2006.cif
>> ) uses this:
>>
>> bond valence = exp((Ro-Rij)/B)
>>
>> where Rij is the metal-ligand distance, and R0, N and B are parameters
>> specific to the metal-ligand pair.  Obviously these parameter sets may
>> give different results depending on which formula you are using.
>>
>> See the IUCr monograph by I.D.Brown: "The Chemical Bond in Inorganic
>> Chemistry: The Bond Valence Model", chapter 3 for more info.
>>
>> Cheers
>>
>> -- Ian
>>
>> On Tue, Dec 21, 2010 at 8:55 AM, Robbie Joosten
>>  wrote:
>> > You could also try the original WASP here (also for coloured Indo-dutch
>> > catholics): http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl or you can
>> > use the latest WHAT_CHECK that also has an implementation of Nayal and Di
>> > Cera's algorithm.
>> >
>> > Cheers,
>> > Robbie
>> >
>> >> Date: Mon, 20 Dec 2010 22:59:53 +
>> >> From: paul.ems...@bioch.ox.ac.uk
>> >> Subject: Re: [ccp4bb] Mg2+ or water
>> >> To: CCP4BB@JISCMAIL.AC.UK
>> >>
>> >> On 20/12/10 21:48, Robbie Joosten wrote:
>> >> >
>> >> >
>> >> > Also note that Mg2+ is significantly smaller than water. It fits in
>> >> > places where water cannot go. This doesn't look like a magnesium site
>> >> > on first glance.
>> >>
>> >> I tend to agree with Robbie. I wonder what WASP would say... (if you
>> >> use Coot, you can try the "Highly Coordinated Waters" validation test -
>> >> a symmetry-enhanced implementation of the Nayal & Di Cera (1996)
>> >> algorithm).
>> >>
>> >> Paul.
>> >
>>
>>

Re: [ccp4bb] Mg2+ or water

[Oganesyan, Vaheh]

Using different web servers on your refined structure is good thing to
do. But for distinguishing metal ion, specifically Mg, from water was
done unambiguously before these programs existed. The simple rule is two
fold: 1. distance; 2. coordination. For Mg ion distances are between 2
and 2.2 A and coordination is almost always nearly perfect octahedron.
Neither of those criteria met in your structure. Hence the verdict - it
is water.

 

 

 Vaheh  



From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
jlliu liu
Sent: Monday, December 20, 2010 4:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Mg2+ or water

 

Hi All,

I am refining a structure and encountered a problem of modeling a
difference density as water or Mg2+, and would like to hear opinions
from the community. It has the following coordinations (attached): the
water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate
group from the ligand, it also forms salt bridge/H-bonding interaction
with a Glu residue from the protein, it is also within hydrogen bonding
distance to the main chain N of another protein residue. In provious
publication, it was modelled as a Mg2+ and the author reasoned the dual
salt-bridge stabilizes the liganding binding, also the Mg2+ is present
in the protein solution for crystallization. For my case, I have no Mg2+
present in the protein buffer, also modelling it with water refines
perfectly with no indication of positive difference density even at 2.0
sigma cut off. Should I modelled this density as water or as Mg2+. Your
opinions are appreciated.

JL
 




To the extent this electronic communication or any of its attachments contain 
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you have received this electronic communication in error, please reply to the 
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cooperation.

Re: [ccp4bb] Mg2+ or water

[Ibrahim Moustafa]

I agree. It is very unlikely to be Magnesium ion; may be an ordered water!

 I'd recommend to have a look at the following paper which discusses the
different properties of Mg2+, Mn2+, and Zn2+.

   Charles W. Bock, Amy Kaufman Katz, George D. Markham, and Jenny P.
Glusker. Manganese as a replacement for Magnesium and Zinc: Functional
comparison of the divalent ions. (J. Am. Chem. Soc. 1999, 121, 7360-7372)

   Ibrahim

On 12/20/10 6:14 PM, "Dima Klenchin"  wrote:

>> Sorry, the attachment is in here.
> 
> Doesn't look like Mg2+ at all. Distances are too long, Mg is never
> coordinated by amides and if it were Mg you would have seen waters around it.
> 
> Looks like tightly bound water to me.
> 
> - Dima
> 
> 
> 
>> On Mon, Dec 20, 2010 at 4:16 PM, jlliu liu
>> <jlliu20022...@gmail.com> wrote:
>> Hi All,
>> 
>> I am refining a structure and encountered a problem of modeling a
>> difference density as water or Mg2+, and would like to hear opinions from
>> the community. It has the following coordinations (attached): the
>> water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate
>> group from the ligand, it also forms salt bridge/H-bonding interaction
>> with a Glu residue from the protein, it is also within hydrogen bonding
>> distance to the main chain N of another protein residue. In provious
>> publication, it was modelled as a Mg2+ and the author reasoned the dual
>> salt-bridge stabilizes the liganding binding, also the Mg2+ is present in
>> the protein solution for crystallization. For my case, I have no Mg2+
>> present in the protein buffer, also modelling it with water refines
>> perfectly with no indication of positive difference density even at 2.0
>> sigma cut off. Should I modelled this density as water or as Mg2+. Your
>> opinions are appreciated.
>> 
>> JL
>> 
>> 
>> 
>> Content-type: image/png; name=367-mgtest.png
>> Content-disposition: attachment; filename=367-mgtest.png
>> X-Attachment-Id: f_ghy0k5e31
>> 
> 

Re: [ccp4bb] Mg2+ or water

[John Bruning]

Hi,

I'm not sure if it has been mentioned yet, but you could take an
experimental approach if you still have crystals.  You could soak them with
EDTA/EGTA and if the density disappears that is good evidence it was Mg (or
other divalent cation), if not then putatively water.  You could also soak
with Mn (assuming the Mn will bind the Mg site) and look for anomolous
signal.


John

Re: [ccp4bb] Mg2+ or water

[Paula Salgado]

Another option would be to collect data at the Mn K edge (1.89A) - at this
wavelength Mg has a weak anomalous signal that you should still be able to
detect. I've used Mn K edge to successfully distinguish  between Mg, Mn and
Ca ions as well as identify them as ions and not waters by looking at peaks
in the anomalous maps since peak intensity relates to anomalous signal
arising from different ions at different wavelengths.

For details, see:

http://scripts.iucr.org/cgi-bin/paper?S0907444904026800

http://www.cell.com/structure/retrieve/pii/S0969212604000243

On 21 December 2010 15:04, John Bruning  wrote:

> Hi,
>
> I'm not sure if it has been mentioned yet, but you could take an
> experimental approach if you still have crystals.  You could soak them with
> EDTA/EGTA and if the density disappears that is good evidence it was Mg (or
> other divalent cation), if not then putatively water.  You could also soak
> with Mn (assuming the Mn will bind the Mg site) and look for anomolous
> signal.
>
>
> John
>
>

[ccp4bb] AKTA Prime dead/delay volume...and a transmembrane protein...

[James Pauff]

Hello all,

Does anyone have a rough estimate (or has anyone actually determined) an 
average dead/delay volume between buffers run on an AKTA Prime FPLC?  We are 
attempting to overexpress/isolate a smaller His-tagged transmembrane protein, 
and require running several detergent buffers in succession over the column.  
This obviously creates fractions that are just dead volume/ddH2O between 
buffers, and we would like to narrow in on where to look for the protein prior 
to analyzing the fractions (i.e. how many fractions can we discard?).  Should 
we run ddH2O and then the first mL into 'waste' before running each buffer over 
the column (and collecting fractions)?  Just not used to this system yet!  
Thank you!

Jim


  

[ccp4bb] Postdoctoral position at LBNL -- Free electron laser software methods

[Nicholas Sauter]

The Lawrence Berkeley National Laboratory has an opportunity for a
Computational Biologist Postdoctoral Fellow as part of a project that
will involve writing the software to process X-ray diffraction
patterns from protein crystals analyzed on the free-electron laser at
Linac Coherent Light Source. The successful candidate will be the
primary developer of new methods for this data processing.  While
protein crystallography methods for traditional rotation geometry
experiments are well-established, LCLS data will be acquired with
completely different optics and geometry, and at 100-fold higher rate.
 Both the underlying physics and the wide range of user needs will
need to be accounted for.  Durable computer code will then be
implemented in an object-oriented framework, using both C++ and Python
scripting.

We are seeking applicants with a recent Ph.D. in Computational
Biology, Computer Science, Biophysics, Applied Mathematics, or related
areas.  Clear written expression in the form of co-authored
publications and/or computer documentation is a must.  Key success
factors include a proficient understanding of network architecture and
parallel processing, and experience with forefront approaches such as
GPU computing. GUI development experience is also important, as a
focus will be on creating systems to interpret the results in real
time.

