Re: [ccp4bb] large R-Rfree difference in "final" structure

[ccp4]

 Hi, Ian Tickle has asked the most pertinent question - there are many
reasons for highish FreeR factors. Only some are due to errors or missing
molecules or other problems. How many "Free" reflections are there? When
the sample is very small (< 500 say) you can get different results from a
different Free R set.

And remember that the numbers you quote are overall values, 
It is worth looking at the plot of R and Free R against resolution. They
should stay reasonably parallel, but if the data becomes very weak at the
resolution limit the the FreeR can shoot up relative to the overall Rvalue.

 
I use COOT validation to search the difference maps for peaks and holes. 
If there is something missing from your model you should see it in that
list..
Eleanor
 
On Wed, 13 Jul 2011 20:37:51 +0100, Matthias Zebisch
 wrote:
> Hi Careina,
> 
>  our lab we once had the problem, that the asymmetric unit contained 8 
> molecules,
> whereas 7 had only been modeled. Somehow the 8th monomer had evaded 
> detection.
> So be careful not to miss density.
> 
> Matthias
> 
> 
> On 7/13/2011 7:54 PM, Robbie Joosten wrote:
>> Hi Careina,
>>
>>
>>
>> Assuming you don't suffer from a very poor data parameter ratio that
>> would lead to such a large R-free/R, you need to improve your
refinement.
>> If you have NCS you should use local NCS restraints. You could also try
>> jelly-body restraints, although they may not work at your resolution.
>>
>>
>>
>> Cheers,
>>
>> Robbie
>>
>> 
>>> Date: Wed, 13 Jul 2011 08:38:38 -0700
>>> From: careinaedgo...@yahoo.com
>>> Subject: [ccp4bb] large R-Rfree difference in "final" structure
>>> To: CCP4BB@JISCMAIL.AC.UK
>>>
>>> Dear ccp4 bulletin board
>>>
>>> I just have a slight concern regarding my Rwork Rfree difference. I
>>> have a structure that I have solved. I am reasonably content that it
is
>>> complete because it has refined well, it no longer has bad geometries
>>> and contacts and all the rotamers, ramachandra, bond lengths etc are
>>> good. It gives favourable scores on molprobity and procheck. My only
>>> concern is the R factor difference. The resolution of the structure is
>>> 2.3A. The R factor is 0.24 after refinement but the Rfree is 0.33
which
>>> seems to me to be rather high. Should I be concerned?
>>>
>>> During refinement Rfree only drops from about 0.36 to 0.33 while the R
>>> factor drops from 0.31 to 0.24.. I have removed automatic weighting in
>>> refmac in order to constrain my bond lengths and angles during a
couple
>>> of rounds of refinement. This did not have any effect on the R
factors,
>>> however. I am fairly content that the space group I have chosen is
>>> correct so I am not sure what else could cause the big difference in R
>>> factors? There is no twinning.
>>>
>>> Can I be satisfied that my structure is correct despite the high R
free
>>> or should I be doing other checks/ trying other things before I can
>>> submit this structure?
>>>
>>> Thank you for any help
>>> Careina

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

[ccp4]

I would run DM first to extend the experimental phases to the limit of
your resolution..
Then if you run REFMAC to refine your MR model, using the experimental
phases as part of the input the output phases from REFMAC will include
contributions from both the MR and the DM/experimental phasing.  This will
be weighted according to the FOMs for the refined model R factors and the
experimental ABCDs
( But make sure your MR and experimental phases are on the same origin and
hand! Easiuest way to do this is to get a mtz file with the Model phases
alone, "CAD"ed with the experimental phases then run phasematch - a
reflection utility. This tells you if you need to shift the origin for
either MR or exptl phases and applies the correction..) 

Of course the part of the map with the model phases will look better, and
at 3.1A you will have trouble rebuilding the remaining stuff but good luck!

