Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Frank von Delft]

Seems reasonable:  P21 with beta=90 is pretty common, and it often ends 
up twinned as well.


Most importantly, your R-factors support the hypothesis.

(Only keep in mind that R-factors are lower in twinned refinement - look 
on Garib's webpage for the presentation where he describes that -- 
sorry, I don't remember the exact slides.)


phx


On 27/03/2013 23:40, Andrey Nascimento wrote:


Dear all,


As I said in the latest topic, I could not model the third molecule. 
But when I superpose the two trimmers found in P1 MR solution (link 
below), I get the first two molecules perfect aligned and the third 
molecule inverted! (It is also possible to see the 2-fold axis and the 
third molecule lying on it!)


I tried to run a MR with a model with two alternative positions and 
adjusted occupancy for the third molecule, but the Rfactor/free get 
higher (> 40%) and the map becomes worse – even the good ones 
(molecules 1 and 2) and for third molecule it remains bad (or worse).


A procedure that “solved” the problem (decreased the Rfactor/free and 
gave good maps for third molecule) was the following: I integrated and 
scaled the data in P21, then I ran the sfcheck and it showed a twinned 
data (probably because of the (pseudo) higher symmetry present – 
P21212). So, I detwinned the data (with detwinn) and run a MR with 
detwinned data that gave a very good solution with tree molecules in 
ASU (it have never happened before!). After the MR I refined this MR 
solution against the original P21 data (without detwinn procedure) 
with amplitude based twin refinement in Refmac5 and, finally, it gave 
a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the 
first round of refinement. I think that procedure probably discard 
reflections related to other positions making increasing the signal of 
the most frequent position.


Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf

Is there some problem in procedure described? If so, does anybody have 
a suggestion how can I model these disorder? Moreover, it seems to be 
a long range disorder (multiples positions along the all lattice), 
since even in P1 the maps for this third molecule are very bad.


Thank you for all the suggestions.

Cheers,

Andrey


2013/3/25 Eleanor Dodson >


First - I dont think you have a 3rd molecule where you have put it
- or at least not one with full occupancy. Those maps are a clear
indication that something is wrong. What is the Matthews
coefficient for the numbers in the asymmetric unit?

Presumably your processing gave you a lattice which fitted the
diffraction spots? ie you didnt miss a set of observations? You
should see that at the data processing stage, and the different
integration programs also try to report it. If there is
non-crystallographic translation that can confuse things a bit;
some classes of reflections might be systematically weak, but you
can find if there is such a phenomena by doing a patterson. Or run
ctruncate after merging the data - it checks this, and so does
Xtriage.  All these options will also check for twinning. If there
is NCT then that could explain the high Rfactor.

Are the spots nicely shaped? There are some cases of sheared
crystals, which usually show up in distorted diffraction spots.

If this is so and you have integrated the data according to an
orthogonal lattice, there is nothing to stop you merging those
observations in a low symmetry. Pointless gives you good
statistics on the scoring for different symmetry operators.
You can either run MR again in that symmetry - check all SGS
consistent with the pointgroup, or try to work out how to position
your P22121 solution in the new SG.  There may well be 2n+1 copies
of your molecule when you double the size of the asymmetric unit
-  all hard to check without more information.
Good luck Eleanor




On 22 March 2013 17:54, Andrey Nascimento
mailto:andreynascime...@gmail.com>>
wrote:

Dear all,

I have tried the procedure recommended by Zbyszek, expanding
data from a higher symmetry and keeping the R-free set. But
the map for third molecule (new molecule placed) are still
very bad, even when a tried to reprocess data in P1 or P2 (P 1
21 1). The previous placed molecule (present in P2 21 21 ASU)
and its symmetry related on P21 shows a very good map, but the
third molecule are almost completely wrong (~50 residues in
470 are placed in quite good map) and map does not have
connectivity to build a new molecule (even in lower sigmas,
0.8-1.0). I have tried automatic model building (AutoBuild and
ARP/wARP) but they cannot build anything that make some sense
or build a random chains without any sense.


I do not have an extensive knowledge of crystallography, but I
hav

Re: [ccp4bb] delete subject

[Frank von Delft]

"mistake"?  I beg to differ, violently:  a student had an honest 
question and did exactly what the ccp4bb exists for:  posted his 
question there.  Moreover, when asking he showed he had thought about 
it, and provided complete background -- that's what we all want, right?


 * I disagree with Tassos:  the email was not rude, on not one of the
   counts he listed.  (I concede he may have had a bad day... I had one
   on Monday :-)
 * I disagree (slightly) with Tim:  the teasing was not malicious.
 * And I share Mark's dream...

Students:  please don't stop asking your questions here!!!

phx.




On 27/03/2013 22:47, VAN RAAIJ , MARK JOHAN wrote:

Dear Tom,
don't feel too bad about it - everyone can make a mistake.
Some of the replies give crystallographic tips that may be useful to 
other beginning and not-so-beginning crystallographers. Although I 
agree the attachments to the first mail would perhaps better be 
deleted from the records.

Greetings,
Mark


Quoting Tom Van den Bergh :

Is it possible to delete my post: refinement protein structure from 
ccp4 bb, i get too many bad reactions. I think its bettter to just 
delete the whole topic.


Greetings,

Tom

[ccp4bb] another mystery density (link to picture no attachment)

[Laurie Betts]

Here is a link to a picture of some mystery difference density that I find
in a low resolution (2.8 but high overall B and high avg. ADPs) nestled in
an area where a beta strand buckles out.

I have circled three N-H groups that seem to be pointing at it.  Two are
backbone amides and one is a histidine.

The crystallization medium is 6 M ammonium nitrate, Tris buffer, and
glycerol for cryoprotection.

It looks too elongated to be a metal it almost looks as if the histidine
has an adduct.

The top side of the pocket is hydrophobic (pro and Leu).

Can anyone give a possibility other than evil sausage density?

Laurie Betts

https://docs.google.com/file/d/0Bx8-qQA_HKegekZKSng1ZEV2MHM/edit

Re: [ccp4bb] delete subject

[mjvdwoerd]

Earlier today, I thought this and did not write it. It is a slightly different 
theme on your suggestion:

I hear  there are now (but have not seen examples of)  "journals" (web sites) 
where you do exactly what Tom did: you put your data there, which "proves" that 
you did the work (first) and you do not worry about the fact that you are 
making it public before formal publication, because making data public is the 
reason why you got the data in the first place. And nobody can claim to have 
done the work, because everybody knows that someone else was first - the web 
site is "proof". The results are not peer-reviewed of course (even though, in 
the case of CCP4, things are inherently peer-reviewed to some extent, that is 
what he asked us to do).  And I hear that there are now journals that will 
accept references to such web sites.

