Re: [ccp4bb] Unknown density

[Rafael Marques]

Dear John

Ethanol would be my first guess,

Best wishes


__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"


De: CCP4 bulletin board  em nome de John Smith 

Enviado: quinta-feira, 11 de julho de 2024 09:35
Para: CCP4BB@JISCMAIL.AC.UK 
Assunto: [ccp4bb] Unknown density

Dear all!
We have solved a structure at 2.2 A resolution. We see this interesting density 
near a Tyrosine. It is not fully aligned, like pi stacking. Otherwise, 
negatively charged or polar residues surround the smaller 'head'.

Does anybody have any idea?

Thanks for the help!

Best
John




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Re: [ccp4bb] Unknown density in Trypsin

[a . perrakis]

Dear Mahmoud,

The first thing to do is to make a list of (a) all your protein buffer 
components, (b) all your crystallisation conditions components, and (c) all the 
cryo-protection chemicals, and try to consider all of those as possibilities, 
but then again indeed it is most likely glycerol anyway ;-)

A,

On 12 Sep 2023, at 14:48, Maria Håkansson 
mailto:maria.hakans...@saromics.com>> wrote:

Hi Mahmoud,

It looks like glycerol (residue GOL in Coot)  to me. Add it under File/Get 
Monomer GOL.
There should be hydrogen bonds, at least one for it to turn up as well-ordered 
as below.

Good Luck!

Best regards,
Maria


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Mahmoud RIZK mailto:mahmoud.r...@esrf.fr>>
Date: Tuesday, 12 September 2023 at 14:40
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Unknown density in Trypsin
Hi everyone,
I am encountering a strong clear density within a structure of trypsin at 1.3 
Å, please find attached a 360 rotation video of it, alternatively I uploaded it 
on YouTube: https://youtube.com/shorts/tHovB55lpio?feature=share
the map shown here is a difference map at 3 sigmas.
here is the its site with respect to the protein (image below):

(PS: this blob has been detected within different trypsin structures from 
several collected data.)
(I tried 
checkmyblob.bioreproducibility.org<http://checkmyblob.bioreproducibility.org> 
without any success)

I would greatly appreciate any suggestions on how to identify and characterize 
this blob. Thank you in advance for your assistance!

With best regards,
Mahmoud




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Re: [ccp4bb] unknown density in 1.6 A structure

[Peer Mittl]

Dear Nick,

I like your idea and your comments on this substance. 1,2-hexandiol 
supposed to be chiral but probably it is used as a racemic mixture in 
cosmetics. There is even a crystal structure in the PDB (4EUS) and an 
associated 3 letter code. I´ll give it a try because it seems to fit 
nicely the ED map. I just have to find out how to handle racemics in 
buster refinement. Thank you all for your helpful comments.


Have a nice day.
-Peer

Am 03.06.2021 um 13:00 schrieb Nicholas Keep:
How about an overlay of the structure you have with a second ligand 
conformation where the OH and CH2OH are swapped? ie two alternative 
conformations of the ligand.  That might remove the last green density


1,2-hexanediol is apparently widely used in cosmetics and 
moisturisers.  Did someone somehow touch the protein or 
crystallisation tray with recently moisturised hands?  Even so it must 
have very high affinity for your protein


Best wishes

Nick





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Re: [ccp4bb] unknown density in 1.6 A structure

[Sevvana, Madhumati]

Hi Peer,
Reminds me of a lipocalin structure I solved which co-purified fatty acid with 
it. I could model fatty acids and we along with our collaborators used several 
complementary biophysical methods followed by functional assays to identify the 
ligand.
https://www.sciencedirect.com/science/article/abs/pii/S002228360901078X
[https://ars.els-cdn.com/content/image/1-s2.0-S0022283609X00422-cov150h.gif]<https://www.sciencedirect.com/science/article/abs/pii/S002228360901078X>
Serendipitous Fatty Acid Binding Reveals the Structural Determinants for Ligand 
Recognition in Apolipoprotein 
M<https://www.sciencedirect.com/science/article/abs/pii/S002228360901078X>
Apolipoprotein M (ApoM) is a 25-kDa HDL-associated apolipoprotein and a member 
of the lipocalin family of proteins. Mature apoM retains its signal pep…
www.sciencedirect.com
Thank you.
All the best,
Madhu


Madhumati Sevvana, Ph.D.
Assistant Research Scientist
Structural Virology lab of Prof. Richard Kuhn
Hockmeyer Hall of Structural Biology
Department of Biological Sciences
Purdue University, West Lafayette IN




From: CCP4 bulletin board  on behalf of Peer Mittl 

Sent: Thursday, June 3, 2021 06:50
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] unknown density in 1.6 A structure

I'm struggling with the electron density shown in the attachment. The
substance does not seem to come from the crystallization condition nor
from the mounting buffer. The 1.6 A maps are rather clear, because the
compound binds into the hydrophobic core, which seems to be loosely packed.

-Peer





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Re: [ccp4bb] unknown density in 1.6 A structure

[Andrew Mesecar]

Dear Peer,

This reminds me of my colleague Michael Rossmann's discovery of a Pocket
Factor which is a fatty acid-like molecule residing in a hydrophobic pocket
underneath the "canyon" in enteroviruses. See
https://link.springer.com/article/10.1007/s13238-014-0092-6.  You likely
have an endogenous ligand that binds and stays bound during purification.
You may want to do some MS on your purified protein to see if you can
identify it.  Your density should provide enough information on a mass
range to look for.

Andy Mesecar

On Thu, Jun 3, 2021 at 6:50 AM Peer Mittl  wrote:

> I'm struggling with the electron density shown in the attachment. The
> substance does not seem to come from the crystallization condition nor
> from the mounting buffer. The 1.6 A maps are rather clear, because the
> compound binds into the hydrophobic core, which seems to be loosely packed.
>
> -Peer
>
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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-- 
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Head, Department of Biochemistry
Walther Professor of Cancer Structural Biology
Deputy Director, Purdue Center for Cancer Research
E-Mail: amese...@purdue.edu
_
*Department of Biochemistry Contact Information:*
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[ccp4bb] AW: [ccp4bb] unknown density in 1.6 A structure

[Meyer, Timo]

Hi Peer,


this density reminds me of something I saw in one of my own structures as well 
and which also was not a component that should have been in there (PDB ID 
6SMT). I never truly found out what it was, but I modeled it as 2-ethylhexanol, 
which is known to be a precursor for the synthesis and also degradation product 
of the wide-spread plasticizer DEHP, although I found out, that at least our 
crystallization plates do not contain this component.


Best,

Timo


Von: CCP4 bulletin board  im Auftrag von Peer Mittl 

Gesendet: Donnerstag, 3. Juni 2021 12:50:00
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] unknown density in 1.6 A structure

I'm struggling with the electron density shown in the attachment. The
substance does not seem to come from the crystallization condition nor
from the mounting buffer. The 1.6 A maps are rather clear, because the
compound binds into the hydrophobic core, which seems to be loosely packed.

-Peer





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Re: [ccp4bb] unknown density in 1.6 A structure

[Dom Bellini - MRC LMB]

Hi Peer,

If I understand you correctly you are saying that you do not know what 
substance may be in the density; then perhaps you only need to extend by 
an extra atom to create a new kind of symmetrical molecule that will fit 
the density?


BW,

D

On 03/06/2021 11:55, SHEPARD William wrote:

Hi Peer,

Have you tried to model the hydroxyl O1 disordered at the carbonyl
position? Check the H-bonding pattern to the end groups, this might give
some clues.

Cheers,
Bill


Le 03/06/21 12:50, « Peer Mittl »  a écrit :


I'm struggling with the electron density shown in the attachment. The
substance does not seem to come from the crystallization condition nor

>from the mounting buffer. The 1.6 A maps are rather clear, because the

compound binds into the hydrophobic core, which seems to be loosely
packed.

-Peer





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--
Dom Bellini, Xray Crystallography Facility (1S205)
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH
Phone 01223 267839



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Re: [ccp4bb] unknown density in 1.6 A structure

[SHEPARD William]

Hi Peer, 

Have you tried to model the hydroxyl O1 disordered at the carbonyl
position? Check the H-bonding pattern to the end groups, this might give
some clues. 

Cheers, 
Bill


Le 03/06/21 12:50, « Peer Mittl »  a écrit :

>I'm struggling with the electron density shown in the attachment. The
>substance does not seem to come from the crystallization condition nor
>from the mounting buffer. The 1.6 A maps are rather clear, because the
>compound binds into the hydrophobic core, which seems to be loosely
>packed.
>
>-Peer
>
>
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
>This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
>mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
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Re: [ccp4bb] unknown density

[Debanu Das]

Hi,

This may be of interest in this discussion (the abstract is enlightening in
itself):

"Water polygons in high‐resolution protein crystal structures"
Jonas Lee, Sung‐Hou Kim
https://pubmed.ncbi.nlm.nih.gov/19551896/

Download software here (you can analyze input pdb file to look at water
polygon structures): https://sourceforge.net/projects/pdbwaterpolygon/

Thanks,
Debanu

On Sun, Apr 4, 2021 at 10:27 AM Debanu  wrote:

> Hi,
>
> This may be of interest in this discussion (the abstract is enlightening
> in itself):
>
> "Water polygons in high‐resolution protein crystal structures"
> Jonas Lee, Sung‐Hou Kim
> https://pubmed.ncbi.nlm.nih.gov/19551896/
>
> Download software here (you can analyze input pdb file to look at water
> polygon structures): https://sourceforge.net/projects/pdbwaterpolygon/
>
> Thanks,
> Debanu
>
> On Tue, Mar 23, 2021 at 2:15 AM Barone, Matthias 
> wrote:
>
>> can confirm jon´s comment. I find these in virtually every high-res
>> structure. some of them wobble a bit given the AA close by, such as Arg.
>>
>>
>> Dr. Matthias Barone
>>
>> AG Kuehne, Rational Drug Design
>>
>> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
>> Robert-Rössle-Strasse 10
>> 13125 Berlin
>>
>> Germany
>> Phone: +49 (0)30 94793-284
>> --
>> *From:* CCP4 bulletin board  on behalf of Jon
>> Cooper <488a26d62010-dmarc-requ...@jiscmail.ac.uk>
>> *Sent:* Monday, March 22, 2021 5:38:40 PM
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* Re: [ccp4bb] unknown density
>>
>> Definitely water pentamer, no doubt at all ;-0
>> Cheers, Jon.C.
>>
>> Sent from ProtonMail mobile
>>
>>
>>
>>  Original Message 
>> On 22 Mar 2021, 14:16, Mark J. van Raaij < mjvanra...@cnb.csic.es>
>> wrote:
>>
>>
>> The ring looks too big to be imidazole or a nucleotide or a carbohydrate,
>> so it’s probably mainly water molecules.
>> Perhaps partially replaced by PEG to explain the density between them
>> (i.e. water molecules in most copies of the protein and PEG in some other
>> copies). I’ve seen horse-shoe shaped PEG in a high-res structure before,
>> PEGs in several confirmations might explain a circle.
>> Practically speaking, I’d first model five waters and see if they refine
>> well.
>>
>> Mark
>>
>> On 22 Mar 2021, at 14:58, Sam Tang  wrote:
>>
>> Hello fellow colleagues
>>
>> Hope you are all well while the pandemics persists. I just wonder if
>> anyone may have an idea what this density (looking like a pentagon) might
>> be. The data was collected to 1.8 A and crystal was grown in Bis-tris +
>> PEG3350. Imidazole residual? Nucleotide (the protein itself is
>> nucleotide-binding, but shouldn't be at this particular site)?
>>
>>
>> https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing
>>
>> Thanks!
>>
>> BRS
>>
>> Sam
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>
>>
>>



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Re: [ccp4bb] unknown density

[Debanu]

Hi,

This may be of interest in this discussion (the abstract is enlightening in
itself):

"Water polygons in high‐resolution protein crystal structures"
Jonas Lee, Sung‐Hou Kim
https://pubmed.ncbi.nlm.nih.gov/19551896/

Download software here (you can analyze input pdb file to look at water
polygon structures): https://sourceforge.net/projects/pdbwaterpolygon/

Thanks,
Debanu

On Tue, Mar 23, 2021 at 2:15 AM Barone, Matthias 
wrote:

> can confirm jon´s comment. I find these in virtually every high-res
> structure. some of them wobble a bit given the AA close by, such as Arg.
>
>
> Dr. Matthias Barone
>
> AG Kuehne, Rational Drug Design
>
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
>
> Germany
> Phone: +49 (0)30 94793-284
> --
> *From:* CCP4 bulletin board  on behalf of Jon
> Cooper <488a26d62010-dmarc-requ...@jiscmail.ac.uk>
> *Sent:* Monday, March 22, 2021 5:38:40 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] unknown density
>
> Definitely water pentamer, no doubt at all ;-0
> Cheers, Jon.C.
>
> Sent from ProtonMail mobile
>
>
>
>  Original Message 
> On 22 Mar 2021, 14:16, Mark J. van Raaij < mjvanra...@cnb.csic.es> wrote:
>
>
> The ring looks too big to be imidazole or a nucleotide or a carbohydrate,
> so it’s probably mainly water molecules.
> Perhaps partially replaced by PEG to explain the density between them
> (i.e. water molecules in most copies of the protein and PEG in some other
> copies). I’ve seen horse-shoe shaped PEG in a high-res structure before,
> PEGs in several confirmations might explain a circle.
> Practically speaking, I’d first model five waters and see if they refine
> well.
>
> Mark
>
> On 22 Mar 2021, at 14:58, Sam Tang  wrote:
>
> Hello fellow colleagues
>
> Hope you are all well while the pandemics persists. I just wonder if
> anyone may have an idea what this density (looking like a pentagon) might
> be. The data was collected to 1.8 A and crystal was grown in Bis-tris +
> PEG3350. Imidazole residual? Nucleotide (the protein itself is
> nucleotide-binding, but shouldn't be at this particular site)?
>
>
> https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing
>
> Thanks!
>
> BRS
>
> Sam
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
>
>



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Re: [ccp4bb] unknown density

[Barone, Matthias]

can confirm jon´s comment. I find these in virtually every high-res structure. 
some of them wobble a bit given the AA close by, such as Arg.


