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  June 2015

CCP4BB June 2015

Options

Subscribe or Unsubscribe

Subscribe or Unsubscribe

Log In

Log In

Get Password

Get Password

Subject:

Should I refine a low-resolution dataset 6 A

From:

Kerstin Keller <[log in to unmask]>

Reply-To:

Kerstin Keller <[log in to unmask]>

Date:

Tue, 9 Jun 2015 13:19:29 +0200

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (54 lines)

Hi Experts,

Last year I collected a dataset at 6 A of 2600 aa multi-domain protein. 

Resolution range 50 - 6 (6.7-6)
Wilson B    370 A^2
Reflection 34120 (12240)
Rmeas 0.2 (4.4)
Rpim 0.06(1.2)
I-sigI  9 (1)
CC1/2   0.9 (0.5)
 
I have the following questions in my mind.

1. Does it make any sense to solve the structure at _this_ resolution? It is not completely novel protein, there are known structures with about 54 % identity to this. The fold is known to be the same. 

2. Doing a molecular replacement with "Phaser" using EM-model gives me a unique solution. And I can see a reasonable electron density map. Tried again with AMORE - The amore-build-output model also gives the same solution. In both cases, the solutions are unique. There are no so-called translational symmetry.

3. If I do a Refmac restrained refinement, though I get R/Rfree in around 30.1/35.5 the stereochemistry is very poor (18 % outliers). I had to enable tight WEIGHT MATRIX (1e-7). Here at this resolution does it make any sense to a restrained refinement?

4. If I do only a rigid body with Refmac, the R-factor/Rfree are at around 41 %, and in many places model does not fit density. When I manually correct these and refine there is basically no change in R-factor/R-free (it even worsens in cases).

>From: Bernhard Rupp (Hofkristallrat a.D.) <[log in to unmask]>>Date: 27 April 2015 at 21:54
>Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!
>To: [log in to unmask]
>What we cannot tell sans supporting density is whether it is a more accurate model, although I have>rarely seen an improvement in geometry giving worse density fit. Usually a mess remains a mess - 
>there is (at this resolution) no free lunch. The key question is again – does the model justify 

5. I did try DEN, reference model restraints, PROSMART(refmac) etc which have no improvement at all - R/Rfree stuck at 41 %. 

6. Since my group is EM-group, I wonder when EM-maps of 6 A are published, why are X-ray data at the same resolution not being published? What happens to these datasets?

7. Can I _just_ do a molecular replacement and just mutate residues (based on a sequence alignment - There are large numbers of deletions and hence sequence registers are different/unknown) and deposit it as a model in the PDB? Should I put the side-chains or it is meaning less at this resolution? Why in the EM-field they are allowed to deposit such coordinates with side-chains?

8. As [log in to unmask] points out 

>Particularly in Molecular Replacement structures, and here particularly in those with multi->segment/domain models, there are almost always parts that fit well and others>that fit poorly -  with simply not enough data at the given resolution to improve the poor parts>sans additional phase information. Bias issues have been discussed and need not be iterated here.
Since my protein also has multiple catalytic domains, some of them better resolved and others terrible. What about bias here at 6 A resolution? Very large problem, indeed.

Apologies for the long email, and any suggestion will be gratefully received.

Many thanks,
Kerstin



---------- Forwarded message ----------
From: Bernhard Rupp (Hofkristallrat a.D.) <[log in to unmask]>
Date: 27 April 2015 at 21:54
Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!
To: [log in to unmask]

I’d be very careful at judging low resolution structures. This is a tricky businessrequiring a lot more info than just the PDB validation report. The 3+ to 4 Aresolution range is a particularly deceptive one: The crystallographer does not have much data given the model parameters (perhaps consulting his figure showingdeterminacy for coordinate refinement might help)http://www.ruppweb.org/Garland/gallery/Ch12/pages/Biomolecular_Crystallography_Fig_12-11.htm At this resolution one has about enough data to keep enthusiasm up but at the same time it isnot quite yet bad enough to throw up the hands and admit that that one is de facto modelling with a few X-ray restraints (i.e. data), requiring correspondingly suitable refinement protocols (and discipline,aka mental restraints in addition to stereochemical restraints). One is easily spoiled by looking exceptional 2A structures of huge complexes, butnature (I do not mean the journal but the same time would not exclude it) is often cruel. Particularly in Molecular Replacement structures, and here particularly in those with multi-segment/domain models, there are almost always parts that fit well and othersthat fit poorly -  with simply not enough data at the given resolution to improve the poor parts sans additional phase information. Bias issues have been discussed and need not be iterated here. Pavel is correct in pointing out that a model with better geometry is also a more plausible model.What we cannot tell sans supporting density is whether it is a more accurate model, although I haverarely seen an improvement in geometry giving worse density fit. Usually a mess remains a mess - there is (at this resolution) no free lunch. The key question is again – does the model justify the specific conclusions drawn from it? If a poor model is better than no model at all, be it, as longas this is recognized and not used as an excuse for careless work. Facile dictu, difficile factu.
 		 	   		  

Top of Message | Previous Page | Permalink

JiscMail Tools


RSS Feeds and Sharing


Advanced Options


Archives

October 2019
September 2019
August 2019
July 2019
June 2019
May 2019
April 2019
March 2019
February 2019
January 2019
December 2018
November 2018
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