Applicants are invited to post a cover latter and CV for job listing
#25178 at http://jobs.lbl.gov/details.asp?jid=25178&p=1

Re: [ccp4bb] AKTA Prime dead/delay volume...and a transmembrane protein...

[Christian Roth]

Hi Jim,

If I remember correctly the delay volume from the pumps to the top of the 
column is roughly 8 ml for Äkta Purifier, Explorer and also Prime. The actual 
volume from the bottom of the column to the fractionator, or more important 
from the UV cell to the fractionator depends on the tubing and must be 
calulated by hand. Normally this is already done and you could find this value 
in the software in the fractionation part. The standard systems Explorer, 
Purifier starts with the fractionation after this dead volume. So you can wait 
roughly 8 ml plus your column volume, start the fractionation and than your 
protein should be in one of the first fractions, assuming a step gradient. 
But be cautious it is just a rough estimation!!!

Christian 

Am Dienstag 21 Dezember 2010 16:17:13 schrieb James Pauff:
> Hello all,
> 
> Does anyone have a rough estimate (or has anyone actually determined) an
>  average dead/delay volume between buffers run on an AKTA Prime FPLC?  We
>  are attempting to overexpress/isolate a smaller His-tagged transmembrane
>  protein, and require running several detergent buffers in succession over
>  the column.  This obviously creates fractions that are just dead
>  volume/ddH2O between buffers, and we would like to narrow in on where to
>  look for the protein prior to analyzing the fractions (i.e. how many
>  fractions can we discard?).  Should we run ddH2O and then the first mL
>  into 'waste' before running each buffer over the column (and collecting
>  fractions)?  Just not used to this system yet!  Thank you!
> 
> Jim
> 

Re: [ccp4bb] Bfactor is zero?

[Joseph Cockburn]

Hi,
I saw the same thing once and the cause was that the crystal had been
hideously over-exposed during data collection. As a result, essentially
all the spots at lower than 2.5A resolution were overloaded. The Wilson
plot was thus more or less flat at medium to high resolution and
accordingly the Wilson B was very low (less than 1A**2). As in your case,
the first indication of the problem was that the atomic B-factors were
refining to ridiculously low values (although of course REFMAC was only
doing the right thing).
Hope that helps,
Joe



> Hi Bill,
>
> if you put a water oxygen in place where a heavier atom is, then water
> oxygen's B-factor will refine to a value close to zero. This is the
> feature
> that we currently use as one of many criteria to develop automatic
> identification and building of metals.
>
> Overall Wilson B-factor of 0.6A**2 tells that there is something weird
> about
> the data. What is the resolution?
>
> Pavel.
>
> PS> As Nat mentioned, PHENIX related questions are best to send to PHENIX
> (and not CCP4) mailing list:
> http://www.phenix-online.org/
>
>
> On Mon, Dec 20, 2010 at 7:34 AM, Zhibing Lu  wrote:
>
>> Hi All,
>> Recently I solved a structure in which some water molecules have
>> Bfactors
>> at 0  and overall wilson Bfactor is 0.654 based on PHENIX refinement. Is
>> it
>> possible?
>> Bill Lu
>>
>

[ccp4bb] I am very sorry for trouble by sending picture,and won't do it again

I am very sorry for trouble by sending picture,and won't do it again

Re: [ccp4bb] Mg2+ or water

[William G. Scott]

On Dec 20, 2010, at 1:16 PM, jlliu liu wrote:

> it is also within hydrogen bonding distance to the main
> chain N of another protein residue.


This also strongly suggests it is not Mg2+, which prefers hard ligands such as 
charged oxygen, rather than softer ligands like uncharged backbone nitrogens 
(the interactions are primarily electrostatic, rather than orbital-mediated).

[ccp4bb] Postdoctoral position at the University of Toronto

[Jinrong Min]

A Post-doctoral position in chromatin structural  biology is available in my 
group. My lab aims to characterize proteins and protein/DNA or RNA complexes  
by 
X-ray crystallography in  combination with other biochemical and biophysical 
techniques.


Applicants should have a Ph.D. in structural biology, biochemistry,  
molecular/cell biology with 0-4 years postdoctoral research experience. 
Interested candidates are invited to send their CV with 3 references to: 
Jinrong 
Min 

by email: jr@utoronto.ca  

Jinrong Min
Principal Investigator, Chromatin Biology and Epigenetics
Structural Genomics Consortium
Assistant Professor, Department of Physiology
University of Toronto
101 College St., Toronto
On M5G 1L7, Canada
http://www.sgc.utoronto.ca/Chromatin