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, March 22, 2021 5:38:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unknown density

Definitely water pentamer, no doubt at all ;-0
Cheers, Jon.C.

Sent from ProtonMail mobile



 Original Message 
On 22 Mar 2021, 14:16, Mark J. van Raaij < mjvanra...@cnb.csic.es> wrote:

The ring looks too big to be imidazole or a nucleotide or a carbohydrate, so 
it’s probably mainly water molecules.
Perhaps partially replaced by PEG to explain the density between them (i.e. 
water molecules in most copies of the protein and PEG in some other copies). 
I’ve seen horse-shoe shaped PEG in a high-res structure before, PEGs in several 
confirmations might explain a circle.
Practically speaking, I’d first model five waters and see if they refine well.

Mark

On 22 Mar 2021, at 14:58, Sam Tang 
mailto:samtys0...@gmail.com>> wrote:

Hello fellow colleagues

Hope you are all well while the pandemics persists. I just wonder if anyone may 
have an idea what this density (looking like a pentagon) might be. The data was 
collected to 1.8 A and crystal was grown in Bis-tris + PEG3350. Imidazole 
residual? Nucleotide (the protein itself is nucleotide-binding, but shouldn't 
be at this particular site)?

https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing

Thanks!

BRS

Sam



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Re: [ccp4bb] unknown density

[Pearce, N.M. (Nick)]

This doesn’t surprise me in the slightest, given what how the two maps are 
calculated.

The polder map is essentially the same as your "simple omit map” without the 
bulk solvent contribution that is present in the omit-refine map. Since this is 
an approximately constant contribution, if you recontour the Polder map to a 
higher contour you should be able to get the two maps to look basically the 
same.

Nick

On 23 Mar 2021, at 09:36, Sam Tang 
mailto:samtys0...@gmail.com>> wrote:

Dear colleagues

Thanks a lot for the comments. I echo that this pentagon-like density may 
likely be water molecules. What had puzzled us a bit was that the output of 
Polder map which gave some interlinked density.

I should also mention that this site is close to a predicted peptide binding 
site and that is what we are actually looking for. (To me the strong positive 
density in Polder map is not indicative of an amino acid residue as well.)

My Polder map looks like this:
https://drive.google.com/file/d/10dGpdcDhrp_ld6wPH3Er7iZ3KVgU2RCN/view?usp=sharing

BRS

Sam

On Tue, 23 Mar 2021 at 04:43, Bernhard Rupp 
mailto:hofkristall...@gmail.com>> wrote:
Textbook knowledge 😊
http://www.ruppweb.org/Garland/gallery/Ch12/pages/Biomolecular_Crystallography_Fig_12-28.htm

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org<mailto:b...@hofkristallamt.org>
+1 925 209 7429
+43 676 571 0536
-
Doors and corners – that’s where they get you
-





From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Sharan Karade
Sent: Monday, March 22, 2021 13:10
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] unknown density

Hi

Please look at the pic attached. found similar pentagon water network.



On Mon, Mar 22, 2021 at 12:40 PM Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk<mailto:488a26d62010-dmarc-requ...@jiscmail.ac.uk>>
 wrote:
Definitely water pentamer, no doubt at all ;-0
Cheers, Jon.C.

Sent from ProtonMail mobile



 Original Message 
On 22 Mar 2021, 14:16, Mark J. van Raaij < 
mjvanra...@cnb.csic.es<mailto:mjvanra...@cnb.csic.es>> wrote:

The ring looks too big to be imidazole or a nucleotide or a carbohydrate, so 
it’s probably mainly water molecules.
Perhaps partially replaced by PEG to explain the density between them (i.e. 
water molecules in most copies of the protein and PEG in some other copies). 
I’ve seen horse-shoe shaped PEG in a high-res structure before, PEGs in several 
confirmations might explain a circle.
Practically speaking, I’d first model five waters and see if they refine well.

Mark


On 22 Mar 2021, at 14:58, Sam Tang 
mailto:samtys0...@gmail.com>> wrote:

Hello fellow colleagues

Hope you are all well while the pandemics persists. I just wonder if anyone may 
have an idea what this density (looking like a pentagon) might be. The data was 
collected to 1.8 A and crystal was grown in Bis-tris + PEG3350. Imidazole 
residual? Nucleotide (the protein itself is nucleotide-binding, but shouldn't 
be at this particular site)?

https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing

Thanks!

BRS

Sam



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Re: [ccp4bb] unknown density

[Jon Cooper]

Definitely water pentamer, no doubt at all ;-0
Cheers, Jon.C.

Sent from ProtonMail mobile

 Original Message 
On 22 Mar 2021, 14:16, Mark J. van Raaij wrote:

> The ring looks too big to be imidazole or a nucleotide or a carbohydrate, so 
> it’s probably mainly water molecules.
> Perhaps partially replaced by PEG to explain the density between them (i.e. 
> water molecules in most copies of the protein and PEG in some other copies). 
> I’ve seen horse-shoe shaped PEG in a high-res structure before, PEGs in 
> several confirmations might explain a circle.
> Practically speaking, I’d first model five waters and see if they refine well.
>
> Mark
>
>> On 22 Mar 2021, at 14:58, Sam Tang  wrote:
>>
>> Hello fellow colleagues
>>
>> Hope you are all well while the pandemics persists. I just wonder if anyone 
>> may have an idea what this density (looking like a pentagon) might be. The 
>> data was collected to 1.8 A and crystal was grown in Bis-tris + PEG3350. 
>> Imidazole residual? Nucleotide (the protein itself is nucleotide-binding, 
>> but shouldn't be at this particular site)?
>>
>> https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing
>>
>> Thanks!
>>
>> BRS
>>
>> Sam
>>
>> ---
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
> ---
>
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Re: [ccp4bb] unknown density

[Mark J. van Raaij]

The ring looks too big to be imidazole or a nucleotide or a carbohydrate, so 
it’s probably mainly water molecules.
Perhaps partially replaced by PEG to explain the density between them (i.e. 
water molecules in most copies of the protein and PEG in some other copies). 
I’ve seen horse-shoe shaped PEG in a high-res structure before, PEGs in several 
confirmations might explain a circle.
Practically speaking, I’d first model five waters and see if they refine well.

Mark 

> On 22 Mar 2021, at 14:58, Sam Tang  wrote:
> 
> Hello fellow colleagues
> 
> Hope you are all well while the pandemics persists. I just wonder if anyone 
> may have an idea what this density (looking like a pentagon) might be. The 
> data was collected to 1.8 A and crystal was grown in Bis-tris + PEG3350. 
> Imidazole residual? Nucleotide (the protein itself is nucleotide-binding, but 
> shouldn't be at this particular site)?
> 
> https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing
>  
> 
> 
> Thanks!
> 
> BRS
> 
> Sam
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 
> 



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Re: [ccp4bb] unknown density

[Tanner, John J.]

Could be 5 water molecules. Pentagons of water molecules are common in high 
resolution structures. Add 5 waters and see if the inter-water distances are 
appropriate for hydrogen bonding  (2.5-3.2 A).

From: CCP4 bulletin board  on behalf of Sam Tang 

Date: Monday, March 22, 2021 at 9:00 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] unknown density
WARNING: This message has originated from an External Source. This may be a 
phishing expedition that can result in unauthorized access to our IT System. 
Please use proper judgment and caution when opening attachments, clicking 
links, or responding to this email.
Hello fellow colleagues

Hope you are all well while the pandemics persists. I just wonder if anyone may 
have an idea what this density (looking like a pentagon) might be. The data was 
collected to 1.8 A and crystal was grown in Bis-tris + PEG3350. Imidazole 
residual? Nucleotide (the protein itself is nucleotide-binding, but shouldn't 
be at this particular site)?

https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing<https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fdrive.google.com%2Ffile%2Fd%2F1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ%2Fview%3Fusp%3Dsharing&data=04%7C01%7C%7Ca3b0c198e3e14bd9405408d8ed3acf6a%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637520184290892971%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=2kZEUkgOzddNoFf57Rb0ZBj8CwmamIuYxqoo1BWrVMM%3D&reserved=0>

Thanks!

BRS

Sam



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[ccp4bb] unknown density

[Sam Tang]

Hello fellow colleagues

Hope you are all well while the pandemics persists. I just wonder if anyone
may have an idea what this density (looking like a pentagon) might be. The
data was collected to 1.8 A and crystal was grown in Bis-tris + PEG3350.
Imidazole residual? Nucleotide (the protein itself is nucleotide-binding,
but shouldn't be at this particular site)?

https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing

Thanks!

BRS

Sam



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Re: [ccp4bb] Unknown density

[Robbie Joosten]

Hi Nick,

It sometimes helps tweaking the solvent mask settings. In Refmac the command 
for the option is 'solvent optimise'. PDB-REDO uses this function as well.

Cheers,
Robbie

On 31 May 2019 10:53, Nicholas Keep  wrote:

There is no blue (2fo-fc) density at least at the current contour level
in your difference density.  That probably indicates what you are seeing
is noise rather than a real feature.

I am also seeing more intense (above 7 sigma in some cases) positive
difference density without actual density in my current refmac refined
structure than I am used to.

I ascribed this to being in P1 with significant anisotropy leading to
ripples???, but if this is widespread then there may have been tweaks to
the software.  Anyone else want to comment on this

Best wishes

Nick

--
Prof Nicholas H. Keep
Executive Dean of School of Science
Professor of Biomolecular Science
Crystallography, Institute for Structural and Molecular Biology,
Department of Biological Sciences
Birkbeck,  University of London,
Malet Street,
Bloomsbury
LONDON
WC1E 7HX

email n.k...@mail.cryst.bbk.ac.uk
Telephone 020-7631-6852  (Room G54a Office)
   020-7631-6800  (Department Office)
Fax   020-7631-6803
If you want to access me in person you have to come to the crystallography 
entrance
and ring me or the department office from the internal phone by the door



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Re: [ccp4bb] Unknown density

[Nicholas Keep]

There is no blue (2fo-fc) density at least at the current contour level 
in your difference density.  That probably indicates what you are seeing 
is noise rather than a real feature.


I am also seeing more intense (above 7 sigma in some cases) positive 
difference density without actual density in my current refmac refined 
structure than I am used to.


I ascribed this to being in P1 with significant anisotropy leading to 
ripples???, but if this is widespread then there may have been tweaks to 
the software.  Anyone else want to comment on this


Best wishes

Nick

--
Prof Nicholas H. Keep
Executive Dean of School of Science
Professor of Biomolecular Science
Crystallography, Institute for Structural and Molecular Biology,
Department of Biological Sciences
Birkbeck,  University of London,
Malet Street,
Bloomsbury
LONDON
WC1E 7HX

email n.k...@mail.cryst.bbk.ac.uk
Telephone 020-7631-6852  (Room G54a Office)
  020-7631-6800  (Department Office)
Fax   020-7631-6803
If you want to access me in person you have to come to the crystallography 
entrance
and ring me or the department office from the internal phone by the door



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Re: [ccp4bb] Unknown density

[Daniel M. Himmel, Ph. D.]

Oops.  I meant to say that Ca2+ usually has seven coordinating atoms.
Mg2+ usually has six.  Sorry for the error.

-Daniel

On Wed, Mar 7, 2018 at 2:27 PM, Daniel M. Himmel, Ph. D. <
danielmhim...@gmail.com> wrote:

> Rajesh,
>
> Ca2+ usually is coordinated by five atoms, whereas Mg2+ usually is
> coordinated by six.  I've seen that a lot in structures that I've
> determined.
>
> -Daniel
>
>
> On Wed, Mar 7, 2018 at 2:14 PM, Eleanor Dodson <176a9d5ebad7-dmarc-
> requ...@jiscmail.ac.uk> wrote:
>
>> You just calculate a anom map - it doesnt need you to dselect Ca or Mg or
>> anything else..
>> Judge the peak heights by the signal you get over any S sites in the
>> model. The relative signal you expect depends on your wavelength of course.
>> Eleanor
>>
>>
>> On 7 March 2018 at 19:09, Rajesh Kumar  wrote:
>>
>>> Hi Ivan,
>>>
>>> https://csgid.org/csgid/metal_sites/
>>>
>>> suggest that Mg++ is the correct metal. Ca++ is not acceptable.
>>>
>>> I am going to validate it by calculating anomalous peak for Ca++ too.
>>>
>>> Thank you
>>> Rajesh
>>>
>>>
>>> On Wed, Mar 7, 2018 at 1:06 PM, Ivan Shabalin <
>>> iva...@iwonka.med.virginia.edu> wrote:
>>>
 Hi, Rajesh,

 It does look like Ca to me. It has around 2.4 A distances to waters and
 quite many electrons (18), which correspond to rather wide and high density
 peak in the middle.