Freely sharing unpublished data on a public forum might well be the future, 
even if in our corner of science this is not yet commonplace. 

The pivotal point to Tom is that he can learn from the suggestions that have 
been made. I hope he will. I actually hope that he will follow up on the 
suggestions (privately maybe).  Unlike some, I do not feel that it was bad to 
find a big file in my inbox, this is what "move to" is for. I think my reaction 
was "ouch, he did not want to do what he just did and it cannot be undone". But 
maybe this is not true. There is definitely value in sharing preliminary data, 
especially for junior people. To have such a function as part of CCP4 might be 
a very good suggestion, but I agree with you that perhaps it should not land in 
its full glory in everyone's mailbox.

Mark

 

 

-Original Message-
From: William G. Scott 
To: CCP4BB 
Sent: Wed, Mar 27, 2013 6:09 pm
Subject: Re: [ccp4bb] delete subject


Dear Tom et al:

Although arriving too late to participate in the snark-fest, it occurred to me 
that maybe this is almost exactly how we should solve structures and educate 
graduate students (or others).

Instead of attachments, the relevant files could be shared via dropbox.  Those 
of generous spirit could help solve, refine, correct, critique or otherwise 
improve structures before formal peer review.  (If everyone knows the source of 
the data, it is far less likely to be ripped off, not more.)

It might cut down on the number of mistakes (or worse) that appear in the PDB 
and journals, new mentorships and collaborations might be established, in 
exceptional cases co-authorship, or more generally, an acknowledgement could be 
offered.

For students like mine who are comparatively isolated in a small institution 
somewhat off the beaten path, it would be a real asset and advantage to them 
not 
to have to rely only upon my limited abilities and increasingly obsolete 
knowledge.

We should all be able to learn from one anther without fear of reproach.

All the best,

Bill


William G. Scott
Professor
Department of Chemistry and Biochemistry
and The Center for the Molecular Biology of RNA
228 Sinsheimer Laboratories
University of California at Santa Cruz
Santa Cruz, California 95064
USA

 


On Mar 27, 2013, at 3:36 PM, Tom Van den Bergh 
 
wrote:

> Is it possible to delete my post: refinement protein structure from ccp4 bb, 
> i 
get too many bad reactions. I think its bettter to just delete the whole topic.
> 
> Greetings,
> 
> Tom

 

Re: [ccp4bb] delete subject

[Antony Oliver]

Dear Tom,

I'm sure the files can be easily removed from the server, if that is what you 
wish / want to happen. A quick email to the administrators at 
c...@ccp4.ac.uk should do the trick.

Reading around all the all leg-pulling / "other comments" aside - from your 
post you've got actually got a really good number of useful suggestions and 
comments that should help you along the path to solve and refine your structure 
yourself.

Best of luck with your data.  With regards,

Tony.



Is it possible to delete my post: refinement protein structure from ccp4 bb, i 
get too many bad reactions. I think its bettter to just delete the whole topic.

Greetings,

Re: [ccp4bb] delete subject

[William G. Scott]

Dear Tom et al:

Although arriving too late to participate in the snark-fest, it occurred to me 
that maybe this is almost exactly how we should solve structures and educate 
graduate students (or others).

Instead of attachments, the relevant files could be shared via dropbox.  Those 
of generous spirit could help solve, refine, correct, critique or otherwise 
improve structures before formal peer review.  (If everyone knows the source of 
the data, it is far less likely to be ripped off, not more.)

It might cut down on the number of mistakes (or worse) that appear in the PDB 
and journals, new mentorships and collaborations might be established, in 
exceptional cases co-authorship, or more generally, an acknowledgement could be 
offered.

For students like mine who are comparatively isolated in a small institution 
somewhat off the beaten path, it would be a real asset and advantage to them 
not to have to rely only upon my limited abilities and increasingly obsolete 
knowledge.

We should all be able to learn from one anther without fear of reproach.

All the best,

Bill


William G. Scott
Professor
Department of Chemistry and Biochemistry
and The Center for the Molecular Biology of RNA
228 Sinsheimer Laboratories
University of California at Santa Cruz
Santa Cruz, California 95064
USA

 


On Mar 27, 2013, at 3:36 PM, Tom Van den Bergh 
 wrote:

> Is it possible to delete my post: refinement protein structure from ccp4 bb, 
> i get too many bad reactions. I think its bettter to just delete the whole 
> topic.
> 
> Greetings,
> 
> Tom

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Andrey Nascimento]

Dear all,


As I said in the latest topic, I could not model the third molecule. But
when I superpose the two trimmers found in P1 MR solution (link below), I
get the first two molecules perfect aligned and the third molecule
inverted! (It is also possible to see the 2-fold axis and the third
molecule lying on it!)



I tried to run a MR with a model with two alternative positions and
adjusted occupancy for the third molecule, but the Rfactor/free get higher
(> 40%) and the map becomes worse – even the good ones (molecules 1 and 2)
and for third molecule it remains bad (or worse).



A procedure that “solved” the problem (decreased the Rfactor/free and gave
good maps for third molecule) was the following: I integrated and scaled
the data in P21, then I ran the sfcheck and it showed a twinned data
(probably because of the (pseudo) higher symmetry present – P21212). So, I
detwinned the data (with detwinn) and run a MR with detwinned data that
gave a very good solution with tree molecules in ASU (it have never
happened before!). After the MR I refined this MR solution against the
original P21 data (without detwinn procedure) with amplitude based twin
refinement in Refmac5 and, finally, it gave a good statistics (R factor /
free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think
that procedure probably discard reflections related to other positions
making increasing the signal of the most frequent position.



Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf



Is there some problem in procedure described? If so, does anybody have a
suggestion how can I model these disorder? Moreover, it seems to be a long
range disorder (multiples positions along the all lattice), since even in
P1 the maps for this third molecule are very bad.



Thank you for all the suggestions.