Eleanor
 

On Tue, 12 Jul 2011 21:15:24 -0400, "Bosch, Juergen" 
wrote:
> http://www.globalphasing.com/sharp/
> or you could extend your phases using DM if you have more than one
> molecule in the asu.
> I'm sure there is also a way to convince solve/resolve to do what you
want.
> If you see enough in your map, just build a backbone model and use that
to
> generate a crude mask for DM (in case you can apply NCS averaging).
> 
> Jürgen
> 
> On Jul 12, 2011, at 9:08 PM, Jiamu Du wrote:
> 
> Dear All,
> I am now working on a low resolution phase determination (around 3.3 A
> with Se anomalous signal around 3.8 A).
> I can find the Se site and get the phase, but the density map is not so
> good.
> Some part of the protein (about 1/3) has a homologue model which is also
> can be found using Phaser. The homologue region has a good map while
other
> region only show a poor map.
> I think the combination of experimental phase and MR phase might improve
> the map. Is there anybody can help find which program can work on this?
> 
> Thanks.
> --
> Jiamu Du, Ph.D.
> Postdoctoral Research Fellow
> Laboratory of Structural Biology
> Memorial Sloan-Kettering Cancer Center
> RRL 269, 430 E 67th Street
> New York, NY, 10021
> E-mail: d...@mskcc.org
> Tel: (217) - 417 - 9897
> 
> 
> ..
> 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] Off topic - transformation problems

[Renos Savva]

You may also want to try 0.5 - 2% (w/v) glucose in your plates alongside 
transformation into the NEB T7 Express strains:


http://www.neb.com/nebecomm/products/productC3013.asp

http://www.neb.com/nebecomm/products/faqproductC3013.asp

LysY gives you higher final ODs [no lysozyme activity] than using 
pLysS/E and is compatible with the T7 promoter in your pET20b.


Renos


On 14/07/2011 03:33, Dima Klenchin wrote:

In this case, pGEX4T3 vector also expressed inserts constitutively.
http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm



Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low
leak expression from PTAC promoter. Definitely lower than the relatively
leaky T7 promoter in pET20, which does not express laqI^q. Switching to
a plasmid from higher pET series that do have laqI^q (e.g. pET31) should
reduce the leakiness.


I also thought that target protein may have tocicity on host strain.
But, why pGEX4T3-target protein was not show the same phenomena.
Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and
BL21(DE3)pLysS.


pLysS should help with repression under non-inducing conditions.

If it is true toxicity issue, you might need to switch to plates with
synthetic medium that does not contain any traces of lactose.

- Dima

[ccp4bb] Influence of symmetry related molecules on a protomer conformation in crystal structures

[sukanta mondal]

I know, in PyMOL using 'symexp' possible to generate symmetry related
molecules for a given crystal structure. But I'm looking for some
program/software (for batch) by which I can find out the number
of symmetry related molecules (distance cutoff <= 5A) interacting with
a given chain in a crystal structure.

Thanking you,
suku
NIBIO, Osaka

Re: [ccp4bb] large R-Rfree difference in "final" structure

[Herman . Schreuder]

Dear Careina,
As other have mentioned, my feeling is that something is suspicious.
However the problem might be very difficult or impossible to catch and
other structures have been deposited with similar R/Rfrees...
 
What I would check in addition to what has been mentioned, is if some
twinning is present. With say a twin-factor of 10-20% processing may
proceed sort of ok and refinement as well with a somewhat higher Rfree.
You may also suffer from disorder. You mention that you model is
complete: does that mean that the complete sequence of your protein has
been fitted, or does this mean that no more positive difference density
is left? If a significant portion of your protein is not visible, this
may still compromise your Rfactors. In the latter case, I don't think
you can do much about it, except offering this as an explanation for
your high R/Rfree.
 
Best,
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Careina Edgooms
Sent: Wednesday, July 13, 2011 5:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] large R-Rfree difference in "final" structure


Dear ccp4 bulletin board

I just have a slight concern regarding my Rwork Rfree
difference. I have a structure that I have solved. I am reasonably
content that it is complete because it has refined well, it no longer
has bad geometries and contacts and all the rotamers, ramachandra, bond
lengths etc are good. It gives favourable scores on molprobity and
procheck. My only concern is the R factor difference. The resolution of
the structure is 2.3A. The R factor is 0.24 after refinement but the
Rfree is 0.33 which seems to me to be rather high. Should I be
concerned?