 Mg has distances at 2.07, and 10 electrons (should be a smaller peak).

 Na has 2.41 distances to waters, 10 electrons (should be a smaller
 peak).

 After you place a metal and refine it, try using CheckMyMetal to
 validate it:

 https://csgid.org/csgid/metal_sites/


 It takes into account preferable coordination geometries, coordination
 bond distances, and compares ADPs to average ADP for the surrounding atoms.

 If the dataset is very good, and the wavelength of X-ray is rather
 long, you might even get anomalous signal for Ca:

 http://skuld.bmsc.washington.edu/scatter/AS_periodic.html

 Ivan



 With best regards,
 Ivan Shabalin, Ph.D.
 Research Scientist,
 Department of Molecular Physiology and Biological Physics,
 University of Virginia,
 1340 Jefferson Park Avenue
 ,
 Pinn Hall,Room 4223,
 Charlottesville, VA 22908

 On 03/06/2018 05:19 PM, Rajesh Kumar wrote:

> Dear All,
>
> Have you had experience with this kind of density? I am wandering what
> this could be?
>
> Thank you very much for the help.
>
> -Rajesh
>
>
>
>
>
 

>>>
>>>
>>
>

Re: [ccp4bb] Unknown density

[Degano Massimo]

I would not rely only on coordination numbers, since Ca2+ can be also 
hepta-coordinated. You can look at the high resolution structures of 
parvalbumin and nucleoside hydrolases. And, although not up to date, this site 
is also useful http://mespeus.bch.ed.ac.uk/tanna/ . The anomalous map 
suggestion is probably the best way to go.

Max

--
Dr. Massimo Degano
Biocrystallography Unit
Dept. of Immunology, Transplantation, and Infectious Diseases
DIBIT Fondazione San Raffaele
via Olgettina 58
20132 Milan - Italy
email: degano.mass...@hsr.it
phone: +39-0226437152
fax: +39-0226434153
skype: maxdegano
ORCID: -0002-0787-1883


On 7 Mar 2018, at 20:27, Daniel M. Himmel, Ph. D. 
mailto:danielmhim...@gmail.com>> wrote:

Rajesh,

Ca2+ usually is coordinated by five atoms, whereas Mg2+ usually is
coordinated by six.  I've seen that a lot in structures that I've determined.

-Daniel


On Wed, Mar 7, 2018 at 2:14 PM, Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
 wrote:
You just calculate a anom map - it doesnt need you to dselect Ca or Mg or 
anything else..
Judge the peak heights by the signal you get over any S sites in the model. The 
relative signal you expect depends on your wavelength of course.
Eleanor


On 7 March 2018 at 19:09, Rajesh Kumar 
mailto:rajesh.p...@gmail.com>> wrote:
Hi Ivan,

https://csgid.org/csgid/metal_sites/

suggest that Mg++ is the correct metal. Ca++ is not acceptable.

I am going to validate it by calculating anomalous peak for Ca++ too.

Thank you
Rajesh


On Wed, Mar 7, 2018 at 1:06 PM, Ivan Shabalin 
mailto:iva...@iwonka.med.virginia.edu>> wrote:
Hi, Rajesh,

It does look like Ca to me. It has around 2.4 A distances to waters and quite 
many electrons (18), which correspond to rather wide and high density peak in 
the middle.

Mg has distances at 2.07, and 10 electrons (should be a smaller peak).

Na has 2.41 distances to waters, 10 electrons (should be a smaller peak).

After you place a metal and refine it, try using CheckMyMetal to validate it:

https://csgid.org/csgid/metal_sites/


It takes into account preferable coordination geometries, coordination bond 
distances, and compares ADPs to average ADP for the surrounding atoms.

If the dataset is very good, and the wavelength of X-ray is rather long, you 
might even get anomalous signal for Ca:

http://skuld.bmsc.washington.edu/scatter/AS_periodic.html

Ivan



With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park 
Avenue,
 Pinn Hall,Room 4223,
Charlottesville, VA 22908

On 03/06/2018 05:19 PM, Rajesh Kumar wrote:
Dear All,

Have you had experience with this kind of density? I am wandering what this 
could be?

Thank you very much for the help.

-Rajesh












Rispetta l'ambiente: non stampare questa mail se non ? necessario.
Respect the environment: if it's not necessary, don't print this mail.

Re: [ccp4bb] Unknown density

[Daniel M. Himmel, Ph. D.]

Rajesh,

Ca2+ usually is coordinated by five atoms, whereas Mg2+ usually is
coordinated by six.  I've seen that a lot in structures that I've
determined.

-Daniel


On Wed, Mar 7, 2018 at 2:14 PM, Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> You just calculate a anom map - it doesnt need you to dselect Ca or Mg or
> anything else..
> Judge the peak heights by the signal you get over any S sites in the
> model. The relative signal you expect depends on your wavelength of course.
> Eleanor
>
>
> On 7 March 2018 at 19:09, Rajesh Kumar  wrote:
>
>> Hi Ivan,
>>
>> https://csgid.org/csgid/metal_sites/
>>
>> suggest that Mg++ is the correct metal. Ca++ is not acceptable.
>>
>> I am going to validate it by calculating anomalous peak for Ca++ too.
>>
>> Thank you
>> Rajesh
>>
>>
>> On Wed, Mar 7, 2018 at 1:06 PM, Ivan Shabalin <
>> iva...@iwonka.med.virginia.edu> wrote:
>>
>>> Hi, Rajesh,
>>>
>>> It does look like Ca to me. It has around 2.4 A distances to waters and
>>> quite many electrons (18), which correspond to rather wide and high density
>>> peak in the middle.
>>>
>>> Mg has distances at 2.07, and 10 electrons (should be a smaller peak).
>>>
>>> Na has 2.41 distances to waters, 10 electrons (should be a smaller peak).
>>>
>>> After you place a metal and refine it, try using CheckMyMetal to
>>> validate it:
>>>
>>> https://csgid.org/csgid/metal_sites/
>>>
>>>
>>> It takes into account preferable coordination geometries, coordination
>>> bond distances, and compares ADPs to average ADP for the surrounding atoms.
>>>
>>> If the dataset is very good, and the wavelength of X-ray is rather long,
>>> you might even get anomalous signal for Ca:
>>>
>>> http://skuld.bmsc.washington.edu/scatter/AS_periodic.html
>>>
>>> Ivan
>>>
>>>
>>>
>>> With best regards,
>>> Ivan Shabalin, Ph.D.
>>> Research Scientist,
>>> Department of Molecular Physiology and Biological Physics,
>>> University of Virginia,
>>> 1340 Jefferson Park Avenue
>>> ,
>>> Pinn Hall,Room 4223,
>>> Charlottesville, VA 22908
>>>
>>> On 03/06/2018 05:19 PM, Rajesh Kumar wrote:
>>>
 Dear All,

 Have you had experience with this kind of density? I am wandering what
 this could be?

 Thank you very much for the help.

 -Rajesh





>>> 
>>>
>>
>>
>

Re: [ccp4bb] Unknown density

[Eleanor Dodson]

You just calculate a anom map - it doesnt need you to dselect Ca or Mg or
anything else..
Judge the peak heights by the signal you get over any S sites in the model.
The relative signal you expect depends on your wavelength of course.
Eleanor


On 7 March 2018 at 19:09, Rajesh Kumar  wrote:

> Hi Ivan,
>
> https://csgid.org/csgid/metal_sites/
>
> suggest that Mg++ is the correct metal. Ca++ is not acceptable.
>
> I am going to validate it by calculating anomalous peak for Ca++ too.
>
> Thank you
> Rajesh
>
>
> On Wed, Mar 7, 2018 at 1:06 PM, Ivan Shabalin  edu> wrote:
>
>> Hi, Rajesh,
>>
>> It does look like Ca to me. It has around 2.4 A distances to waters and
>> quite many electrons (18), which correspond to rather wide and high density
>> peak in the middle.
>>
>> Mg has distances at 2.07, and 10 electrons (should be a smaller peak).
>>
>> Na has 2.41 distances to waters, 10 electrons (should be a smaller peak).
>>
>> After you place a metal and refine it, try using CheckMyMetal to validate
>> it:
>>
>> https://csgid.org/csgid/metal_sites/
>>
>>
>> It takes into account preferable coordination geometries, coordination
>> bond distances, and compares ADPs to average ADP for the surrounding atoms.
>>
>> If the dataset is very good, and the wavelength of X-ray is rather long,
>> you might even get anomalous signal for Ca:
>>
>> http://skuld.bmsc.washington.edu/scatter/AS_periodic.html
>>
>> Ivan
>>
>>
>>
>> With best regards,
>> Ivan Shabalin, Ph.D.
>> Research Scientist,
>> Department of Molecular Physiology and Biological Physics,
>> University of Virginia,
>> 1340 Jefferson Park Avenue
>> ,
>> Pinn Hall,Room 4223,
>> Charlottesville, VA 22908
>>
>> On 03/06/2018 05:19 PM, Rajesh Kumar wrote:
>>
>>> Dear All,
>>>
>>> Have you had experience with this kind of density? I am wandering what
>>> this could be?
>>>
>>> Thank you very much for the help.
>>>
>>> -Rajesh
>>>
>>>
>>>
>>>
>>>
>> 
>>
>
>

Re: [ccp4bb] Unknown density

[Rajesh Kumar]

Hi Ivan,

https://csgid.org/csgid/metal_sites/

suggest that Mg++ is the correct metal. Ca++ is not acceptable.

I am going to validate it by calculating anomalous peak for Ca++ too.

Thank you
Rajesh

On Wed, Mar 7, 2018 at 1:06 PM, Ivan Shabalin <
iva...@iwonka.med.virginia.edu> wrote:

> Hi, Rajesh,
>
> It does look like Ca to me. It has around 2.4 A distances to waters and
> quite many electrons (18), which correspond to rather wide and high density
> peak in the middle.
>
> Mg has distances at 2.07, and 10 electrons (should be a smaller peak).
>
> Na has 2.41 distances to waters, 10 electrons (should be a smaller peak).
>
> After you place a metal and refine it, try using CheckMyMetal to validate
> it:
>
> https://csgid.org/csgid/metal_sites/
>
>
> It takes into account preferable coordination geometries, coordination
> bond distances, and compares ADPs to average ADP for the surrounding atoms.
>
> If the dataset is very good, and the wavelength of X-ray is rather long,
> you might even get anomalous signal for Ca:
>
> http://skuld.bmsc.washington.edu/scatter/AS_periodic.html
>
> Ivan
>
>
>
> With best regards,
> Ivan Shabalin, Ph.D.
> Research Scientist,
> Department of Molecular Physiology and Biological Physics,
> University of Virginia,
> 1340 Jefferson Park Avenue
> ,
> Pinn Hall,Room 4223,
> Charlottesville, VA 22908
>
> On 03/06/2018 05:19 PM, Rajesh Kumar wrote:
>
>> Dear All,
>>
>> Have you had experience with this kind of density? I am wandering what
>> this could be?
>>
>> Thank you very much for the help.
>>
>> -Rajesh
>>
>>
>>
>>
>>
> 
>

Re: [ccp4bb] Unknown density

[Ivan Shabalin]

Hi, Rajesh,

It does look like Ca to me. It has around 2.4 A distances to waters and 
quite many electrons (18), which correspond to rather wide and high 
density peak in the middle.


Mg has distances at 2.07, and 10 electrons (should be a smaller peak).

Na has 2.41 distances to waters, 10 electrons (should be a smaller peak).

After you place a metal and refine it, try using CheckMyMetal to 
validate it:


https://csgid.org/csgid/metal_sites/


It takes into account preferable coordination geometries, coordination 
bond distances, and compares ADPs to average ADP for the surrounding atoms.


If the dataset is very good, and the wavelength of X-ray is rather long, 
you might even get anomalous signal for Ca:


http://skuld.bmsc.washington.edu/scatter/AS_periodic.html

Ivan



With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908

On 03/06/2018 05:19 PM, Rajesh Kumar wrote:

Dear All,

Have you had experience with this kind of density? I am wandering what 
this could be?


Thank you very much for the help.

-Rajesh

Re: [ccp4bb] Unknown density

[Prem Prakash]

Hi Rajesh,

It might be water molecules present at the symmetry axis, one more water
molecule at the centre (exactly at the axis, with occupancy of 0.5). If you
put three water molecule you probably may find the other three
symmetry-mates  water molecules in opposite sides. OR One water molecule at
the symmetry axis.surrounded by 5 different water molecules. Or You can try
As Garib mentioned it may be a six anions (or water) coordinated with some
metals possibly in crystallization or protein buffer. Please try refining
both cases parallely. I once encountered such case in my structure.
Probably it may help.