Cheers,

Andrey

2013/3/25 Eleanor Dodson 

> First - I dont think you have a 3rd molecule where you have put it - or at
> least not one with full occupancy. Those maps are a clear indication that
> something is wrong. What is the Matthews coefficient for the numbers in the
> asymmetric unit?
>
> Presumably your processing gave you a lattice which fitted the diffraction
> spots? ie you didnt miss a set of observations? You should see that at the
> data processing stage, and the different integration programs also try to
> report it. If there is non-crystallographic translation that can confuse
> things a bit; some classes of reflections might be systematically weak, but
> you can find if there is such a phenomena by doing a patterson. Or run
> ctruncate after merging the data - it checks this, and so does Xtriage.
> All these options will also check for twinning. If there is NCT then that
> could explain the high Rfactor.
>
> Are the spots nicely shaped? There are some cases of sheared crystals,
> which usually show up in distorted diffraction spots.
>
> If this is so and you have integrated the data according to an orthogonal
> lattice, there is nothing to stop you merging those observations in a low
> symmetry. Pointless gives you good statistics on the scoring for different
> symmetry operators.
> You can either run MR again in that symmetry - check all SGS consistent
> with the pointgroup, or try to work out how to position your P22121
> solution in the new SG.  There may well be 2n+1 copies of your molecule
> when you double the size of the asymmetric unit -  all hard to check
> without more information.
> Good luck Eleanor
>
>
>
>
> On 22 March 2013 17:54, Andrey Nascimento wrote:
>
>> Dear all,
>>
>> I have tried the procedure recommended by Zbyszek, expanding data from a
>> higher symmetry and keeping the R-free set. But the map for third molecule
>> (new molecule placed) are still very bad, even when a tried to reprocess
>> data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21
>> 21 ASU) and its symmetry related on P21 shows a very good map, but the
>> third molecule are almost completely wrong (~50 residues in 470 are placed
>> in quite good map) and map does not have connectivity to build a new
>> molecule (even in lower sigmas, 0.8-1.0). I have tried automatic model
>> building (AutoBuild and ARP/wARP) but they cannot build anything that make
>> some sense or build a random chains without any sense.
>>
>>
>> I do not have an extensive knowledge of crystallography, but I have been
>> thinking about some questions:
>>
>>
>> If the third molecule (the bad one) is lying on the 2-fold symmetry axis
>> on P 2 21 21, and since it does not have an intrinsic 2-fold symmetry axis
>> (like protein molecule), how can I merge the structure factors (or
>> intensities) related by symmetry and expand to lower symmetry afterwards?
>> In this case the molecule lying on the 2-fold symmetry axis will have the
>> structure factors wrongly merged, since the molecule is not symmetric, is
>> it ok?
>>
>>
>> If the third molecule is lying on the 2-fold symmetry axis on P 2 21 21,
>> and only another tw

Re: [ccp4bb] off topic, BN PAGE

[Zhijie Li]

Hi Careina,

BN PAGE can be affected by many factors. Considering the complexity and the 
chance of winning and the amount of information you gain even when you win, I 
do not recommend fighting it. 
BN PAGE, like other gel-based methods, requires that your complex is fairly 
stable - not having a weak affinity and not a high dissociation rate. Also the 
complex formation should be compatible with the gel running condition: the pH 
and salts and other things. Coomassie itself may compete for some hydrophobic 
surfaces or positively charged residues on the proteins - in such case there's 
little you can do. 
If you see a band corresponding to the expected complex weight, then 
congratulations, BN gel might be your tool (with cautions though as you can 
also get false positives with BN gel). But if not, then my suggestion is to 
move to other techniques such as gel filtration, analytical 
ultracentrifugation, BiaCore, ITC and so on. I had a case in which I could 
confidently show complex formation with gel-filtration and Biacore, but not 
with BN gel.

Zhijie


From: Careina Edgooms 
Sent: Wednesday, March 27, 2013 5:24 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off topic, BN PAGE


Hi


Has anyone found the coomassie in a BN PAGE to be interfering with the 
oligomeric structure of their protein? If so, how did you deal with this?
Thanks


Careina

Re: [ccp4bb] delete subject

[VAN RAAIJ , MARK JOHAN]

Dear Tom,
don't feel too bad about it - everyone can make a mistake.
Some of the replies give crystallographic tips that may be useful to  
other beginning and not-so-beginning crystallographers. Although I  
agree the attachments to the first mail would perhaps better be  
deleted from the records.

Greetings,
Mark


Quoting Tom Van den Bergh :

Is it possible to delete my post: refinement protein structure from  
ccp4 bb, i get too many bad reactions. I think its bettter to just  
delete the whole topic.


Greetings,

Tom

[ccp4bb] delete subject

[Tom Van den Bergh]

Is it possible to delete my post: refinement protein structure from ccp4 bb, i 
get too many bad reactions. I think its bettter to just delete the whole topic.

Greetings,

Tom

Re: [ccp4bb] refinement protein structure

[Bosch, Juergen]

Hi Tom,

some suggestions for you:

You should calculate a selfrotation function and see if you can learn something 
from it.
You should definitely run Matthews Coefficient and see if you get a brilliant 
idea there

And congratulations to your first crystal structure it looks really great, for 
a starter this is the right resolution to play with and get exposed to all the 
programs used in the crystallographic community.

You got my other email off-the board but for the record keepers here, in case 
you were offended by my early April fools joke I do apologize. And the second 
part in the private email still holds true.

Jürgen

P.S. may I use your data in an X-ray workshop ?

On Mar 27, 2013, at 5:26 PM, Alexander Aleshin wrote:

But the amount of time spent on turning a protein into a publishable structural 
data is pretty much same, if not larger. There are no low hanging fruits any 
more.

On Mar 27, 2013, at 11:50 AM, Frank von Delft wrote:

Is it too much to dream that Tom has set a trail-blazing precedent and 
demonstrated to us all how unnecessary it is be anal about our oh-so-precious 
data and structures that in the year 2013 are almost completely useless without 
a huge dollop of other experimental data...?



On 27/03/2013 18:32, Anastassis Perrakis wrote:
I think it will be the first time in 15 years I will disagree with Tim.

I personally  found the posting of Tom van der Bergh irritatingly disrespectful 
in many levels.

1. It does not respect my mailbox capacity
2. It does not respect CCP4 developers posting output from phenix.refine
3. It does not respect his supervisors and colleagues who (right now) look like 
fools (to me)
4. It does not respect himself, as I actually suspect he is a proactive 
motivated student who came out as a bit of a fool

These said, I am rather easily irritated these days, so I will not comment on 
the irritable character of the email.

As for the answers, some were funny, some were informative, some funny and 
informative.
Not too much political correctness please, because we will soon start calling 
disordered loops
positionally challenged polypeptide segments (*).