During refinement Rfree only drops from about 0.36 to 0.33 while
the R factor drops from 0.31 to 0.24.. I have removed automatic
weighting in refmac in order to constrain my bond lengths and angles
during a couple of rounds of refinement. This did not have any effect on
the R factors, however. I am fairly content that the space group I have
chosen is correct so I am not sure what else could cause the big
difference in R factors? There is no twinning. 

Can I be satisfied that my structure is correct despite the high
R free or should I be doing other checks/ trying other things before I
can submit this structure?

Thank you for any help
Careina

Re: [ccp4bb] Influence of symmetry related molecules on a protomer conformation in crystal structures

[vandana kukshal]

hi ,
in CCP 4 package u can run CONTACT program use NCONT to find symmetry
contacts only. u can Fix contact distance  minimum and maximum also .




On Thu, Jul 14, 2011 at 5:18 PM, sukanta mondal wrote:

> I know, in PyMOL using 'symexp' possible to generate symmetry related
> molecules for a given crystal structure. But I'm looking for some
> program/software (for batch) by which I can find out the number
> of symmetry related molecules (distance cutoff <= 5A) interacting with
> a given chain in a crystal structure.
>
> Thanking you,
> suku
> NIBIO, Osaka
>



-- 
Vandana Kukshal
Postdoc Fellow
structure biology group
international Center For Genetic Engineering and Biotechnolgy
India

Re: [ccp4bb] Off topic - transformation problems

[Eric Larson]

On Thu, 14 Jul 2011, Wonjin Bae wrote:


Hi, all

Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and 
pGEX4T3 vector.
Then, these two construct were transformed to BL21(DE3) expression host.
DNA sequencing results were accurate.

In case of pGEX, many colony was formed and shown the high level of expression.
But, colony was not shown in pET20b case. (no one colony)
In case of mock-pET20a vector transformed very well.


Simplest explanation - wrong antibiotic used on the pET20b case?



What's the problems? I never experieced transfomation problems.
Does anyone know how to solve the this problems?

Thanks a lot !!
Genie,




Eric T. Larson, PhD
Biomolecular Structure Center
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195

email: larso...@u.washington.edu

Re: [ccp4bb] Off topic - transformation problems

[Eric Larson]

oops - I just caught that the empty pET20 did transform well so the other 
suggestions about toxicity are probably more accurate.

On Thu, 14 Jul 2011, Eric Larson wrote:


On Thu, 14 Jul 2011, Wonjin Bae wrote:


Hi, all

Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and 
pGEX4T3 vector.

Then, these two construct were transformed to BL21(DE3) expression host.
DNA sequencing results were accurate.

In case of pGEX, many colony was formed and shown the high level of 
expression.

But, colony was not shown in pET20b case. (no one colony)
In case of mock-pET20a vector transformed very well.


Simplest explanation - wrong antibiotic used on the pET20b case?



What's the problems? I never experieced transfomation problems.
Does anyone know how to solve the this problems?

Thanks a lot !!
Genie,




Eric T. Larson, PhD
Biomolecular Structure Center
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195

email: larso...@u.washington.edu

[ccp4bb] Multi crystal averaging : data on same scale before averaging?

[Francis E Reyes]

Hi all

Walking through multi xtal averaging with RAVE. I finally got a good  
mask and optimized NCS for my xtal forms. However, in the CRAVE manual  
I see this


- the reflections in the input MTZ files *MUST* have been put on the  
same temperature factor scale prior to cross-crystal averaging (see  
the DATAMAN manual on how to do this) !!!


Scale the separate datasets together? Wouldn't this just be a mess  
since the crystal should be non isomorphous to each other?


Or does this say that within each dataset, all the data should be on  
the same scale (which would be if I used scala to scale) ?


Am I interpreting this correctly?

Thanks!

F



-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] Multi crystal averaging : data on same scale before averaging?

[Edward A. Berry]

If I recall correctly DATAMAN does Wilson scaling in which the scale
and B-factor are adjusted so the average reflection intensity in
resolution bins are the same. I suspect it may not be required if
all the data have been put on an approximately absolute scale by
e.g. truncate (although that doesn't adjust the B-factor).