Good luck

Prem

On Wed, Mar 7, 2018 at 4:37 AM, Abhik Mukhopadhyay 
wrote:

> Hi Rajesh,
>
> You may find this database is also useful while refining your model
>
> http://mespeus.bch.ed.ac.uk/MESPEUS_10/
>
> and for calcium
>
> http://mespeus.bch.ed.ac.uk/MESPEUS_10/_5.jsp
>
> Abhik
>
> On Tue, Mar 6, 2018 at 10:59 PM, Patrick Loll  wrote:
>
>> Calcium likes to form octahedral complexes with water (or other
>> oxygen-containing) ligands. This looks like a classic example.
>>
>> After you model and refine this, you’ll want to check water-metal
>> distances, to make sure they are appropriate for calcium. There is a nice
>> literature on such things, which I of course don’t have at my fingertips;
>> but I think Wladek Minor has done some data-mining in metal-containing
>> protein structures, and Amy Katz and Jenny Glusker have a number of papers
>> that are relevant. There are more, of course—a little time in the “library”
>> is warranted.
>>
>> Cheers,
>>
>> Pat Loll
>>
>> > On 6 Mar 2018, at 5:19 PM, Rajesh Kumar  wrote:
>> >
>> > Dear All,
>> >
>> > Have you had experience with this kind of density? I am wandering what
>> this could be?
>> >
>> > Thank you very much for the help.
>> >
>> > -Rajesh
>> >
>> >
>> > 
>> >
>>
>> 
>> ---
>> Patrick J. Loll, Ph. D.
>> Professor of Biochemistry & Molecular Biology
>> Drexel University College of Medicine
>> Room 10-102 New College Building
>> 245 N. 15th St
>> .,
>> Mailstop 497
>> Philadelphia, PA  19102-1192  USA
>>
>> (215) 762-7706
>> pjl...@gmail.com
>> pj...@drexel.edu
>>
>
>


-- 
With kind regards,

Prem Prakash
PhD Research Scholar
Protein Crystallography Lab
Biosciences and Bioengineering
IIT Bombay

Re: [ccp4bb] Unknown density

[Abhik Mukhopadhyay]

Hi Rajesh,

You may find this database is also useful while refining your model

http://mespeus.bch.ed.ac.uk/MESPEUS_10/

and for calcium

http://mespeus.bch.ed.ac.uk/MESPEUS_10/_5.jsp

Abhik

On Tue, Mar 6, 2018 at 10:59 PM, Patrick Loll  wrote:

> Calcium likes to form octahedral complexes with water (or other
> oxygen-containing) ligands. This looks like a classic example.
>
> After you model and refine this, you’ll want to check water-metal
> distances, to make sure they are appropriate for calcium. There is a nice
> literature on such things, which I of course don’t have at my fingertips;
> but I think Wladek Minor has done some data-mining in metal-containing
> protein structures, and Amy Katz and Jenny Glusker have a number of papers
> that are relevant. There are more, of course—a little time in the “library”
> is warranted.
>
> Cheers,
>
> Pat Loll
>
> > On 6 Mar 2018, at 5:19 PM, Rajesh Kumar  wrote:
> >
> > Dear All,
> >
> > Have you had experience with this kind of density? I am wandering what
> this could be?
> >
> > Thank you very much for the help.
> >
> > -Rajesh
> >
> >
> > 
> >
>
> 
> ---
> Patrick J. Loll, Ph. D.
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
>
> (215) 762-7706
> pjl...@gmail.com
> pj...@drexel.edu
>

Re: [ccp4bb] Unknown density

[Patrick Loll]

Calcium likes to form octahedral complexes with water (or other 
oxygen-containing) ligands. This looks like a classic example. 

After you model and refine this, you’ll want to check water-metal distances, to 
make sure they are appropriate for calcium. There is a nice literature on such 
things, which I of course don’t have at my fingertips; but I think Wladek Minor 
has done some data-mining in metal-containing protein structures, and Amy Katz 
and Jenny Glusker have a number of papers that are relevant. There are more, of 
course—a little time in the “library” is warranted.

Cheers,

Pat Loll

> On 6 Mar 2018, at 5:19 PM, Rajesh Kumar  wrote:
> 
> Dear All,
> 
> Have you had experience with this kind of density? I am wandering what this 
> could be?
> 
> Thank you very much for the help.
> 
> -Rajesh
> 
> 
> 
> 

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu

Re: [ccp4bb] Unknown density

[Garib Murshudov]

You could try to put CA (as you have CA in your crystallisation condition) and 
waters for coordinating atoms. If after refinement distances are around 2.3-2.4 
(if I remember correctly) and B values of atoms (CA and waters) are consistent, 
i.e. similar to each other then CA is good interpretation. Similarly you could 
use MG (but since this atoms does not seem to be involved in structure or 
function it is unlikely to be part of your protein, it should come from 
crystallisation, purification etc, so MG is less likely than CA) and the 
distances are around 2-2.1A and (again) B values are consistent then MG is good.

Regards,
Garib

On 6 Mar 2018, at 22:37, Hussain Gps  wrote:

> Hi Rajesh,
> 
> Can you please let everyone know the buffer/reagents involved throughout the 
> process.
> 
> Also, how strong is the density? In other words, until what contour level you 
> see it in the Fo-Fc map?
> 
> Anyways, looking at the shape, it looks well coordinated - could be 
> cacodylate? Or water, am not sure.
> 
> Cheers,
> Hussain 
> 
>> On 7 Mar 2018, at 9:19 am, Rajesh Kumar  wrote:
>> 
>> Dear All,
>> 
>> Have you had experience with this kind of density? I am wandering what this 
>> could be?
>> 
>> Thank you very much for the help.
>> 
>> -Rajesh
>> 
>> 
>> 
>> 

Dr Garib N Murshudov
MRC-LMB
Francis Crick Avenue
Cambridge 
CB2 0QH UK
Web http://www.mrc-lmb.cam.ac.uk, 
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/

Re: [ccp4bb] Unknown density

[Hussain Gps]

Hi Rajesh,

Can you please let everyone know the buffer/reagents involved throughout the 
process.

Also, how strong is the density? In other words, until what contour level you 
see it in the Fo-Fc map?

Anyways, looking at the shape, it looks well coordinated - could be cacodylate? 
Or water, am not sure.

Cheers,
Hussain 

> On 7 Mar 2018, at 9:19 am, Rajesh Kumar  wrote:
> 
> Dear All,
> 
> Have you had experience with this kind of density? I am wandering what this 
> could be?
> 
> Thank you very much for the help.
> 
> -Rajesh
> 
> 
> 
> 

Re: [ccp4bb] Unknown density

[Rajesh Kumar]

Hi Hussain,

Crystallization condition is 200 mM CaCl2 and 20% PEG 3350.

I some what realized that it could be Ca++ with coordinated waters. But I
really wanted to have
second opinion from our group.

Thank you every one.

-Rajesh





On Tue, Mar 6, 2018 at 5:37 PM, Hussain Gps  wrote:

> Hi Rajesh,
>
> Can you please let everyone know the buffer/reagents involved throughout
> the process.
>
> Also, how strong is the density? In other words, until what contour level
> you see it in the Fo-Fc map?
>
> Anyways, looking at the shape, it looks well coordinated - could be
> cacodylate? Or water, am not sure.
>
> Cheers,
> Hussain
>
> > On 7 Mar 2018, at 9:19 am, Rajesh Kumar  wrote:
> >
> > Dear All,
> >
> > Have you had experience with this kind of density? I am wandering what
> this could be?
> >
> > Thank you very much for the help.
> >
> > -Rajesh
> >
> >
> > 
> >
>

Re: [ccp4bb] Unknown density

[Phil Evans]

Mg2+(H20)6 ?

> On 6 Mar 2018, at 22:19, Rajesh Kumar  wrote:
> 
> Dear All,
> 
> Have you had experience with this kind of density? I am wandering what this 
> could be?
> 
> Thank you very much for the help.
> 
> -Rajesh
> 
> 
> 
> 

Re: [ccp4bb] Unknown density

[Garib Murshudov]

It looks like 6 coordinated metal atom with coordinated (possibly) with waters. 
The simplest case could be NA. You could use anomalous difference map to see if 
there is any anomalous signal there.

Regards
Garib


On 6 Mar 2018, at 22:19, Rajesh Kumar  wrote:

> Dear All,
> 
> Have you had experience with this kind of density? I am wandering what this 
> could be?
> 
> Thank you very much for the help.
> 
> -Rajesh
> 
> 
> 
> 

Dr Garib N Murshudov
MRC-LMB
Francis Crick Avenue
Cambridge 
CB2 0QH UK
Web http://www.mrc-lmb.cam.ac.uk, 
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/

Re: [ccp4bb] Unknown density

[Pavel Natashin]

Dear all,

Thank you very much for help!

The problem was solved very easy (thanks Dominik A. Herbs). I just used another 
program for refinement (Refmac5 instead Phenix.refine) and this "noisy" density 
almost disappeared.

http://www.4sync.com/photo/--e2gHrG/Density-Refmac5.html

Thanks a lot for help again!

Best regards,

Pavel V. Natashin

 
PhD student
Photobiology Laboratory
Institute of Biophysics
Russian Academy of Sciences Siberian Branch
Krasnoyarsk 660036, Russia

National Laboratory of Biomacromolecules
Institute of Biophysics Chinese Academy of Sciences
Beijing 100101, China

Re: [ccp4bb] Unknown density

[Albert Guskov]

Dear Pavel,
you should also always consider your cryoprotectant molecules, so let's say
if you used PEG for cryoprotection, there's a very high probability you may
see it in your structure.  It's very hard to say from the snapshots, but
for the patch of density depicted in fig.2 I would try PEG (of course only
if you had it).
All the best,
Albert


2013/2/25 Antony Oliver 

> Dear Pavel,
>
> Is this density located on a symmetry axis?
> There is quite often a lot of "noise" around these regions - and it may
> not, in fact, be possible to model this satisfactorily.
>
> Tony.
>
>
> ---
> Dr Antony W Oliver
> Senior Research Fellow
> CR-UK DNA Repair Enzymes Group
> Genome Damage and Stability Centre
> Science Park Road
> University of Sussex
> Falmer, Brighton, BN1 9RQ
>
> email: antony.oli...@sussex.ac.uk
> tel (office): +44 (0)1273 678349
> tel (lab): +44 (0)1273 677512
>
> On Feb 25, 2013, at 10:03 AM, Natashin Pavel wrote:
>
> > Hello everyone, > >
> > I’m working on a structure with a 1.72Å resolution data and almost
> finished it. But there is a piece of unknown density between two protein
> molecules.
> > The following pictures show this density from two sides:
> >
> > http://www.4sync.com/photo/WHewU7Ek/Density_1.html >
> >
> > http://www.4sync.com/photo/RM3IEesw/Density_-_2.html
> >
> > This density is located between two protein molecules and is not
> connected to either of them. >
> > I have tried to fit several small organic molecules from crystallization
> conditions and protein sample buffer, also tried ligand database search
> using “Phenix ligand identification” software, but no satisfactory results.
> > Crystallization condition: DL-Malic acid >
> > Sample Buffer: Bis-Tris, EDTA >
> > Reagents from protein purification step (also checked): Tris, Urea,
> Triton-X100, DTT. > >
> >
> > I will very much appreciate to hear any suggestions and ideas on what to
> do.
> >
> > Best regerds,
> >
> > Pavel V. Natashin
> >
> >
> > PhD student
> > Photobiology Laboratory
> > Institute of Biophysics
> > Russian Academy of Sciences Siberian Branch
> > Krasnoyarsk 660036, Russia
> >
> > National Laboratory of Biomacromolecules
> > Institute of Biophysics Chinese Academy of Sciences
> > Beijing 100101, China
>
>

Re: [ccp4bb] Unknown density

[Antony Oliver]

Dear Pavel,

Is this density located on a symmetry axis?  
There is quite often a lot of "noise" around these regions - and it may not, in 
fact, be possible to model this satisfactorily.

Tony.


---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Feb 25, 2013, at 10:03 AM, Natashin Pavel wrote:

> Hello everyone,
> 
> 
> I’m working on a structure with a 1.72Å resolution data and almost finished 
> it. But there is a piece of unknown density between two protein molecules. 
> The following pictures show this density from two sides:
> 
> http://www.4sync.com/photo/WHewU7Ek/Density_1.html
> 
> 
> http://www.4sync.com/photo/RM3IEesw/Density_-_2.html
> 
> This density is located between two protein molecules and is not connected to 
> either of them.
> 
> I have tried to fit several small organic molecules from crystallization 
> conditions and protein sample buffer, also tried ligand database search using 
> “Phenix ligand identification” software, but no satisfactory results. 
> Crystallization condition: DL-Malic acid
> 
> Sample Buffer: Bis-Tris, EDTA
> 
> Reagents from protein purification step (also checked): Tris, Urea, 
> Triton-X100, DTT. 
> 
> 
> 
> I will very much appreciate to hear any suggestions and ideas on what to do.
> 
> Best regerds, 
> 
> Pavel V. Natashin
> 
>  
> PhD student
> Photobiology Laboratory 
> Institute of Biophysics
> Russian Academy of Sciences Siberian Branch 
> Krasnoyarsk 660036, Russia 
> 
> National Laboratory of Biomacromolecules
> Institute of Biophysics Chinese Academy of Sciences
> Beijing 100101, China

Re: [ccp4bb] unknown density

[Shiva Bhowmik]

My suggestion would be to refine with full glu and then put either O and C
atoms at the region corresponidng to the extra electron density.

