JiscMail Logo
Email discussion lists for the UK Education and Research communities

Help for CCP4BB Archives


CCP4BB Archives

CCP4BB Archives


CCP4BB@JISCMAIL.AC.UK


View:

Message:

[

First

|

Previous

|

Next

|

Last

]

By Topic:

[

First

|

Previous

|

Next

|

Last

]

By Author:

[

First

|

Previous

|

Next

|

Last

]

Font:

Proportional Font

LISTSERV Archives

LISTSERV Archives

CCP4BB Home

CCP4BB Home

CCP4BB  August 2018

CCP4BB August 2018

Options

Subscribe or Unsubscribe

Subscribe or Unsubscribe

Log In

Log In

Get Password

Get Password

Subject:

Re: Unidentified large blobs in the electron density

From:

Dale Tronrud <[log in to unmask]>

Reply-To:

Dale Tronrud <[log in to unmask]>

Date:

Sun, 5 Aug 2018 10:35:04 -0700

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (113 lines)

   Positive difference map features indicate a likelihood that atoms
should be placed in that location.  Putting atoms in that density, any
atoms, will lower the R value.  This does not mean that your
interpretation is correct.

   The fact that you, the person who has seen this map most clearly,
can't decide between a PO4 and a Ade means that this density is
ambiguous.  These are two very different shapes!

   A constellation of isolated blobs is about the worst thing to figure
out.  It is quite possible that you have a partially occupied something
with ordered water molecules when that thing is not present.  When
looking for partially occupied things you have to contour the map at a
much lower level, but not take what you see too seriously.  Remember you
are looking for something at, say, 50% occupancy, with 50% occupied
water molecules sitting on top.

   That said, I don't understand you difference maps.  You built a ADN
into your blobs, and that molecule is quite a bit larger than your
blobs, yet you have a huge amount of positive difference density
covering your ADN.  How is this possible?  The goal of refinement is to
make the difference map go to zero at the location of atoms.  There is
something about these maps that you are not telling us.

   When interpreting blobs the first and most important thing to
consider is what is in the crystal.  If it doesn't contain PO4 there is
no reason to test PO4 as a possibility.  If you didn't add ADN then you
are hypothesizing that it was carried all the way through purification
which means that has to bind fairly strongly to survive even at partial
occupancy.  Is this location on your protein a nucleotide binding site?
These things are easily recognized just from the structure of the protein.

   Does the hydrogen bonding and charge-charge interactions of your
model make sense?  It is hard to tell in a flat picture, but I don't see
many hydrogen bonds to your ADN model.  If I compare your PO4 model to
the structure I see in the ADN map, I see that there is a ASP right next
to that blob.  You can't put a PO4 next to an ASP.

   Since the map is confusing you have to use as much information from
it as possible (lower contours) but add in as much information from
other sources as possible.  What's in the crystal?  What is your
cryoprotectant?  Is this part the the protein a known binding motif for
something?  What is the function of this protein and what sorts of
compounds might be expected to bind to it?  Is this an enzyme and is the
spot anywhere near the active site?  Do you know where the active site is?

   Once you build a model you have to test it.  You should be your worst
critic!  As I said, a drop in R value is meaningless.  Does the
chemistry make sense?  Can you explain why that molecule is there?  Does
it have a purpose?  Can you perform an experiment that confirms your
model?  Can you soak more of that compound into the crystal and see an
increase in occupancy?  Can you analyze the crystal by some other means
to detect the compound?  Mass Spec?  If PO4, can you detect the presence
of Phosphorus?  The validation has to be designed based on your model.

   You can get better maps than simple Fo-Fc maps.  I really like the
maps produced by Buster and have great success with getting better views
that way.  It is possible that the "Polder" map from Phenix might help -
I'm not too clear on the difference between it and the Buster map.

   Load as much information into your head as you can, get the best
possible map(s), and start running though the possibilities!  And be
willing to accept that you may never figure it out.  Don't build a model
you don't believe.

   I once spent about twenty years trying to figure out a blob (not full
time!).  I got a nice paper about it in the end.

Dale Tronrud


On 8/5/2018 7:00 AM, Preeti Preeti wrote:
> Dear CCP4 member
> 
> I am  solving a protein structure with Resolution 2.2 Angstrom. I could
> see some blobs (2fo-fc @1sigma) and fo-fc @ 2.5 sigma) and need your
> suggestions on these extra electron densities. In addition to this, in
> one of the large blob I have added the phosphate group.
>  This is a nucelotide binding protein however structure I am showing
> here was of native protein crystal without any nucleotide soaking and
> also no phosphate buffer was used at any time during the purification as
> well as crystallization process.  
> Furthermore in one such blob I tried fitting the adenine moiety though
> it is not fitting exactly in the map, it decreased the Rfree value
> significantly, 
> 
> kindly suggest me what it should be corresponding to?
> 
> Also please let me know if any other structure information required
> regarding this protein data or this blob density
> 
> Thanks a lot in advance
> 
> 
> 
> 
> 
> 
> 
> Preeti
> 
> 
> 
> ------------------------------------------------------------------------
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
> 

########################################################################

To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Top of Message | Previous Page | Permalink

JiscMail Tools


RSS Feeds and Sharing


Advanced Options


Archives

October 2018
September 2018
August 2018
July 2018
June 2018
May 2018
April 2018
March 2018
February 2018
January 2018
December 2017
November 2017
October 2017
September 2017
August 2017
July 2017
June 2017
May 2017
April 2017
March 2017
February 2017
January 2017
December 2016
November 2016
October 2016
September 2016
August 2016
July 2016
June 2016
May 2016
April 2016
March 2016
February 2016
January 2016
December 2015
November 2015
October 2015
September 2015
August 2015
July 2015
June 2015
May 2015
April 2015
March 2015
February 2015
January 2015
December 2014
November 2014
October 2014
September 2014
August 2014
July 2014
June 2014
May 2014
April 2014
March 2014
February 2014
January 2014
December 2013
November 2013
October 2013
September 2013
August 2013
July 2013
June 2013
May 2013
April 2013
March 2013
February 2013
January 2013
December 2012
November 2012
October 2012
September 2012
August 2012
July 2012
June 2012
May 2012
April 2012
March 2012
February 2012
January 2012
December 2011
November 2011
October 2011
September 2011
August 2011
July 2011
June 2011
May 2011
April 2011
March 2011
February 2011
January 2011
December 2010
November 2010
October 2010
September 2010
August 2010
July 2010
June 2010
May 2010
April 2010
March 2010
February 2010
January 2010
December 2009
November 2009
October 2009
September 2009
August 2009
July 2009
June 2009
May 2009
April 2009
March 2009
February 2009
January 2009
December 2008
November 2008
October 2008
September 2008
August 2008
July 2008
June 2008
May 2008
April 2008
March 2008
February 2008
January 2008
December 2007
November 2007
October 2007
September 2007
August 2007
July 2007
June 2007
May 2007
April 2007
March 2007
February 2007
January 2007


JiscMail is a Jisc service.

View our service policies at https://www.jiscmail.ac.uk/policyandsecurity/ and Jisc's privacy policy at https://www.jisc.ac.uk/website/privacy-notice

Secured by F-Secure Anti-Virus CataList Email List Search Powered by the LISTSERV Email List Manager