If you do end up scaling your data in dataman, be sure to go back
to the original data for refinement once you solve the structure,
or your B-factors will not be right.

eab


Francis E Reyes wrote:

Hi all

Walking through multi xtal averaging with RAVE. I finally got a good
mask and optimized NCS for my xtal forms. However, in the CRAVE manual
I see this

- the reflections in the input MTZ files *MUST* have been put on the
same temperature factor scale prior to cross-crystal averaging (see
the DATAMAN manual on how to do this) !!!

Scale the separate datasets together? Wouldn't this just be a mess
since the crystal should be non isomorphous to each other?

Or does this say that within each dataset, all the data should be on
the same scale (which would be if I used scala to scale) ?

Am I interpreting this correctly?

Thanks!

F



-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

[ccp4bb] NMR resources at PDBe (pdbe.org/nmr)

[Gerard DVD Kleywegt]

Hi all,

The Protein Data Bank in Europe (PDBe; http://pdbe.org/) continues to improve 
its services to the scientific community. As part of our recent website 
update, we have reorganised and simplified the way information about NMR 
entries is shown. We have also released Vivaldi (Visualisation and Validation 
Display; http://pdbe.org/vivaldi), an interactive tool that allows you to 
inspect available validation reports and check for restraint violations or RDC 
fits. For an example, try http://pdbe.org/vivaldi/2keo


The pages describing the NMR resources at PDBe (http://pdbe.org/nmr) have been 
revised and streamlined. They allow you to search the VASCO, OLDERADO, RECOORD 
and logRECOORD databases (see below) and also provide information about 
deposition of NMR structures and accompanying data, as well as some work 
related to the CCPN framework. The NMR statistics page shows the current 
status of NMR-related information in the PDB and at PDBe 
(http://www.ebi.ac.uk/pdbe-apps/nmr/statistics).


On the PDBe atlas pages of a particular NMR entry (e.g., http://pdbe.org/2keo 
- cytochrome-b5-like domain of the HERC2 E3 ligase - or try 
http://pdbe.org/hasnmr for a randomly picked NMR entry), you can access 
available experimental information by clicking on the "Experiment" link in the 
navigation menu on the left of the page. Alternatively, whenever you see a 
PDBprint (http://pdbe.org/pdbprints) for an NMR entry, you can click on the 
"NMR" icon to get to the NMR experimental information page. Finally, if you 
know the PDB code, you can go there directly (e.g., 
http://pdbe.org/2keo/experimental). When you click a "View" link in the table 
on the experimental information page, it will redirect you to a predefined 
Vivaldi view (e.g., restraints or validation reports).


The experimental information page allows you to navigate to the corresponding 
Biological Magnetic Resonance Bank (BMRB) entry containing assigned chemical 
shifts. (Note: experimentally determined chemical shifts are now mandatory 
when depositing NMR structures). Sometimes, more than one BMRB entry relates 
to the same PDB entry, for instance in the case of close homologues or 
alternate protein constructs (>97% sequence identity). You can download 
deposited restraints from the PDB archive or navigate to the remediated 
restraints from the NMR Restraints Grid (NRG) at BMRB. For most NMR entries 
there is an in-depth validation report from the NRG-CING database, hosted at 
the Radboud University of Nijmegen.


VASCO - Validation of assigned chemical shifts through coordinates - is a 
database of 2000+ validation reports, which can be downloaded as text files or 
viewed interactively in Vivaldi. OLDERADO reports on domain composition, 
clustering and representative models of a deposited ensemble are available for 
most NMR entries that contain proteins. They can be viewed in Vivaldi or in a 
tabular format. Two databases of recalculated ensembles are hosted by PDBe: 
RECOORD, with 500+ entries, which compares four common structure-determination 
protocols, and logRECOORD, with 300+ entries, which uses a "log-normal" 
potential for interpreting NOE data.