On Wed, May 18, 2011 at 1:41 PM, Borhani, David <
david.borh...@deshawresearch.com> wrote:

>  Glutamate methyl ester? (or ethyl ester, which I’m not sure has ever been
> documented…hard to tell how many extra heavy atoms given the view direction
> in the image you supplied). Or the methyl/ethyl amides.
>
>
>
> Dave
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Bosch,
> Juergen
> *Sent:* Wednesday, 18 May 2011 14:06
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] unknown density
>
>
>
> I take the lysine back, just realized wrong color coding it's an oxygen and
> not a nitrogen closer to the extra density :-)
>
> Jürgen
>
>
>
> On May 18, 2011, at 2:03 PM, Bosch, Juergen wrote:
>
>
>
>  But that's a lysine, so maybe a PTM ? Acetylated perhaps ?
>
>
>
> Jürgen
>
>
>
> On May 18, 2011, at 1:56 PM, Shiva Bhowmik wrote:
>
>
>
>  Hi Cedric,
>
>
>
> I presume you collected the datset at a synchrtron source. It could be free
> radical generated due to X-ray radiation has modified the glutamate
> residues. I have seen this happening for Ser residues.
>
>
>
> Cheers,
>
>
>
> Shiva
>
> On Wed, May 18, 2011 at 9:08 AM, cedric bauvois 
> wrote:
>
> Hi, everyone,
>
> I am working on the refinement of a structure  at about 1.4 Ang and found
> some clear extra density connected to a glutamate (see figure), but I don't
> know what it should be. The crystal came out from Tris-HCl buffer with
> peg600, and protein buffer contains sodium-phosphate.
>
> Does anyone figure out what this density can be ?
> Thanks for any help you can give.
>
>  Cédric
>
>
>
>
>
> ..
>
> Jürgen Bosch
>
> Johns Hopkins Bloomberg School of Public Health
>
> Department of Biochemistry & Molecular Biology
>
> Johns Hopkins Malaria Research Institute
>
> 615 North Wolfe Street, W8708
>
> Baltimore, MD 21205
>
> Phone: +1-410-614-4742
>
> Lab:  +1-410-614-4894
>
> Fax:  +1-410-955-3655
>
> http://web.mac.com/bosch_lab/
>
>
>
>
>
>
>
>
>
> ..
>
> Jürgen Bosch
>
> Johns Hopkins Bloomberg School of Public Health
>
> Department of Biochemistry & Molecular Biology
>
> Johns Hopkins Malaria Research Institute
>
> 615 North Wolfe Street, W8708
>
> Baltimore, MD 21205
>
> Phone: +1-410-614-4742
>
> Lab:  +1-410-614-4894
>
> Fax:  +1-410-955-3655
>
> http://web.mac.com/bosch_lab/
>
>
>
>
>
>
>

Re: [ccp4bb] unknown density

[Borhani, David]

Glutamate methyl ester? (or ethyl ester, which I'm not sure has ever been 
documented...hard to tell how many extra heavy atoms given the view direction 
in the image you supplied). Or the methyl/ethyl amides.

Dave

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bosch, 
Juergen
Sent: Wednesday, 18 May 2011 14:06
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unknown density

I take the lysine back, just realized wrong color coding it's an oxygen and not 
a nitrogen closer to the extra density :-)
Jürgen

On May 18, 2011, at 2:03 PM, Bosch, Juergen wrote:


But that's a lysine, so maybe a PTM ? Acetylated perhaps ?

Jürgen

On May 18, 2011, at 1:56 PM, Shiva Bhowmik wrote:


Hi Cedric,

I presume you collected the datset at a synchrtron source. It could be free 
radical generated due to X-ray radiation has modified the glutamate residues. I 
have seen this happening for Ser residues.

Cheers,

Shiva
On Wed, May 18, 2011 at 9:08 AM, cedric bauvois 
mailto:cbauv...@gmail.com>> wrote:
Hi, everyone,

I am working on the refinement of a structure  at about 1.4 Ang and found some 
clear extra density connected to a glutamate (see figure), but I don't know 
what it should be. The crystal came out from Tris-HCl buffer with peg600, and 
protein buffer contains sodium-phosphate.

Does anyone figure out what this density can be ?
Thanks for any help you can give.
 Cédric


..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/




..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

Re: [ccp4bb] unknown density

[Bosch, Juergen]

I take the lysine back, just realized wrong color coding it's an oxygen and not 
a nitrogen closer to the extra density :-)
Jürgen

On May 18, 2011, at 2:03 PM, Bosch, Juergen wrote:

But that's a lysine, so maybe a PTM ? Acetylated perhaps ?

Jürgen

On May 18, 2011, at 1:56 PM, Shiva Bhowmik wrote:

Hi Cedric,

I presume you collected the datset at a synchrtron source. It could be free 
radical generated due to X-ray radiation has modified the glutamate residues. I 
have seen this happening for Ser residues.

Cheers,

Shiva

On Wed, May 18, 2011 at 9:08 AM, cedric bauvois 
mailto:cbauv...@gmail.com>> wrote:
Hi, everyone,

I am working on the refinement of a structure  at about 1.4 Ang and found some 
clear extra density connected to a glutamate (see figure), but I don't know 
what it should be. The crystal came out from Tris-HCl buffer with peg600, and 
protein buffer contains sodium-phosphate.

Does anyone figure out what this density can be ?
Thanks for any help you can give.
 Cédric


..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/




..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

Re: [ccp4bb] unknown density

[Bosch, Juergen]

But that's a lysine, so maybe a PTM ? Acetylated perhaps ?

Jürgen

On May 18, 2011, at 1:56 PM, Shiva Bhowmik wrote:

Hi Cedric,

I presume you collected the datset at a synchrtron source. It could be free 
radical generated due to X-ray radiation has modified the glutamate residues. I 
have seen this happening for Ser residues.

Cheers,

Shiva

On Wed, May 18, 2011 at 9:08 AM, cedric bauvois 
mailto:cbauv...@gmail.com>> wrote:
Hi, everyone,

I am working on the refinement of a structure  at about 1.4 Ang and found some 
clear extra density connected to a glutamate (see figure), but I don't know 
what it should be. The crystal came out from Tris-HCl buffer with peg600, and 
protein buffer contains sodium-phosphate.

Does anyone figure out what this density can be ?
Thanks for any help you can give.
 Cédric


..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

Re: [ccp4bb] unknown density

[Shiva Bhowmik]

Hi Cedric,

I presume you collected the datset at a synchrtron source. It could be free
radical generated due to X-ray radiation has modified the glutamate
residues. I have seen this happening for Ser residues.

Cheers,

Shiva

On Wed, May 18, 2011 at 9:08 AM, cedric bauvois  wrote:

> Hi, everyone,
>
> I am working on the refinement of a structure  at about 1.4 Ang and found
> some clear extra density connected to a glutamate (see figure), but I
> don't know what it should be. The crystal came out from Tris-HCl buffer with
> peg600, and protein buffer contains sodium-phosphate.
>
> Does anyone figure out what this density can be ?
> Thanks for any help you can give.
>
>  Cédric

Re: [ccp4bb] unknown density

[Robbie Joosten]

Dear Prescilla,
 
Hermans suggestion look pretty convincing. Try to refine it and see if the 
difference density disappears. I'd replace the C8 atom with oxygen because you 
need it to coordinate the magnesium ion. The residue near C11 can also tell you 
whether that atom chould be polar or not. I'm guessing it's a HIS so the C11 
atom should be a hydrogen bond donor or acceptor. The Residue next to the HIS 
(ASP or ASN) may tell you more about the protonation of the HIS. This is a fun 
game.
 
Cheers,
Robbie Joosten


> Date: Fri, 1 Oct 2010 17:21:31 +0200
> From: herman.schreu...@sanofi-aventis.com
> Subject: Re: [ccp4bb] unknown density
> To: CCP4BB@JISCMAIL.AC.UK
>
> Hi Priscalla and Flip,
>
> I did not bother to find out what format the maps had, I just loaded
> them in coot with no problems. I fitted a cyclohexane ring with three
> atoms attached to it (see attached pdb). It fits quite well, but there
> are two cautions:
>
> 1) it is impossible to deduce the type of atoms from this map, so the
> carbons may be oxygen, nitrogen, whatever.
> 2) The molecule might have a disordered attachment or binding mode and
> may be larger as what is fitted.
>
> It will be a nice puzzle to find out (biochemically, literature) what
> exactly is bound, but it is clearly some ligand molecule and not PEG.
>
> Good luck!
> Herman
>
> -Original Message-
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
> Priscila Oliveira de Giuseppe
> Sent: Friday, October 01, 2010 4:39 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] unknown density
>
> Hi Flip,
>
> Following your suggestion, I send the link to download the screenshots
> of the ligand density:
> http://twitpic.com/2tn35i
>
> Best regards
>
> Priscila
>
>
>
> 
> From: Flip Hoedemaeker [fhoe...@googlemail.com] On Behalf Of Flip
> Hoedemaeker [f...@formulatrix.com]
> Sent: Friday, October 01, 2010 9:18 AM
> To: Priscila Oliveira de Giuseppe
> Subject: Re: [ccp4bb] unknown density
>
> Hi Priscilla,
>
> I don't think a lot of people will take the trouble to find out for
> themselves which format your map files have... Its better to send screen
> shots, better still to put your screenshots on a social media page
> (facebook, twitpic, whatever) and email the link.
>
> Flip
>
> On 10/1/2010 13:12, Priscila Oliveira de Giuseppe wrote:
>> Hi, everyone,
>>
>> I solved a structure at 1.7 A. After fitting all residues into the
>> model, I found an extra density at the catalytic site. The components
>> of the crystallization condition did not explain it (crystallization
>> condition: 40% PEG200, 0.1 M Tris-HCl, protein buffer: PBS)
>>
>> Neither substrates nor products of this enzyme fitted this density.
>>
>> I modelled two PEG molecules with half occupancy. They fitted very
> well, but I am not convinced that this is the best explanation.
>>
>> Does anyone figure out what this density can be? Attached to this
> e-mail I send the maps and a pdb file with the residues surrounding the
> ligand density.
>>
>>
>>
>> Thanks
>>
>> Priscila   

Re: [ccp4bb] unknown density

[Herman . Schreuder]

Hi Priscalla and Flip,

I did not bother to find out what format the maps had, I just loaded
them in coot with no problems. I fitted a cyclohexane ring with three
atoms attached to it (see attached pdb). It fits quite well, but there
are two cautions:

1) it is impossible to deduce the type of atoms from this map, so the
carbons may be oxygen, nitrogen, whatever.
2) The molecule might have a disordered attachment or binding mode and
may be larger as what is fitted.

It will be a nice puzzle to find out (biochemically, literature) what
exactly is bound, but it is clearly some ligand molecule and not PEG.

Good luck!
Herman 

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Priscila Oliveira de Giuseppe
Sent: Friday, October 01, 2010 4:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unknown density

Hi Flip,

Following your suggestion, I send the link to download the screenshots
of the ligand density:
http://twitpic.com/2tn35i

Best regards

Priscila




From: Flip Hoedemaeker [fhoe...@googlemail.com] On Behalf Of Flip
Hoedemaeker [f...@formulatrix.com]
Sent: Friday, October 01, 2010 9:18 AM
To: Priscila Oliveira de Giuseppe
Subject: Re: [ccp4bb] unknown density

Hi Priscilla,

I don't think a lot of people will take the trouble to find out for
themselves which format your map files have... Its better to send screen
shots, better still to put your screenshots on a social media page
(facebook, twitpic, whatever) and email the link.

Flip

On 10/1/2010 13:12, Priscila Oliveira de Giuseppe wrote:
>   Hi, everyone,
>
>   I solved a structure at 1.7 A. After fitting all residues into the 
> model, I found an extra density at the catalytic site. The components 
> of the crystallization condition did not explain it (crystallization 
> condition: 40% PEG200, 0.1 M Tris-HCl, protein buffer: PBS)
>
> Neither substrates nor products of this enzyme fitted this density.
>
> I modelled two PEG molecules with half occupancy. They fitted very
well, but I am not convinced that this is the best explanation.
>
>   Does anyone figure out what this density can be? Attached to this
e-mail I send the maps and a pdb file with the residues surrounding the
ligand density.
>
>
>
> Thanks
>
> Priscila


UKN.pdb
Description: UKN.pdb

Re: [ccp4bb] unknown density

[Priscila Oliveira de Giuseppe]

Hi Flip,

Following your suggestion, I send the link to download the screenshots of the 
ligand density:
http://twitpic.com/2tn35i

Best regards

Priscila




From: Flip Hoedemaeker [fhoe...@googlemail.com] On Behalf Of Flip Hoedemaeker 
[f...@formulatrix.com]
Sent: Friday, October 01, 2010 9:18 AM
To: Priscila Oliveira de Giuseppe
Subject: Re: [ccp4bb] unknown density

Hi Priscilla,

I don't think a lot of people will take the trouble to find out for
themselves which format your map files have... Its better to send screen
shots, better still to put your screenshots on a social media page
(facebook, twitpic, whatever) and email the link.