Tassos

(*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
meeting...!

Dear so-far-posters,

I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.

No regards to the ones addressed,
Tim

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] refinement protein structure

[Alexander Aleshin]

But the amount of time spent on turning a protein into a publishable structural 
data is pretty much same, if not larger. There are no low hanging fruits any 
more. 

On Mar 27, 2013, at 11:50 AM, Frank von Delft wrote:

> Is it too much to dream that Tom has set a trail-blazing precedent and 
> demonstrated to us all how unnecessary it is be anal about our oh-so-precious 
> data and structures that in the year 2013 are almost completely useless 
> without a huge dollop of other experimental data...?
> 
> 
> 
> On 27/03/2013 18:32, Anastassis Perrakis wrote:
>> I think it will be the first time in 15 years I will disagree with Tim.
>> 
>> I personally  found the posting of Tom van der Bergh irritatingly 
>> disrespectful in many levels.
>> 
>> 1. It does not respect my mailbox capacity
>> 2. It does not respect CCP4 developers posting output from phenix.refine
>> 3. It does not respect his supervisors and colleagues who (right now) look 
>> like fools (to me)
>> 4. It does not respect himself, as I actually suspect he is a proactive 
>> motivated student who came out as a bit of a fool
>> 
>> These said, I am rather easily irritated these days, so I will not comment 
>> on the irritable character of the email.
>> 
>> As for the answers, some were funny, some were informative, some funny and 
>> informative.
>> Not too much political correctness please, because we will soon start 
>> calling disordered loops
>> positionally challenged polypeptide segments (*).
>> 
>> Tassos
>> 
>> (*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
>> meeting...!
>> 
>>> Dear so-far-posters,
>>> 
>>> I do not know Tom Van den Bergh, nor do I know his background, nor the
>>> history of the data, nor the reasons why he may have sent it to this
>>> list (although I think he did it to ask for help), but I find these
>>> answers irritatingly disrespectful and nasty.
>>> 
>>> No regards to the ones addressed,
>>> Tim

Re: [ccp4bb] refinement protein structure

[Ravi Nookala]

Dear Tom,
It goes to show...there are sinners and saints in all walks of life!
C'mon, there is a lesson to be learnt in this somewhere!
Amen
Ravi
On 27/03/2013 18:32, Anastassis Perrakis wrote:

I think it will be the first time in 15 years I will disagree with Tim.

I personally  found the posting of Tom van der Bergh irritatingly disrespectful 
in many levels.

1. It does not respect my mailbox capacity
2. It does not respect CCP4 developers posting output from phenix.refine
3. It does not respect his supervisors and colleagues who (right now) look like 
fools (to me)
4. It does not respect himself, as I actually suspect he is a proactive 
motivated student who came out as a bit of a fool

These said, I am rather easily irritated these days, so I will not comment on 
the irritable character of the email.

As for the answers, some were funny, some were informative, some funny and 
informative.
Not too much political correctness please, because we will soon start calling 
disordered loops
positionally challenged polypeptide segments (*).

Tassos

(*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
meeting...!


Dear so-far-posters,

I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.

No regards to the ones addressed,
Tim


--
Dr. Ravi Nookala
Dept. of Biochemistry
University of Cambridge
+44 (0)1223766033 (Office)
+44 (0)7505808969 (Mobile)
http://uk.linkedin.com/pub/ravi-nookala/5/a87/b04

Re: [ccp4bb] refinement protein structure

[Frank von Delft]

Is it too much to dream that Tom has set a trail-blazing precedent and 
demonstrated to us all how unnecessary it is be anal about our 
oh-so-precious data and structures that in the year 2013 are almost 
completely useless without a huge dollop of other experimental data...?




On 27/03/2013 18:32, Anastassis Perrakis wrote:

I think it will be the first time in 15 years I will disagree with Tim.

I personally  found the posting of Tom van der Bergh irritatingly disrespectful 
in many levels.

1. It does not respect my mailbox capacity
2. It does not respect CCP4 developers posting output from phenix.refine
3. It does not respect his supervisors and colleagues who (right now) look like 
fools (to me)
4. It does not respect himself, as I actually suspect he is a proactive 
motivated student who came out as a bit of a fool

These said, I am rather easily irritated these days, so I will not comment on 
the irritable character of the email.

As for the answers, some were funny, some were informative, some funny and 
informative.
Not too much political correctness please, because we will soon start calling 
disordered loops
positionally challenged polypeptide segments (*).

Tassos

(*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
meeting...!


Dear so-far-posters,

I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.

No regards to the ones addressed,
Tim

Re: [ccp4bb] refinement protein structure

[Anastassis Perrakis]

I think it will be the first time in 15 years I will disagree with Tim.

I personally  found the posting of Tom van der Bergh irritatingly disrespectful 
in many levels.

1. It does not respect my mailbox capacity
2. It does not respect CCP4 developers posting output from phenix.refine
3. It does not respect his supervisors and colleagues who (right now) look like 
fools (to me)
4. It does not respect himself, as I actually suspect he is a proactive 
motivated student who came out as a bit of a fool

These said, I am rather easily irritated these days, so I will not comment on 
the irritable character of the email.

As for the answers, some were funny, some were informative, some funny and 
informative.
Not too much political correctness please, because we will soon start calling 
disordered loops 
positionally challenged polypeptide segments (*). 

Tassos

(*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
meeting...!

> Dear so-far-posters,
> 
> I do not know Tom Van den Bergh, nor do I know his background, nor the
> history of the data, nor the reasons why he may have sent it to this
> list (although I think he did it to ask for help), but I find these
> answers irritatingly disrespectful and nasty.
> 
> No regards to the ones addressed,
> Tim

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

Dear Tom, 

I think that we've all actually been rather gently teasing you - admittedly a 
difficult concept to put across via the medium of e-mail.
In fact, I think we've all offered sensible constructive suggestions, and 
indeed pointed out what you should try next.

Apologies for any inadvertent offence - none intended. 

Tony.


> Dear so-far-posters,
> 
> I do not know Tom Van den Bergh, nor do I know his background, nor the
> history of the data, nor the reasons why he may have sent it to this
> list (although I think he did it to ask for help), but I find these
> answers irritatingly disrespectful and nasty.