Finally, you may be interested in some recent NMR-related papers co-authored 
by current and former PDBe staff:


1. Lemak et al., "A novel strategy for NMR resonance assignment and protein 
structure determination", Journal of Biomolecular NMR 49, 27-38, 2011 
(http://dx.doi.org/10.1007/s10858-010-9458-0)


2. Bernard et al., "Bayesian estimation of NMR restraint potential and weight: 
a validation on a representative set of protein structures", Proteins 79, 
1525-1537, 2011 (http://dx.doi.org/10.1002/prot.22980)


3. Penkett et al., "Straightforward and complete deposition of NMR data to the 
PDBe", Journal of Biomolecular NMR 48, 85-92, 2010 
(http://dx.doi.org/10.1007/s10858-010-9439-3)


4. Fogh et al., "MEMOPS: data modelling and automatic code generation", 
Journal of Integrative Bioinformatics 7, 123, 2010 
(http://dx.doi.org/10.2390/biecoll-jib-2010-123)


5. Rieping & Vranken, "Validation of archived chemical shifts through atomic 
coordinates", Proteins 78, 2482-2489, 2010 
(http://dx.doi.org/10.1002/prot.22756)


We hope that expert and non-expert users alike will find the NMR resources at 
PDBe useful. As always, we welcome constructive criticism, comments, 
suggestions, bug reports, etc. through the feedback button at the top of any 
PDBe web page.


--Gerard

---
Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK
ger...@ebi.ac.uk . pdbe.org
Secretary: Pauline Haslam  pdbe_ad...@ebi.ac.uk

Re: [ccp4bb] Multi crystal averaging : data on same scale before averaging?

[Gerard DVD Kleywegt]

This is correct - see http://xray.bmc.uu.se/usf/dataman_man.html#S27

At the time I wrote this (1993...), I was a recovering NMRtist and The Other 
Gerard was doing a sabbatical in Uppsala and in fact in the office next to 
mine. He pointed out that I had to do this and also provided code.


--The other other Gerard



On Thu, 14 Jul 2011, Edward A. Berry wrote:


If I recall correctly DATAMAN does Wilson scaling in which the scale
and B-factor are adjusted so the average reflection intensity in
resolution bins are the same. I suspect it may not be required if
all the data have been put on an approximately absolute scale by
e.g. truncate (although that doesn't adjust the B-factor).

If you do end up scaling your data in dataman, be sure to go back
to the original data for refinement once you solve the structure,
or your B-factors will not be right.

eab


Francis E Reyes wrote:

Hi all

Walking through multi xtal averaging with RAVE. I finally got a good
mask and optimized NCS for my xtal forms. However, in the CRAVE manual
I see this

- the reflections in the input MTZ files *MUST* have been put on the
same temperature factor scale prior to cross-crystal averaging (see
the DATAMAN manual on how to do this) !!!

Scale the separate datasets together? Wouldn't this just be a mess
since the crystal should be non isomorphous to each other?

Or does this say that within each dataset, all the data should be on
the same scale (which would be if I used scala to scale) ?

Am I interpreting this correctly?

Thanks!

F



-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder






Best wishes,

--Gerard

**
   Gerard J.  Kleywegt

http://xray.bmc.uu.se/gerard/  mailto:ger...@xray.bmc.uu.se
**
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
**

[ccp4bb] output individual redundancies

[Ed Pozharski]

I am looking for a way to output redundancy per individual reflection,
preferably for scala but if that is not possible then maybe for
scalepack.  

>From my (admittedly quick) look at the scala manual it seems that I can
use something like UNMERGED output option to exclude outliers and then
would need to write a bit of code to calculate the redundancies.  But I
hope that I missed something and there is a secret keyword that would
add redundancies to the merged mtz file.

Cheers,

Ed.

 


-- 
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] output individual redundancies

[Ethan Merritt]

On Thursday, July 14, 2011 02:55:26 pm Ed Pozharski wrote:
> I am looking for a way to output redundancy per individual reflection,
> preferably for scala but if that is not possible then maybe for
> scalepack.  

If you read the unmerged file from scalepack into ccp4 using
combat, it creates a data column with label M/ISYM that I think is
what you are asking for.  You can use the "Import Unmerged Data (Combat)"
tab in the ccp4i GUI.