Flip

On 10/1/2010 13:12, Priscila Oliveira de Giuseppe wrote:
>   Hi, everyone,
>
>   I solved a structure at 1.7 A. After fitting all residues into the model, I 
> found an extra density at the catalytic site. The components of the 
> crystallization condition did not explain it (crystallization condition: 40% 
> PEG200, 0.1 M Tris-HCl, protein buffer: PBS)
>
> Neither substrates nor products of this enzyme fitted this density.
>
> I modelled two PEG molecules with half occupancy. They fitted very well, but 
> I am not convinced that this is the best explanation.
>
>   Does anyone figure out what this density can be? Attached to this e-mail I 
> send the maps and a pdb file with the residues surrounding the ligand density.
>
>
>
> Thanks
>
> Priscila

Re: [ccp4bb] unknown density

[Vellieux Frederic]

Hi,

I did a little bit of "modeling" in your density (starting from a 
nicotinamide ring, the positioned nicotinamide is enclosed). The middle 
part looks suspiciously like a 6 membered ring. Could it be a molecule 
in a half-chair conformation? There is only the blob that is 
perpendicular to the ring that would remain unexplained...


If it is indeed a molecule in a half-chair conformation then you'd need 
to ask a chemist what it could be.


Note that I did not try to distort the nicotinamide ring to a half-chair 
conformation


Blobology is a difficult branch of our trade... I've had a case where I 
had to state in the publication that a sodium (I think it was) ion had 
been introduced in the model to explain a density feature but that it 
wasn't known what this spherical blob of density in fact corresponded to.


Fred.

Priscila Oliveira de Giuseppe wrote:

 Hi, everyone,

 I solved a structure at 1.7 A. After fitting all residues into the model, I 
found an extra density at the catalytic site. The components of the 
crystallization condition did not explain it (crystallization condition: 40% 
PEG200, 0.1 M Tris-HCl, protein buffer: PBS)

Neither substrates nor products of this enzyme fitted this density.

I modelled two PEG molecules with half occupancy. They fitted very well, but I 
am not convinced that this is the best explanation.

 Does anyone figure out what this density can be? Attached to this e-mail I 
send the maps and a pdb file with the residues surrounding the ligand density.



Thanks

Priscila




nicotinamide.pdb
Description: Protein Databank data

Re: [ccp4bb] unknown density

[Mayer, Mark (NIH/NICHD) [E]]

I'd support BR's position. Why the compelling urge to fill every blob with 
atoms? Density should only be modeled when its interpretable. One can comment 
about the blob in a paper, and perhaps even show maps for the region, 
indicating that there might be something there if its believed to be a 
functionally important region. One could deposit without building in this site, 
but say that combinations of disorder ions/water etc all fit the density 
equally well.

From: Bostjan Kobe [b.k...@uq.edu.au]
Sent: Wednesday, February 03, 2010 8:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unknown density

I think there is at least one more option here (relevant at least in some rare 
cases):
Identifying what that something is likely to be can be significant and may 
advance your career

Of course it is important to present supporting or otherwise evidence for the 
interpretation. As already discussed, this is difficult in a PDB file, and 
that’s why the accompanying publication can be very important.

Bostjan


On 4/02/10 3:54 AM, "Bernhard Rupp"  wrote:

General remark – if I may
Putting nothing in:
no significant effect on model and life in general
Putting ‘something’ potentially wrong and misleading in:
could be detrimental to your career

BR


From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katja 
Schleider
Sent: Wednesday, February 03, 2010 9:18 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unknown density


  Dear all,

 I found some fairly substantial density in the active site of my protein 
structure. But I don´t know what it should be. My crystallisation condition 
consists of lithium sulfate, citrate-phosphate and peg1000. The whole lot 
doesn't make a dashed bit of sense!  Any suggestions to fill this density with 
something?
 Picture is attached.

 Thanks a lot,

 Katja

__
Do You Yahoo!?
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---
Bostjan Kobe
ARC Federation Fellow
Professor of Structural Biology
School of Chemistry and Molecular Biosciences
  and Institute for Molecular Bioscience (Division of Chemistry and Structural 
Biology)
Cooper Road
University of Queensland
Brisbane, Queensland 4072
Australia
Phone: +61 7 3365 2132
Fax: +61 7 3365 4699
E-mail: b.k...@uq.edu.au
URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html
Office: Building 76 Room 329
Notice: If you receive this e-mail by mistake, please notify me, and do not 
make any use of its contents. I do not waive any privilege, confidentiality or 
copyright associated with it. Unless stated otherwise, this e-mail represents 
only the views of the Sender and not the views of The University of Queensland.

Re: [ccp4bb] unknown density

[Bostjan Kobe]

I think there is at least one more option here (relevant at least in some
rare cases):
Identifying what that something is likely to be can be significant and may
advance your career

Of course it is important to present supporting or otherwise evidence for
the interpretation. As already discussed, this is difficult in a PDB file,
and that¹s why the accompanying publication can be very important.

Bostjan


On 4/02/10 3:54 AM, "Bernhard Rupp"  wrote:

> General remark ­ if I may
> Putting nothing in:
> no significant effect on model and life in general
> Putting Œsomething¹ potentially wrong and misleading in:
> could be detrimental to your career
>  
> BR
>  
> 
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katja
> Schleider
> Sent: Wednesday, February 03, 2010 9:18 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] unknown density
>  
>  
>   Dear all,
>  
>  I found some fairly substantial density in the active site of my protein
> structure. But I don´t know what it should be. My crystallisation condition
> consists of lithium sulfate, citrate-phosphate and peg1000. The whole lot
> doesn't make a dashed bit of sense!  Any suggestions to fill this density with
> something?
>  Picture is attached.
>  
>  Thanks a lot,
>  
>  Katja  
> 
> __
> Do You Yahoo!?
> Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden Schutz gegen
> Massenmails. 
> http://mail.yahoo.com
> 

---
Bostjan Kobe
ARC Federation Fellow
Professor of Structural Biology
School of Chemistry and Molecular Biosciences
  and Institute for Molecular Bioscience (Division of Chemistry and
Structural Biology)
Cooper Road
University of Queensland
Brisbane, Queensland 4072
Australia
Phone: +61 7 3365 2132
Fax: +61 7 3365 4699
E-mail: b.k...@uq.edu.au
URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html
Office: Building 76 Room 329
Notice: If you receive this e-mail by mistake, please notify me, and do not
make any use of its contents. I do not waive any privilege, confidentiality
or copyright associated with it. Unless stated otherwise, this e-mail
represents only the views of the Sender and not the views of The University
of Queensland.

Re: [ccp4bb] unknown density

[Dale Tronrud]

Mark J. van Raaij wrote:
> as it appears to bear near the guanidine group of an arginine, I would
> suggest it is a partially occupied and disordered sulphate and/or
> phosphate.
> whether this is practically modellable is another matter, a sulphate at
> occ. 0.5 is an option, or putting a water with a remark in the pdb that
> this water is really modelling partially occupied and disordered
> sulphate and/or phosphate.

   I've found that remarks in PDB files are quite useless.  If you label
an atom as a water molecule, every program that reads that file will make
the assumption that you believed a water molecule was there.  Even most
humans will fail to dig into the header of the PDB file to see if you
had some specific comment about that particular atom.

   Since the PDB file is your interpretation of the electron density,
and the electron density will be available to anyone who wants it, I
don't see the point of marking a spot with a dummy atom when you don't
have an interpretation.

   As for developing an interpretation: I've had good success in
cleaning up such maps using Buster.  You don't need to add dummy
waters in the refinement to remove "vacuum bias", just leave it
alone while you fix everything you can figure out.

Dale Tronrud

> Mark
> 
> Quoting Katja Schleider :
> 
>> Dear all,
>>
>> I found some fairly substantial density in the active site of my 
>> protein structure. But I don´t know what it should be. My 
>> crystallisation condition consists of lithium sulfate, 
>> citrate-phosphate and peg1000. The whole lot doesn't make a dashed 
>> bit of sense!  Any suggestions to fill this density with something?
>> Picture is attached.
>>
>> Thanks a lot,
>>
>> Katja
>>
>>
>> __
>> Do You Yahoo!?
>> Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden 
>> Schutz gegen Massenmails.
>> http://mail.yahoo.com

Re: [ccp4bb] unknown density

[Roger Rowlett]

I agree with many others: this is quite possibly sulfate or phosphate.
If collected at 1.54A the anomalous map should help identify the
presence of sulfur. Sulfate or phosphate may be present at lower than
full occupancy. I would refine everything else you reasonably know in
the model (including water and any other anions, ligands, etc.) and see
if the blob of density sharpens up. Usually a sulfate looks like a
sphere of density, and a sulfate + water may be somewhat elongated. If
the difference map without modeled anion looks reasonable, try a
sulfate of phosphate at 1.0 or 0.5 occupancy to see if the 2Fo-Fc and
Fo-Fc maps still look reasonable. There is plenty of precedent for
sulfate/phosphate binding near Glu and Arg residues. If you have
negative Fo-Fc density when using sulfate at 0.5 occupancy, you may
have no other reasonable choice than to model with water (or nothing at
all), with the acknowledgment that this may not be completely correct,
either.

Cheers.


On 2/3/2010 12:17 PM, Katja Schleider wrote:

  

  
Dear all,

I found some fairly substantial density in the active site of my
protein structure. But I don´t know what it should be. My
crystallisation condition consists of lithium sulfate,
citrate-phosphate and peg1000. The whole lot doesn't make a dashed bit
of sense!  Any suggestions to fill this density with something?
Picture is attached.

Thanks a lot,

Katja

  

  
  
__
Do You Yahoo!?
Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden
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-- 

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

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

Re: [ccp4bb] unknown density

[Bernhard Rupp]

General remark – if I may

Putting nothing in: 

no significant effect on model and life in general

Putting ‘something’ potentially wrong and misleading in:

could be detrimental to your career

 

BR

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katja
Schleider
Sent: Wednesday, February 03, 2010 9:18 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unknown density

 


Dear all,

I found some fairly substantial density in the active site of my protein
structure. But I don´t know what it should be. My crystallisation condition
consists of lithium sulfate, citrate-phosphate and peg1000. The whole lot
doesn't make a dashed bit of sense!  Any suggestions to fill this density
with something?
Picture is attached.

Thanks a lot,

Katja


__
Do You Yahoo!?
Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden Schutz
gegen Massenmails. 
http://mail.yahoo.com 

Re: [ccp4bb] unknown density

[David J. Schuller]

On Wed, 2010-02-03 at 18:26 +0100, Mark J. van Raaij wrote:
> as it appears to bear near the guanidine group of an arginine, I would  
> suggest it is a partially occupied and disordered sulphate and/or  
> phosphate.

If it's a sulphate, a look at the anomalous map should show something.

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

Re: [ccp4bb] unknown density

[Vellieux Frederic]

Hi Katja,

What I personally do in such a situation is to start to fill the density 
by water oxygen atoms, refine a bit (the position plus the isotropic 
temperature factors) and then compute a new map. Usually it has cleared 
up somewhat, thus allowing you to find out what it really is. This is 
what happened to me recently with a whole stretch of polypeptide that 
was in a different position in the MR search model and in the structure. 
After introducing water molecules, refinement and a new map, bingo: the 
whole stretch could be modelled.


And PEG tails can be disordered too.

Fred.

Katja Schleider wrote:

Dear all,

I found some fairly substantial density in the active site of my 
protein structure. But I don´t know what it should be. My 
crystallisation condition consists of lithium sulfate, 
citrate-phosphate and peg1000. The whole lot doesn't make a dashed bit 
of sense!  Any suggestions to fill this density with something?

Picture is attached.

Thanks a lot,

Katja

Re: [ccp4bb] unknown density

[Mark J. van Raaij]

as it appears to bear near the guanidine group of an arginine, I would  
suggest it is a partially occupied and disordered sulphate and/or  
phosphate.
whether this is practically modellable is another matter, a sulphate  
at occ. 0.5 is an option, or putting a water with a remark in the pdb  
that this water is really modelling partially occupied and disordered  
sulphate and/or phosphate.

Mark

Quoting Katja Schleider :


Dear all,

I found some fairly substantial density in the active site of my   
protein structure. But I don´t know what it should be. My   
crystallisation condition consists of lithium sulfate,   
citrate-phosphate and peg1000. The whole lot doesn't make a dashed   
bit of sense!  Any suggestions to fill this density with something?

Picture is attached.

Thanks a lot,

Katja


__
Do You Yahoo!?
Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden   
Schutz gegen Massenmails.

http://mail.yahoo.com

Re: [ccp4bb] Unknown density

[Mark J. van Raaij]

How about a 3-fold disordered metal ion?