> No regards to the ones addressed,
> Tim

Re: [ccp4bb] refinement protein structure

[Roger Rowlett]

Tom,

Welcome to the protein crystallography community. I hope you take all 
the teasing in good humor. And a few protocol lessons about sharing 
data, etc. By now you have discovered that structure factor files and a 
possible MR model are like catnip to structural biologists. I learned 
this when I solved my first structure while on a sabbatical leave. I ask 
one question about a tricky (to me) MR solution, share the structure 
factors with ONE postdoc, and the next thing I know the whole building 
is solving my structure. (Cover ears and go La La La La La.) You have 
taken this to a whole new level! :)


To echo the advice of many others, nothing beats actually looking at the 
solution with a critical eye:


1. is there sensible electron density around the model? (You may want
   to modify the model to poly-Ala or truncate to the nearest similar
   residue--you can do this using CCP4 programs.) If sequence identity
   is really poor a poly-Ala search model may be better. Once you get a
   preliminary solution, I've used Parrot and Buccaneer successfully to
   autobuild most of the structure from a relatively poor starting
   model. But there is nothing wrong with manual rebuilding from a good
   starting point with high homology.
2. Does the solution pack well, with definable solvent channels and
   reasonable protein-protein contacts? (Use symexp in Pymol or turn on
   symmetry molecules in Coot) Sometimes it is easy to discover a
   missing or "extra" molecule in the ASU this way. That's what
   happened with my first structure solution--I thought I had 4 chains
   in the ASU and it was really 6, which was quite obvious from the
   packing of my partial (as it turned out) MR solution. An extra two
   chains fit perfectly in the "hole" in the packing view.
3. Don't forget to do a quick Matthews coefficient calculation before
   MR to get some idea of how many molecules are in the ASU. It's not
   perfect, especially for high copy numbers, but it's a start. It's
   normally easy to tell 1 from 2 molecules in the ASU. Not so much 6
   vs. 8.
4. It's not unusual for raw MR solutions to start at R>40%. But if you
   are on the right track, the initial refinement in Refmac should
   drive that down to 30% or lower pretty quickly. If R doesn't drop
   rapidly, you likely have an issue with the MR solution. Be aware
   that in the latest versions of Refmac, that first refinement may
   take as many as 20 cycles to converge, not the default 10 cycles.
   After that, 20 cycles is usually overkill.

Have fun, and solve your protein structure.

Cheers,


___
Roger S. Rowlett
Gordon & Dorothy Kline 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


On 3/27/2013 12:22 PM, Tom Van den Bergh wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of 
mRFP (monomer red fluorescent protein, sequence in attachment). I have 
done molecular replacement with phaser with model 2VAD of protein 
database. Then i have done some model building phenix.autobuild. (2 
pdb's (overall...), freeR flags and log file attached) When i refine 
with phenix.refine my structure i get a R-value of 0,42 which is still 
way too high. (redfluorescent protein.pdb, .mtz and logfile attached) 
When i look at the structure in coot i find many unmodelled blobs and 
many outliers in density analysis and rotamer analysis. The problem is 
that there are so many problems with my structure, that i dont know 
where to begin. Could you try some refinement for me, because this is 
first structure that i need to solve as a student and i dont have too 
many experience with it.


Greetings,

Tom

Re: [ccp4bb] refinement protein structure

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear so-far-posters,

I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.

No regards to the ones addressed,
Tim

On 03/27/2013 06:43 PM, Bosch, Juergen wrote:
> Sorry it has been accepted already, see attachment, it will soon be
> online see the date for that.
> 
> Jürgen
> 
> .. Jürgen Bosch Johns Hopkins University 
> Bloomberg School of Public Health Department of Biochemistry &
> Molecular Biology Johns Hopkins Malaria Research Institute 615
> North Wolfe Street, W8708 Baltimore, MD 21205 Office:
> +1-410-614-4742 Lab:  +1-410-614-4894 Fax:
> +1-410-955-2926 http://lupo.jhsph.edu
> 
> [cid:C2A945C2-7702-4EAE-B6E5-2825754D728C@sph.ad.jhsph.edu] On Mar
> 27, 2013, at 1:30 PM, Steiner, Roberto wrote:
> 
> we should all chip in a water molecule or two and the second author
> becomes "the CCP4 community"….
> 
> On 27 Mar 2013, at 17:22, Antony Oliver
> mailto:antony.oli...@sussex.ac.uk>> 
> wrote:
> 
> At the risk of being somewhat cheeky - perhaps I could claim second
> author? I too have successfully solved the structure - and I
> totally concur with Phil. Placing a second molecule in the
> asymmetric unit, essentially resolves the perceived R-factor
> problem.
> 
> A good thorough manual inspection and rebuilding is *ALWAYS* good
> practice for newcomers to the field.
> 
> Tony.
> 
> 
> On 27 Mar 2013, at 17:14, Petr Leiman
> mailto:petr.lei...@epfl.ch>> wrote:
> 
> Since this is now public domain knowledge and if this gets ever
> published, Phil has my vote to be the first author!
> 
> Petr
> 
> 
> On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:
> 
> That's quite brave - shipping your entire structure to people that
> could be actual competitors.  But it was fun to play at 1.4
> Angstrom over lunch.
> 
> Practical points:
> 
> * not everyone loves 12Mb of attachments in one email in their
> inbox, so if you do this again please put the files on a webserver
> and point us there
> 
> Structural points:
> 
> * the map looks pretty good, but I think the sequence is
> misassigned in some regions (e.g. A118-A122 etc).  Automation is a
> good tool but a poor master, and extreme caution is required before
> taking the results too literally.  Usually you'd expect a 1.4
> Angstrom to be easy to autobuild but I recently had a sequence
> misassignment at just that resolution. That map was trivial to
> interpret with the correct sequence however - one of the joys of
> working with Arp/wArp at 1.4 Angstrom.
> 
> * the large number of positive difference density blobs and water
> molecules clustered in what otherwise would be the solvent void
> strongly suggest that there's a second molecule present.
> 
> 
> If I take redfluorescentprotein_refine_10.pdb (waters removed) and
> exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two
> molecules, it finds them quite successfully.  (for the record an
> LLG of 15111 using nominal sequence identity of 90%).  I will send
> this to you off-list.  Please note that Phaser is using a different
> origin for this molecular replacement solution so the coordinates
> and your previous map do not overlap.
> 
> This rather nicely explains why your structure had an R-factor in
> the 40's despite being a half-way decent model.  The new MR
> solution has an R-free in the 30's in the phenix.refine job I'm
> running right now.
> 
> 
> Going forward I suggest you utilize the Arp/wArp program to
> autobuild your structure for you, starting from the molecular
> replacement solution (or, perhaps with it stripped to ALA).  While
> you could use Autobuild, this is the CCP4 list and so you should
> use CCP4 programs.
> 
> Phil Jeffrey Princeton
> 
> 
> On 3/27/13 12:22 PM, Tom Van den Bergh wrote: Dear members of
> ccp4bb,
> 
> I need some help with the refinement of my structure of a variant
> of mRFP (monomer red fluorescent protein, sequence in attachment).
> I have done molecular replacement with phaser with model 2VAD of
> protein database. Then i have done some model building
> phenix.autobuild. (2 pdb's (overall...), freeR flags and log file
> attached) When i refine with phenix.refine my structure i get a
> R-value of 0,42 which is still way too high. (redfluorescent
> protein.pdb, .mtz and logfile attached) When i look at the
> structure in coot i find many unmodelled blobs and many outliers in
> density analysis and rotamer analysis. The problem is that there
> are so many problems with my structure, that i dont know where to
> begin. Could you try some refinement for me, because this is first
> structure that i need to solve as a student and i dont have too 
> many experience with it.
> 
> Greetings,
> 
> Tom
> 
> 
> 
> Roberto A. Steiner Group Leader Randall Division of Cell and
> Molecular Biophysics King'