Ethan

 
> >From my (admittedly quick) look at the scala manual it seems that I can
> use something like UNMERGED output option to exclude outliers and then
> would need to write a bit of code to calculate the redundancies.  But I
> hope that I missed something and there is a secret keyword that would
> add redundancies to the merged mtz file.
> 
> Cheers,
> 
> Ed.
> 
>  
> 
> 
> 

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
University of Washington, Seattle 98195-7742

Re: [ccp4bb] occupancy-refinement

[ccp4]

You are right that refining occupancy and B  value at the same time cant
be done with REFMAC, and probably wouldnt work anyway at 2.9A 

However, if you set the ligand occupancy to 0.7 say, and refine the
residue B factors, the occupancy should not be set back to 1.0. It
certainly isnt in the local version of REFMAC. 
You can judge to some extent what occupancy gives the cleanest map by
inspection. 
But at 2.9A the correlation between B and occupancy is very high and it
wilnswer. l be hard to get a definitive answer.
Eleanor
On Wed, 13 Jul 2011 10:31:59 -0500, dhurjati putcha
 wrote:
> Dear CCP4ers,ncy and B value 
> 
> While trying to refine a protein-ligand structure (reso=2.9A) I notice
that
> the density (2Fo-Fc)for the ligand is discontinuous. I also notice that
the
> density for the residues in the ligand binding pocket (LBP) is also very
> feeble. And when I refine the ligand occupancy the density for the
ligand
> appears and covers almost all the molecule, and the occupancy refines to
> <1.0.
> 
> However, I cannot refine the occupancy/B factors for the LBP residue
side
> chains because the occupancy stays at 1.0 (and if I change it manually,
it
> returns to 1) and the B factors (group & individual) remain close to the
> average for the remainder of the molecule. If I omit the LBP residue
side
> chains, I get some Fo-Fc density suggesting that these side chains are
> real.
> 
> 
> Is there previous evidence for such phenomena suggesting multiple
> conformations/dynamics/loose binding? Is there a way to quantify the
> dynamics associated with the side chains (as with the ligand where the
> occupancy is <1.0) so I can argue it out with potential manuscript
> reviewers? The R-factor/R free #s (22%/19%) suggest that the refinement
is
> nearing convergence.
> 
> Best regards,
> 
> Kumar.

Re: [ccp4bb] Same protein, different molecule numbers per ASU

[ccp4]

They must represent a different packing - the volume of Shape 1 is 10% >
than 2*Vol-Shape2 

But it isnt uncommon to get different forms - check your symmetry contacts
(PISA will do that) and see how the two forms pack.
eleanor

On Mon, 11 Jul 2011 18:09:52 +0800, ferrol shariff 
wrote:
> Hi Alenxander,
> Thanks for the reply. Here's the details on the unit cell parameters
> 
> [image: image.png]
> 
> [image: image.png]
> 
> [image: image.png]
> 
> Thank you. :)
> 
> Regards,
> 
> Fairolniza
> On Mon, Jul 11, 2011 at 2:47 PM, Alexandre OURJOUMTSEV
> wrote:
> 
>>  Dear Ferrol,
>>
>> ** **
>>
>> Could you let us know the unit cell parameters for both these crystals
?
>> (did I miss them somewhere in your previous mails?).  I wonder if in
fact
>> this is not the same packing with minor variation.
>>
>> Sacha
>>
>> ** **
>>
>> *De :* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *De la part
>> de*ferrol shariff
>> *Envoyé :* dimanche 10 juillet 2011 10:10
>> *À :* CCP4BB@JISCMAIL.AC.UK
>> *Objet :* [ccp4bb] Same protein, different molecule numbers per ASU
>>
>> ** **
>>
>> Hello and good day to everyone! :)
>>
>> ** **
>>
>> I have some general questions on crystallography work. I hope you don't
>> mind giving me some ideas.
>>
>> I have solved my lipase protein both ground-grown crystals and
>> space-grown
>> crystals with good resolutions (1.4A and 2.2A). They are the same
protein
>> from the same source, same purification methods, and produced crystals
>> from
>> the same crystallization conditions (except the gravity part).
>>
>> From the data, it shows that both of them belong to the same space
group
>> P212121. But they have different number of molecule per asymmetric
unit.
>> Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the
>> moment i have problem explaining this issue. Is it normal to have such
>> results? Same protein with different number of molecule/ASU?
>>
>> I've been trying to get some references on this matter but so far i
don't
>> really get anything that can directly explain it. Furthermore, do i
need
>> to
>> relate this with the gravity effect?
>>
>> I hope you don't mind sharing some experiences on crystallography
>> especially regarding this matter.
>>
>> Thank you very much
>>
>> --
>> FAIROLNIZA
>>
>> 
>>