Mark



On 19 Aug 2009, at 17:07, Sampath Natarajan wrote:


Dear All,

Currently I'm modeling one structure with 2.6A data. I could find an  
unknown density which I located in the interface between the three  
similar subunits. This density shows very strong peak, which appears  
until 10 sigma level in the difference map. It seems to be a metal  
ion. But, already I could find two Zn ions in the active site and  
refined well. The aspartic acid of each subunit has very close  
interaction with this density. You can see the pictures, which I  
have attached with this mail. The crystallization condition is 0.05M  
CaCl2 0.1M Bis-Tris pH 6.5, 30% (v/v) PEG monomethyl ether 550.  
Suggestion about this unknown density will be more helpful to find  
the molecule.


thanks a lot,

Regards,

Sampath

Re: [ccp4bb] Unknown density

[Roger Rowlett]

The density looks an awful lot like a polyatomic anion (sulfate, 
phosphate, etc.). It's hard to tell from the fixed images, but it 
appears to be tetrahedral. Is there any chance a polyatomic anion could 
be in the crystallization solution? Perhaps from the protein stock solution?



Sampath Natarajan wrote:

Dear All,
 
Currently I'm modeling one structure with 2.6A data. I could find an 
unknown density which I located in the interface between the three 
similar subunits. This density shows very strong peak, which appears 
until 10 sigma level in the difference map. It seems to be a metal 
ion. But, already I could find two Zn ions in the active site and 
refined well. The aspartic acid of each subunit has very close 
interaction with this density. You can see the pictures, which I have 
attached with this mail. The crystallization condition is 0.05M CaCl_2 
0.1M Bis-Tris pH 6.5, 30% (/v/v/) PEG monomethyl ether 550. Suggestion 
about this unknown density will be more helpful to find the molecule.
 
thanks a lot,
 
Regards,
 
Sampath

--

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: rrowl...@mail.colgate.edu

Re: [ccp4bb] unknown density

[Savvas Savvides]

Dear Bert,
Your density reminded me a little bit of the paper by Pearson et al.
Biochemistry 39:8575-8584(2000) which describes the water structure in the
catalytic center of urease. I just went back to it and it looks like Figure
3 in that paper may hint some similarities to your case, in that you may
indeed have a mixture of water models.

You did not say if your data comes from cryo-cooled crystals that were
manipulated in similar or different ways, or whether one of the two is a
room temperature data set. One of the largely overlooked issues related to
crystal cryo-cooling concerns the structure attained by the solvent.
Structures of urease at 100K and 298K (pdb entries 1ejw and 1ejx) show
marked differences in the water structure in the active site and elsewhere.
This phenomenon has been observed in other structures as well, and not just
with respect to water structure but also for loops and side-chains. Even two
cryo-cooled crystals that are manipulated in similar ways may exhibit
differences.
Bart Hazes addressed some of these issues in an interesting paper in Acta
Cryst. (2005). D61, 80-87.

Nonetheless, to maximize the chances for the best interpretation possible I
would first improve phase accuracy by completing the solvent model as much
as possible (except from the contested density), and then use
post-refinement residual Fo-Fc density to evaluate possible water models
(and other possible interpretations) for the density in question. 

Best wishes
Savvas

-
Savvas Savvides
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
http://www.lprobe.ugent.be/xray.html



-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Van
Den Berg, Bert
Sent: Tuesday, July 07, 2009 10:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unknown density

Dear all,


I have attached a jpeg of some (2fo-fc) density in the active site of a
protein I'm refining (1.9 A data, Rfree 24%, still need to pick waters etc;
oxygen atom placed in the approximate center for reference). It looks like
there are 3 non-hydrogen atoms sandwiched between an Asp, His and Arg. The
molecule seems hydrogen bonded to all these three residues.
Has anybody any idea what this can be? In a map of another crystal of the
same crystal form at lower res (2.3 A), there is a clear, single water
molecule between the Asp and His. Are they waters at partial occupancies
(there is not enough room for 3 separate waters)? The only other thing I can
come up with is formate, but that wasn't used in the crystallization.

Any hints appreciated!

Thanks, Bert


Bert van den Berg
UMass Medical School
Worcester, MA 01605







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Re: [ccp4bb] unknown density

[Jayashankar]

Hi,

Did you check for the 2N,2O coordination. Does  your protein  has  copper
ion  as  cofactor,  whats the pH of the buffer.
And if pH below 6 we can partially eliminate the speculation of copper ion.

S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.


On Tue, Jul 7, 2009 at 10:13 PM, Van Den Berg, Bert <
lambertus.vandenb...@umassmed.edu> wrote:

> Dear all,
>
>
> I have attached a jpeg of some (2fo-fc) density in the active site of a
> protein I'm refining (1.9 A data, Rfree 24%, still need to pick waters
> etc; oxygen atom placed in the approximate center for reference). It
> looks like there are 3 non-hydrogen atoms sandwiched between an Asp, His
> and Arg. The molecule seems hydrogen bonded to all these three residues.
> Has anybody any idea what this can be? In a map of another crystal of
> the same crystal form at lower res (2.3 A), there is a clear, single
> water molecule between the Asp and His. Are they waters at partial
> occupancies (there is not enough room for 3 separate waters)? The only
> other thing I can come up with is formate, but that wasn't used in the
> crystallization.
>
> Any hints appreciated!
>
> Thanks, Bert
>
>
> Bert van den Berg
> UMass Medical School
> Worcester, MA 01605
>
>
>

Re: [ccp4bb] unknown density

[artem]

Theoretically, citric acid can decompose into beta-ketoglutaric acid, and
the latter may decarboxylate into malonic acid...

Not sure if this happens in aqueous solutions, but if your citrate had
malonate contamination and your enzyme prefers malonate to anything else -
that's where it may have come from.

Pure speculation, of course!

Artem

> Dear all,
> i am working on a structure of M.tb, solved at 2.18 A. In my structure
> there
> is a density near to the active site and we modelled it as a malonate (C3
> O4
> H2) molecule acorrding to the density, which was not used in any of our
> buffer except we used 10 mM DTT, 1.6 M Ammonium sulphate, 100 mM citric
> acid.
>
> Is their any possible way to check for unknown densities or can any one
> suggest me for any possible compound which can be similar to the malonate
> or
> can be formed from above mentioned solutions (byproduct).
>
> Thanks
> Rajan
>
> --
> 
> Current Address:
>
> Rajan Vyas
> Research Scholar
> Deptt. of Biotechnology
> Panjab University Chandigarh, India
> 160014Mob. +919417374197
> Fax: +91-172-2625254
> 
>

[ccp4bb] unknown density

[Rajan]

Dear all,
i am working on a structure of M.tb, solved at 2.18 A. In my structure there
is a density near to the active site and we modelled it as a malonate (C3 O4
H2) molecule acorrding to the density, which was not used in any of our
buffer except we used 10 mM DTT, 1.6 M Ammonium sulphate, 100 mM citric
acid.

Is their any possible way to check for unknown densities or can any one
suggest me for any possible compound which can be similar to the malonate or
can be formed from above mentioned solutions (byproduct).

Thanks
Rajan

-- 

Current Address:

Rajan Vyas
Research Scholar
Deptt. of Biotechnology
Panjab University Chandigarh, India
160014Mob. +919417374197
Fax: +91-172-2625254

Re: [ccp4bb] unknown density for a small molecule

[conancao]

It could be purine or pyrimidine ring stacked between the Phe an Tyr. check pdb 
3etr or 3eub of xanthine oxidase binding purine substrates between two Phe at 
the active site.
 
Hongnan Cao
UCR



Date: Tue, 10 Feb 2009 00:23:54 -0500From: lvmengx...@gmail.comsubject: 
[ccp4bb] unknown density for a small moleculeTo: ccp...@jiscmail.ac.ukdear 
All,When I was refining my structure, I found some unmodeled blobs, shown as 
attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor 0.18, 
refined to 1.9 angstrom ). The protein was expressed in E.coli and purified by 
nickel column and gel filtration, both in tris buffer. The crystallization 
condition has 2 M ammonium sulfate and 0.1 M sodium acetate. The lower part 
looks like a sulfate group, which is held by one Arg, one his, one lys and one 
asn. The latter three residues are from another asymmetric unit. The other end 
of the small molecule is stunk by the rings of Tyr and Phe. It also interacts 
with the OH group of another tyr and one water molecule. Is there a program can 
build small molecule models according to the densities? Or could anyone tell 
what it might be from the density? Thanks a lot! Any suggestions will be 
appreciated!Mengxiao
_
上Windows Live 中国首页，下载最新版 MSN！
http://im.live.cn/

Re: [ccp4bb] unknown density for a small molecule

[Pamela Focia]

Hi Mengxiao,

 The density sure reminds me of a nucleotide. 
Note the pi-pi stacking of the planar part 
between Tyr and Phe, and in the third stereo 
picture you attached, projections at what could 
be the 2 and 6 positions remind me of Guanine... 
plus there are Arg, Lys, etc. pointing toward 
where there could be a phosphate group.  Even if 
the density is not quite 'big enough' to fit it, 
I'd give it a try.


 In addition to components of your 
crystallization conditions, you might consider 
that something was either co-purified with your 
protein, or is contaminating your reagents.


Good luck!

-pamela

At 11:37 AM -0500 2/10/09, mengxiao lv wrote:

Thank you so much for the replies!

I should clarify in my mail that the two images 
are actually from one blob, just viewed from two 
sides. Sorry for the confusing. I enclosed some 
stereo densities for the same blob in this mail.


As Artem said, it is very similar to isopentenyl 
phosphate. The upper part is planar.  I agree 
with Marc and Eleanor's suggestion that  the 
molecule is from the crystallization solution. I 
have tried to model one acetate into the 
density, and it fits well. And the lower part 
can also model into a sulfate. However, I will 
see the positive density for the missed link 
between acetate and sulfate. THe pH for 
crystallization is 4.6. Will acetate form some 
compound with sulfate?


To Jan and Rajesh, I didn't use MES buffer in 
the whole procedures. Also, the ring in MES 
might be too large for the upper part.


I also get suggestions to model a molecule with 
a sugar ring, from Kornelius, Poul, Daniele. The 
images I attached are not clear, and I don't 
think the density is large enough for a five or 
six member ring.


Thanks again for the help! Hope the new images 
will make things clear. I appreciate any 
suggestions!


Mengxiao

On Tue, Feb 10, 2009 at 2:08 AM, Kontopidis 
George 
<<mailto:gkontopi...@vet.uth.gr>gkontopi...@vet.uth.gr> 
wrote:


Dear Mengxiao,

 From my experience I would say that the two e. 
densities (blob1 and blob2) are present the same 
molecule.


What that might be is more difficult to answer.

Based in the concentration you gave us and the 
electron density volume I would say that is more 
likely to be ammonium sulphate (NH4 and SO4).


As you say the lower part looks like SO4 and 
make sense to interact with Arg, Lys and Asn and 
the other end looks like NH4.


But those two group there and give it one round 
of refinement. Check the B factors at the end


Do they make sense (directions of H-bonds, 
distance between NH4 and SO4)? Is the Bfactor 
similar for NH4 and SO4


They should not have a difference greater than 50%.

You could try also Na in stead of NH4



George




From: CCP4 bulletin board 
[mailto:<mailto:CCP4BB@JISCMAIL.AC.UK>ccp...@jiscmail.ac.uk] 
On Behalf Of mengxiao lv

Sent: Tuesday, February 10, 2009 7:24 AM

To: <mailto:CCP4BB@JISCMAIL.AC.UK>CCP4BB@JISCMAIL.AC.UK

Subject: [ccp4bb] unknown density for a small molecule



Dear All,


When I was refining my structure, I found some 
unmodeled blobs, shown as attached images 
(contoured at 3 sigma for Fo-Fc, Rfree 0.21 and 
Rfactor 0.18, refined to 1.9 angstrom ).


The protein was expressed in E.coli and purified 
by nickel column and gel filtration, both in 
tris buffer. The crystallization condition has 2 
M ammonium sulfate and 0.1 M sodium acetate.


The lower part looks like a sulfate group, which 
is held by one Arg, one his, one lys and one 
asn. The latter three residues are from another 
asymmetric unit.


The other end of the small molecule is stunk by 
the rings of Tyr and Phe. It also interacts with 
the OH group of another tyr and one water 
molecule.


Is there a program can build small molecule 
models according to the densities? Or could 
anyone tell what it might be from the density?


Thanks a lot! Any suggestions will be appreciated!