Re: [ccp4bb] refinement protein structure

[Steiner, Roberto]

we should all chip in a water molecule or two and the second author becomes 
"the CCP4 community"….

On 27 Mar 2013, at 17:22, Antony Oliver 
 wrote:

> At the risk of being somewhat cheeky - perhaps I could claim second author?
> I too have successfully solved the structure - and I totally concur with Phil.
> Placing a second molecule in the asymmetric unit, essentially resolves the 
> perceived R-factor problem.
> 
> A good thorough manual inspection and rebuilding is *ALWAYS* good practice 
> for newcomers to the field.
> 
> Tony.
> 
> 
> On 27 Mar 2013, at 17:14, Petr Leiman 
> wrote:
> 
>> Since this is now public domain knowledge and if this gets ever published, 
>> Phil has my vote to be the first author!
>> 
>> Petr
>> 
>> 
>> On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:
>> 
>>> That's quite brave - shipping your entire structure to people that could be 
>>> actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.
>>> 
>>> Practical points:
>>> 
>>> * not everyone loves 12Mb of attachments in one email in their inbox, so if 
>>> you do this again please put the files on a webserver and point us there
>>> 
>>> Structural points:
>>> 
>>> * the map looks pretty good, but I think the sequence is misassigned in 
>>> some regions (e.g. A118-A122 etc).  Automation is a good tool but a poor 
>>> master, and extreme caution is required before taking the results too 
>>> literally.  Usually you'd expect a 1.4 Angstrom to be easy to autobuild but 
>>> I recently had a sequence misassignment at just that resolution. That map 
>>> was trivial to interpret with the correct sequence however - one of the 
>>> joys of working with Arp/wArp at 1.4 Angstrom.
>>> 
>>> * the large number of positive difference density blobs and water molecules 
>>> clustered in what otherwise would be the solvent void strongly suggest that 
>>> there's a second molecule present.
>>> 
>>> 
>>> If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
>>> exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two molecules, 
>>> it finds them quite successfully.  (for the record an LLG of 15111 using 
>>> nominal sequence identity of 90%).  I will send this to you off-list.  
>>> Please note that Phaser is using a different origin for this molecular 
>>> replacement solution so the coordinates and your previous map do not 
>>> overlap.
>>> 
>>> This rather nicely explains why your structure had an R-factor in the 40's 
>>> despite being a half-way decent model.  The new MR solution has an R-free 
>>> in the 30's in the phenix.refine job I'm running right now.
>>> 
>>> 
>>> Going forward I suggest you utilize the Arp/wArp program to autobuild your 
>>> structure for you, starting from the molecular replacement solution (or, 
>>> perhaps with it stripped to ALA).  While you could use Autobuild, this is 
>>> the CCP4 list and so you should use CCP4 programs.
>>> 
>>> Phil Jeffrey
>>> Princeton
>>> 
>>> 
>>> On 3/27/13 12:22 PM, Tom Van den Bergh wrote:
 Dear members of ccp4bb,
 
 I need some help with the refinement of my structure of a variant of
 mRFP (monomer red fluorescent protein, sequence in attachment). I have
 done molecular replacement with phaser with model 2VAD of protein
 database. Then i have done some model building phenix.autobuild. (2
 pdb's (overall...), freeR flags and log file attached) When i refine
 with phenix.refine my structure i get a R-value of 0,42 which is still
 way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
 When i look at the structure in coot i find many unmodelled blobs and
 many outliers in density analysis and rotamer analysis. The problem is
 that there are so many problems with my structure, that i dont know
 where to begin. Could you try some refinement for me, because this is
 first structure that i need to solve as a student and i dont have too
 many experience with it.
 
 Greetings,
 
 Tom
 
> 

Roberto A. Steiner
Group Leader
Randall Division of Cell and Molecular Biophysics
King's College London
roberto.stei...@kcl.ac.uk

Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL
London

Phone 0044 20 78488216
Fax0044 20 78486435

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

At the risk of being somewhat cheeky - perhaps I could claim second author?
I too have successfully solved the structure - and I totally concur with Phil.
Placing a second molecule in the asymmetric unit, essentially resolves the 
perceived R-factor problem.

A good thorough manual inspection and rebuilding is *ALWAYS* good practice for 
newcomers to the field.

Tony.