Re: [ccp4bb] abnormal I/Sigma over phi rotation range

[ccp4]

This seems somewhat weird - your Rmerge values increase with more frames
as explained by Tim, but I cant see why the I/SigI should increase sharply
for the 180 degree set then fall off again.
if you have run Scala look at the scaling plots v batch, and resolution to
see if there are any weird outliers.
Eleanor

On Mon, 11 Jul 2011 10:23:00 +0200, Tim Gruene 
wrote:
> Dear Vennila,
> 
> my guess is that you had a larger number of images in the batch/set
> rge  starting at
> 180 degrees (hence the slightly incereased Rmerge for that batch) and
that
> by
> the end of that run your crystal had suffered from radiation damage
(hence
> the
> large Rmerge for the set starting at 360degrees).
> 
> You should report Rmeas instead of Rmerge and let us know about the
number
> of
> frames and the frame width in each set.
> 
> Cheers, Tim
> 
> 
> On Sun, Jul 10, 2011 at 01:54:07PM +0100, Vennila Natesan wrote:
>> Dear CCP4BB users,
>> 
>> In order to determine the redundancy at which the structure can be
>> solved, I divided the master
>> data set of my protein into five sets with phi rotations of  45,65,90,
>> 180 and 360 degrees. I got
>> the mean I/Sigma value as 22.4(11.4), 21.7(11.0), 22.7(11.2),
60.9(45.2)
>> and 15.6(8.1) respectively.
>> I will be very helpful if i get any idea/possible reasons for the
>> abnormal value for 180 degree dataset.
>> 
>> For the information, the completeness values are
>> 75.7(78.5),92.6(92.7),99.5(97.7),99.6(97.7)and 99.5(96.4)
>> respectively and values inside brackets are for highest resolution
>> shell. There was no noticable change in mosaicity
>> value. The Rmerge values are 2.2,2.4,2.7,3.2, and 5.7 (8.7)
>> respectively.
>> 
>> Thanks in advance
>>

Re: [ccp4bb] low resolution refinement

[ccp4]

  Roberto steiner has told you how to use these new REFMAC5.6 features,
Rob  Nicholls has suggested how to generate secondary structure restraints,
and Martyn Winn given a page to install a new interface to make it easier
to use them..

But with such limited data it isnt surprising that the FreeR climbs
steadily. Scaling Fcalcs and Fobs together at this resolution is tricky,
and you should look at your plots of Rfactor and Fo/Fc at the end of
refinement. 
There may be anomalies at the top or bottom resolution.. 
There really isnt a general rule for a fix. You need to "know your data".
You can still get useful information from the maps.
Eleanor

 

On Sat, 9 Jul 2011 16:59:29 +0800, Qixu Cai  wrote:
> Dear all,
> 
> Recently, I refine two low resolution structures in refmac 5.5. Their 
> resolutions are 3A and 3.5A respectively.
> For 3A structure, after MR by phaser and rigidbody refinement&restraint 
> refinement by refmac5.5, I got R factor 25% and R free 35%. And then 
> each time, after my model building in coot and restraint refinement by 
> refmac 5.5, the R factor stays 25%, but R free increases to 38%, even
39%.
> For 3.5A structure, the R factor stays 27%, but R free increases from 
> 37% to 42% after my slightly model building in coot.
> Could you help me to find the reason?
> 
> Maybe the reason is the overfit of the structure? I found that new 
> version of refmac 5.6 has many new features for low resolution 
> refinement, such as jelly boy, secondary structure restraints. But I 
> don't know how to use these new features in old version ccp4i (6.1.13)?
> 
> I also used phenix.refine with the "reference model" ( I have high 
> resolution model for one domain of the low resolution protein) and 
> "secondary structure restraints", but it seams the same. Any suggestion?
> 
> BTW, is that simulator annealing not suitable for low resolution 
> structure? I used the simulator annealing method of CNS and 
> phenix.refine, but the geometry of the structure is always destroyed 
> seriously.
> 
> Could you help me?
> 
> Thank you very much!