Mengxiao



Content-Type: image/jpeg; name="stereo1.jpg"
Content-Disposition: attachment; filename="stereo1.jpg"
X-Attachment-Id: f_fr0sc9ly0

Attachment converted: gerolsteiner:stereo1.jpg (JPEG/«IC») (001FB8B3)
Content-Type: image/jpeg; name="stereo2.jpg"
Content-Disposition: attachment; filename="stereo2.jpg"
X-Attachment-Id: f_fr0scdg81

Attachment converted: gerolsteiner:stereo2.jpg (JPEG/«IC») (001FB8B4)
Content-Type: image/jpeg; name="stereo3.jpg"
Content-Disposition: attachment; filename="stereo3.jpg"
X-Attachment-Id: f_fr0sci0r2

Attachment converted: gerolsteiner:stereo3.jpg (JPEG/«IC») (001FB8B5)



--


Pamela J. Focia, Ph.D.
Research Assistant Professor

Structural Biology Facility Manager
Robert H. Lurie Comprehensive Cancer Center
in the Departments of:
Molecular Pharmacology and Biological Chemistry, Feinberg  School of Medicine,
and Biochemistry, Molecular Biology & Cell Biology,
Northwestern University
303 E. Chicago Ave., S-215,  Chicago 60611
(312)503-0848   fax (312)503-5349
fo...@northwestern.edu

Re: [ccp4bb] unknown density for a small molecule

[Jayashankar]

Hi,

If you could send in stereo mode of the density you want to get suggestion,
it will be convenient for the well trained eyes.


S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.


On Tue, Feb 10, 2009 at 3:16 PM,  wrote:

> Hi,
>
> It could be all sorts of things, but the one that for some reason is stuck
> in my mind is isopentenyl phosphate (phosphate, not pyrophosphate!). Of
> course w/o seeing the density in 3D this is just a guess.
>
> Artem
>
> > Dear All,
> >
> > When I was refining my structure, I found some unmodeled blobs, shown as
> > attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor
> > 0.18, refined to 1.9 angstrom ).
> >
> > The protein was expressed in E.coli and purified by nickel column and gel
> > filtration, both in tris buffer. The crystallization condition has 2 M
> > ammonium sulfate and 0.1 M sodium acetate.
> >
> > The lower part looks like a sulfate group, which is held by one Arg, one
> > his, one lys and one asn. The latter three residues are from another
> > asymmetric unit.
> >
> > The other end of the small molecule is stunk by the rings of Tyr and Phe.
> > It
> > also interacts with the OH group of another tyr and one water molecule.
> >
> > Is there a program can build small molecule models according to the
> > densities? Or could anyone tell what it might be from the density?
> >
> > Thanks a lot! Any suggestions will be appreciated!
> >
> > Mengxiao
> >
>

Re: [ccp4bb] unknown density for a small molecule

[artem]

Hi,

It could be all sorts of things, but the one that for some reason is stuck
in my mind is isopentenyl phosphate (phosphate, not pyrophosphate!). Of
course w/o seeing the density in 3D this is just a guess.

Artem

> Dear All,
>
> When I was refining my structure, I found some unmodeled blobs, shown as
> attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor
> 0.18, refined to 1.9 angstrom ).
>
> The protein was expressed in E.coli and purified by nickel column and gel
> filtration, both in tris buffer. The crystallization condition has 2 M
> ammonium sulfate and 0.1 M sodium acetate.
>
> The lower part looks like a sulfate group, which is held by one Arg, one
> his, one lys and one asn. The latter three residues are from another
> asymmetric unit.
>
> The other end of the small molecule is stunk by the rings of Tyr and Phe.
> It
> also interacts with the OH group of another tyr and one water molecule.
>
> Is there a program can build small molecule models according to the
> densities? Or could anyone tell what it might be from the density?
>
> Thanks a lot! Any suggestions will be appreciated!
>
> Mengxiao
>

Re: [ccp4bb] unknown density for a small molecule

[Herman . Schreuder]

Dear Mengxia,
the lower part could well be a sulfate ion. For the upper part, it is
difficult to guess from a static picture. Unfortunately, at the
resolution you have there are no programs which can build an unknown
small molecule from scratch according to the density. You have to guess,
fit it and refine and look if it's ok. I would also check whether a
natural ligand of the protein (substrate, product, effector molecule)
would fit. They sometimes remain attached during the whole purification
procedure. A trick to get an idea where heavy atoms like sulfur are
located is to scroll the contour level up. If the density for e.g. the
center disappears very late, when most of the other density is no longer
visible, it must be a sulfur or other heavy atom.
 
Good luck!
Herman




From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On
Behalf Of mengxiao lv
Sent: Tuesday, February 10, 2009 6:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unknown density for a small molecule


Dear All,

When I was refining my structure, I found some unmodeled blobs,
shown as attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and
Rfactor 0.18, refined to 1.9 angstrom ). 

The protein was expressed in E.coli and purified by nickel
column and gel filtration, both in tris buffer. The crystallization
condition has 2 M ammonium sulfate and 0.1 M sodium acetate. 

The lower part looks like a sulfate group, which is held by one
Arg, one his, one lys and one asn. The latter three residues are from
another asymmetric unit. 

The other end of the small molecule is stunk by the rings of Tyr
and Phe. It also interacts with the OH group of another tyr and one
water molecule. 

Is there a program can build small molecule models according to
the densities? Or could anyone tell what it might be from the density? 

Thanks a lot! Any suggestions will be appreciated!

Mengxiao

Re: [ccp4bb] unknown density for a small molecule

[Kontopidis George]

Dear Mengxiao,

 From my experience I would say that the two e. densities (blob1 and blob2)
are present the same molecule.

What that might be is more difficult to answer.

Based in the concentration you gave us and the electron density volume I
would say that is more likely to be ammonium sulphate (NH4 and SO4).

As you say the lower part looks like SO4 and make sense to interact with
Arg, Lys and Asn and the other end looks like NH4.

But those two group there and give it one round of refinement. Check the B
factors at the end 

Do they make sense (directions of H-bonds, distance between NH4 and SO4)? Is
the Bfactor similar for NH4 and SO4 

They should not have a difference greater than 50%. 

You could try also Na in stead of NH4

 

George 

 

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
mengxiao lv
Sent: Tuesday, February 10, 2009 7:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unknown density for a small molecule

 

Dear All,

When I was refining my structure, I found some unmodeled blobs, shown as
attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor
0.18, refined to 1.9 angstrom ). 

The protein was expressed in E.coli and purified by nickel column and gel
filtration, both in tris buffer. The crystallization condition has 2 M
ammonium sulfate and 0.1 M sodium acetate. 

The lower part looks like a sulfate group, which is held by one Arg, one
his, one lys and one asn. The latter three residues are from another
asymmetric unit. 

The other end of the small molecule is stunk by the rings of Tyr and Phe. It
also interacts with the OH group of another tyr and one water molecule. 

Is there a program can build small molecule models according to the
densities? Or could anyone tell what it might be from the density? 

Thanks a lot! Any suggestions will be appreciated!

Mengxiao

Re: [ccp4bb] Unknown density

[David J. Schuller]

Remember that since it is on a special position (2-fold?), you may not
be looking at a single object, but an overlay from two or more
positions.

The anomalous map is very useful; the mystery molecule must have an
anomalous scatterer. It would be even more helpful if you had mentioned
the wavelength of the data. Anyway, the only thing in the list of mother
liquor ingredients you provided that scatters anomalously is calcium,
which fits in with the Asp ligands. The additional ligands for the
calcium might be water or acetate.

So: my guess would be two positions of Ca2+, one right on the axis and
one off the axis (and doubled by the symmetry). You can try placing Ca
atoms at these positions and see what the occupancies and thermal
factors refine to. For example, start with an occupancy of 0.5 at each
position and see if the thermal factors refine to a value comparable to
nearby protein atoms.

Cheers,

-  
===
With the single exception of Cornell, there is not a college in the
United States where truth has ever been a welcome guest - R.G. Ingersoll
===
  David J. Schuller
  modern man in a post-modern world
  MacCHESS, Cornell University
  [EMAIL PROTECTED]



On Mon, 2008-02-25 at 11:54 +0100, Franck Coste wrote:
> As Edward MITCHELL suggested, I made a phased anomalous difference map
> and could clearly see the S atom of my sulfide bridges and 1 Ca atom as
> I espected. Concerning the unknown density, the shape is the same as in
> the fo-fc map  Updated images at :
> http://unknowndensity.blog4ever.com/blog/photo-183078.html
> In yellow is the phased anomalous difference map at 4 sigma.
> Franck.

[ccp4bb] Unknown density

[Franck Coste]

As Edward MITCHELL suggested, I made a phased anomalous difference map
and could clearly see the S atom of my sulfide bridges and 1 Ca atom as
I espected. Concerning the unknown density, the shape is the same as in
the fo-fc map  Updated images at :
http://unknowndensity.blog4ever.com/blog/photo-183078.html
In yellow is the phased anomalous difference map at 4 sigma.
Franck.

[ccp4bb] Unknown density

[Franck Coste]

Dear all,
in my maps (green is fo-fc at 4sigma) I have density that I cannot (I
think) attribute to anything from the crystallization conditions (MPD,
Na-acetate, CaCl2) or the protein storage buffer (Hepes, NaCl, NaN3). 2
images (90 degres) can be seen at :
http://unknowndensity.blog4ever.com/blog/photo-183078.html
The middle of this density (pink cube) is on a special position and is
surrounded by charged residues from 2 symetrical molecules. 
Any ideas will be welcome.
Thanks in advance,
Franck.  

Re: [ccp4bb] unknown density in structure

[Artem Evdokimov]

Oxalate. Sometimes forms when PEGs get old and begin to oxidize. That's some
of the stuff that is responsible for PEG's becoming progressively more
acidic as they age.

No idea if you have PEG in solution or not.

Alternative - CH3-C(=O)-COOH
Disordered dimethyl formamide
Dimethyl acetamide (CH3)2-N-C(=O)-CH3

And so forth.

Artem



-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Wu,
Mousheng
Sent: Thursday, July 26, 2007 6:35 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unknown density in structure

There are four molecules in the asymmetric unit. And this density is
present in every molecules.

-Original Message-
From: ANA MARIA MISIC [mailto:[EMAIL PROTECTED] 
Sent: Thursday, July 26, 2007 5:24 PM
To: Wu, Mousheng 
Subject: Re: [ccp4bb] unknown density in structure

Hi Mousheng - Did you check to see if that falls on a special position?
That can lead to a buildup of electron density, and the symmetrical
shape is suspect.

good luck

Ana M. Misic
Research Assistant
Department of Biomolecular Chemistry
Department of Bacteriology
University of Wisconsin - Madison
420 Henry Mall, Rm 230
Madison, WI 53706

Lab Phone: (608)265-9282
Fax: (608)262-9865

- Original Message -
From: "Wu, Mousheng" <[EMAIL PROTECTED]>
Date: Thursday, July 26, 2007 4:21 pm
Subject: [ccp4bb] unknown density in structure
To: CCP4BB@JISCMAIL.AC.UK


> Hi, everyone,
> 
>  
> 
> I am recently solving a structure at 1.8A. After I fitted all residues
> into the model, I found that there was an extra density in a pocket
> consisted of E, N, R and other hydrophobic residues. The density is
> strong and connected like a butterfly.  I checked the crystallization
> condition. I can not figure out what it is. It seems that it is not
from
> the crystallization components.  
> 
>  
> 
> Does anyone know how to figure out what the density is? Is there any
> program or database I can use to predict the potential ligand?
> 
> Thanks.
> 
>  
> 
> Mousheng 
> 

Re: [ccp4bb] unknown density in structure

[Wu, Mousheng]

There are four molecules in the asymmetric unit. And this density is
present in every molecules.

-Original Message-
From: ANA MARIA MISIC [mailto:[EMAIL PROTECTED] 
Sent: Thursday, July 26, 2007 5:24 PM
To: Wu, Mousheng 
Subject: Re: [ccp4bb] unknown density in structure

Hi Mousheng - Did you check to see if that falls on a special position?
That can lead to a buildup of electron density, and the symmetrical
shape is suspect.

good luck

Ana M. Misic
Research Assistant
Department of Biomolecular Chemistry
Department of Bacteriology
University of Wisconsin - Madison
420 Henry Mall, Rm 230
Madison, WI 53706

Lab Phone: (608)265-9282
Fax: (608)262-9865

- Original Message -
From: "Wu, Mousheng" <[EMAIL PROTECTED]>
Date: Thursday, July 26, 2007 4:21 pm
Subject: [ccp4bb] unknown density in structure
To: CCP4BB@JISCMAIL.AC.UK


> Hi, everyone,
> 
>  
> 
> I am recently solving a structure at 1.8A. After I fitted all residues
> into the model, I found that there was an extra density in a pocket
> consisted of E, N, R and other hydrophobic residues. The density is
> strong and connected like a butterfly.  I checked the crystallization
> condition. I can not figure out what it is. It seems that it is not
from
> the crystallization components.  
> 
>  
> 
> Does anyone know how to figure out what the density is? Is there any
> program or database I can use to predict the potential ligand?
> 
> Thanks.
> 
>  
> 
> Mousheng 
> 

[ccp4bb] unknown density in structure

[Wu, Mousheng]

Hi, everyone,

 

I am recently solving a structure at 1.8A. After I fitted all residues
into the model, I found that there was an extra density in a pocket
consisted of E, N, R and other hydrophobic residues. The density is
strong and connected like a butterfly.  I checked the crystallization
condition. I can not figure out what it is. It seems that it is not from
the crystallization components.  

 

Does anyone know how to figure out what the density is? Is there any
program or database I can use to predict the potential ligand?

Thanks.

 

Mousheng