On 27 Mar 2013, at 17:14, Petr Leiman 
 wrote:

> Since this is now public domain knowledge and if this gets ever published, 
> Phil has my vote to be the first author!
> 
> Petr
> 
> 
> On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:
> 
>> That's quite brave - shipping your entire structure to people that could be 
>> actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.
>> 
>> Practical points:
>> 
>> * not everyone loves 12Mb of attachments in one email in their inbox, so if 
>> you do this again please put the files on a webserver and point us there
>> 
>> Structural points:
>> 
>> * the map looks pretty good, but I think the sequence is misassigned in some 
>> regions (e.g. A118-A122 etc).  Automation is a good tool but a poor master, 
>> and extreme caution is required before taking the results too literally.  
>> Usually you'd expect a 1.4 Angstrom to be easy to autobuild but I recently 
>> had a sequence misassignment at just that resolution. That map was trivial 
>> to interpret with the correct sequence however - one of the joys of working 
>> with Arp/wArp at 1.4 Angstrom.
>> 
>> * the large number of positive difference density blobs and water molecules 
>> clustered in what otherwise would be the solvent void strongly suggest that 
>> there's a second molecule present.
>> 
>> 
>> If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
>> exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two molecules, 
>> it finds them quite successfully.  (for the record an LLG of 15111 using 
>> nominal sequence identity of 90%).  I will send this to you off-list.  
>> Please note that Phaser is using a different origin for this molecular 
>> replacement solution so the coordinates and your previous map do not overlap.
>> 
>> This rather nicely explains why your structure had an R-factor in the 40's 
>> despite being a half-way decent model.  The new MR solution has an R-free in 
>> the 30's in the phenix.refine job I'm running right now.
>> 
>> 
>> Going forward I suggest you utilize the Arp/wArp program to autobuild your 
>> structure for you, starting from the molecular replacement solution (or, 
>> perhaps with it stripped to ALA).  While you could use Autobuild, this is 
>> the CCP4 list and so you should use CCP4 programs.
>> 
>> Phil Jeffrey
>> Princeton
>> 
>> 
>> On 3/27/13 12:22 PM, Tom Van den Bergh wrote:
>>> Dear members of ccp4bb,
>>> 
>>> I need some help with the refinement of my structure of a variant of
>>> mRFP (monomer red fluorescent protein, sequence in attachment). I have
>>> done molecular replacement with phaser with model 2VAD of protein
>>> database. Then i have done some model building phenix.autobuild. (2
>>> pdb's (overall...), freeR flags and log file attached) When i refine
>>> with phenix.refine my structure i get a R-value of 0,42 which is still
>>> way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
>>> When i look at the structure in coot i find many unmodelled blobs and
>>> many outliers in density analysis and rotamer analysis. The problem is
>>> that there are so many problems with my structure, that i dont know
>>> where to begin. Could you try some refinement for me, because this is
>>> first structure that i need to solve as a student and i dont have too
>>> many experience with it.
>>> 
>>> Greetings,
>>> 
>>> Tom
>>> 

Re: [ccp4bb] refinement protein structure

[Petr Leiman]

Since this is now public domain knowledge and if this gets ever published, Phil 
has my vote to be the first author!

Petr


On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:

> That's quite brave - shipping your entire structure to people that could be 
> actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.
> 
> Practical points:
> 
> * not everyone loves 12Mb of attachments in one email in their inbox, so if 
> you do this again please put the files on a webserver and point us there
> 
> Structural points:
> 
> * the map looks pretty good, but I think the sequence is misassigned in some 
> regions (e.g. A118-A122 etc).  Automation is a good tool but a poor master, 
> and extreme caution is required before taking the results too literally.  
> Usually you'd expect a 1.4 Angstrom to be easy to autobuild but I recently 
> had a sequence misassignment at just that resolution. That map was trivial to 
> interpret with the correct sequence however - one of the joys of working with 
> Arp/wArp at 1.4 Angstrom.
> 
> * the large number of positive difference density blobs and water molecules 
> clustered in what otherwise would be the solvent void strongly suggest that 
> there's a second molecule present.
> 
> 
> If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
> exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two molecules, 
> it finds them quite successfully.  (for the record an LLG of 15111 using 
> nominal sequence identity of 90%).  I will send this to you off-list.  Please 
> note that Phaser is using a different origin for this molecular replacement 
> solution so the coordinates and your previous map do not overlap.
> 
> This rather nicely explains why your structure had an R-factor in the 40's 
> despite being a half-way decent model.  The new MR solution has an R-free in 
> the 30's in the phenix.refine job I'm running right now.
> 
> 
> Going forward I suggest you utilize the Arp/wArp program to autobuild your 
> structure for you, starting from the molecular replacement solution (or, 
> perhaps with it stripped to ALA).  While you could use Autobuild, this is the 
> CCP4 list and so you should use CCP4 programs.
> 
> Phil Jeffrey
> Princeton
> 
> 
> On 3/27/13 12:22 PM, Tom Van den Bergh wrote:
>> Dear members of ccp4bb,
>> 
>> I need some help with the refinement of my structure of a variant of
>> mRFP (monomer red fluorescent protein, sequence in attachment). I have
>> done molecular replacement with phaser with model 2VAD of protein
>> database. Then i have done some model building phenix.autobuild. (2
>> pdb's (overall...), freeR flags and log file attached) When i refine
>> with phenix.refine my structure i get a R-value of 0,42 which is still
>> way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
>> When i look at the structure in coot i find many unmodelled blobs and
>> many outliers in density analysis and rotamer analysis. The problem is
>> that there are so many problems with my structure, that i dont know
>> where to begin. Could you try some refinement for me, because this is
>> first structure that i need to solve as a student and i dont have too
>> many experience with it.
>> 
>> Greetings,
>> 
>> Tom
>> 

Re: [ccp4bb] refinement protein structure

[Phil Jeffrey]

That's quite brave - shipping your entire structure to people that could 
be actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.


Practical points:

* not everyone loves 12Mb of attachments in one email in their inbox, so 
if you do this again please put the files on a webserver and point us there


Structural points:

* the map looks pretty good, but I think the sequence is misassigned in 
some regions (e.g. A118-A122 etc).  Automation is a good tool but a poor 
master, and extreme caution is required before taking the results too 
literally.  Usually you'd expect a 1.4 Angstrom to be easy to autobuild 
but I recently had a sequence misassignment at just that resolution. 
That map was trivial to interpret with the correct sequence however - 
one of the joys of working with Arp/wArp at 1.4 Angstrom.


* the large number of positive difference density blobs and water 
molecules clustered in what otherwise would be the solvent void strongly 
suggest that there's a second molecule present.



If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two 
molecules, it finds them quite successfully.  (for the record an LLG of 
15111 using nominal sequence identity of 90%).  I will send this to you 
off-list.  Please note that Phaser is using a different origin for this 
molecular replacement solution so the coordinates and your previous map 
do not overlap.


This rather nicely explains why your structure had an R-factor in the 
40's despite being a half-way decent model.  The new MR solution has an 
R-free in the 30's in the phenix.refine job I'm running right now.



Going forward I suggest you utilize the Arp/wArp program to autobuild 
your structure for you, starting from the molecular replacement solution 
(or, perhaps with it stripped to ALA).  While you could use Autobuild, 
this is the CCP4 list and so you should use CCP4 programs.


Phil Jeffrey
Princeton


On 3/27/13 12:22 PM, Tom Van den Bergh wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of
mRFP (monomer red fluorescent protein, sequence in attachment). I have
done molecular replacement with phaser with model 2VAD of protein
database. Then i have done some model building phenix.autobuild. (2
pdb's (overall...), freeR flags and log file attached) When i refine
with phenix.refine my structure i get a R-value of 0,42 which is still
way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
When i look at the structure in coot i find many unmodelled blobs and
many outliers in density analysis and rotamer analysis. The problem is
that there are so many problems with my structure, that i dont know
where to begin. Could you try some refinement for me, because this is
first structure that i need to solve as a student and i dont have too
many experience with it.

Greetings,

Tom

Re: [ccp4bb] refinement protein structure

[Eugene Osipov]

Dear Tom,
I think that only way for you is to make this structure yourself because it
is only way to learn something (at first you must ask in your lab).
Anyway, as student I can make some brief suggestions and guides for your
refinement
1) Find as much info about your protein as possible, especially look at
previous structure (read 2VAD related structure as first step)
2) Before refinement you must find conservative domains inside your protein
or in other words red fluorescent protein motifs - these residues will be
your starting point
3) As this motif is conserved and probably MR will place them in correct
orientation you must move forth and appropriately mutate incorrect
residues, if their CA fits good. If your density becomes bad you will
remove incorrect residues from this point, later by FOFC map you will place
the rest of them in correct orientation. Now move back from the conserved
motif in the same manner.
4) After this run 8-10 rounds of refinement, generate map. Repeat step 3
Hope this will help you somehow

Important note for you: do not show your data before publication of pdb.

2013/3/27 Tom Van den Bergh 

>  Dear members of ccp4bb,
>
> I need some help with the refinement of my structure of a variant of mRFP
> (monomer red fluorescent protein, sequence in attachment). I have done
> molecular replacement with phaser with model 2VAD of protein database. Then
> i have done some model building phenix.autobuild. (2 pdb's (overall...),
> freeR flags and log file attached) When i refine with phenix.refine my
> structure i get a R-value of 0,42 which is still way too high.
> (redfluorescent protein.pdb, .mtz and logfile attached) When i look at the
> structure in coot i find many unmodelled blobs and many outliers in density
> analysis and rotamer analysis. The problem is that there are so many
> problems with my structure, that i dont know where to begin. Could you try
> some refinement for me, because this is first structure that i need to
> solve as a student and i dont have too many experience with it.
>
> Greetings,
>
> Tom
>
>


-- 
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

Dear Tom,

Q1: Are you really supposed to posting this information here?
Q2: Is there really NOT anyone you can ask in your own department to help you 
with this?

Some initial hints/ideas/suggestions though - although I actually assume this 
is meant to be a learning exercise for you …?

1) Have you got the correct spacegroup?
2) Have you got enough molecules in the asymmetric unit?
3) Do your placed molecules pack together to form a sensible crystal lattice?

Tony.

On 27 Mar 2013, at 16:22, Tom Van den Bergh 
mailto:tom.vandenbe...@student.kuleuven.be>>
 wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of mRFP 
(monomer red fluorescent protein, sequence in attachment). I have done 
molecular replacement with phaser with model 2VAD of protein database. Then i 
have done some model building phenix.autobuild. (2 pdb's (overall...), freeR 
flags and log file attached) When i refine with phenix.refine my structure i 
get a R-value of 0,42 which is still way too high. (redfluorescent protein.pdb, 
.mtz and logfile attached) When i look at the structure in coot i find many 
unmodelled blobs and many outliers in density analysis and rotamer analysis. 
The problem is that there are so many problems with my structure, that i dont 
know where to begin. Could you try some refinement for me, because this is 
first structure that i need to solve as a student and i dont have too many 
experience with it.

Greetings,

Tom

[ccp4bb] Postdoctoral fellowship for membrane protein crystallography

[Spudich, John L]

A postdoctoral position is currently available in the Center for Membrane 
Biology at UT-Houston to study microbial rhodopsins (channelrhodopsins, other 
sensory rhodopsins, and ion transporters) by x-ray crystallography and 
biochemical/biophysical methods. Applicants must be highly motivated and have 
demonstrated experience (i.e. publications) with protein crystallography, e.g. 
recombinant protein expression, purification, crystallization, and diffraction 
data collection.  Experience with membrane proteins is an advantage. The 
position requires a recent Ph.D. in one of the natural sciences relevant to 
structural biology. Salary will be commensurate with experience and will be in 
addition to a fringe benefit package. Our lab in the Center for Membrane 
Biology, Department of Biochemistry & Molecular Biology, University of Texas 
Medical School at Houston, is fully equipped for molecular biology, protein 
chemistry, and protein crystallography. In addition, our University is part of 
the Molecular Biology Consortium that operates its own high flux MAD-capable 
x-ray beamline at ALS Berkeley, California.  Apply to 
john.l.spud...@uth.tmc.edu including your CV and names of 3 references.

--
John L. Spudich, Ph.D.
Robert A. Welch Distinguished Chair in Chemistry
Director, Center for Membrane Biology
Professor, Department of Biochemistry & Molecular Biology
University of Texas Medical School
6431 Fannin Street, MSB 6.130
Houston TX 77030 USA

Re: [ccp4bb] refinement protein structure

[Petr Leiman]

Dear Tom,

As far as I know you are the first person who sent his/her data that are 
supposed to be _private_ to 1000+ CCP4BB subscribers.

The person who suggested you send this type of message to CCP4BB board played a 
very cruel practical joke on you.

To CCP4BB support staff: Is it possible to block emails with 1+MB attachments?

Thank you,

Petr


Prof. Petr Leiman
EPFL
BSP-415
CH-1015 Lausanne
Switzerland

On Mar 27, 2013, at 5:22 PM, Tom Van den Bergh wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of mRFP 
(monomer red fluorescent protein, sequence in attachment). I have done 
molecular replacement with phaser with model 2VAD of protein database. Then i 
have done some model building phenix.autobuild. (2 pdb's (overall...), freeR 
flags and log file attached) When i refine with phenix.refine my structure i 
get a R-value of 0,42 which is still way too high. (redfluorescent protein.pdb, 
.mtz and logfile attached) When i look at the structure in coot i find many 
unmodelled blobs and many outliers in density analysis and rotamer analysis. 
The problem is that there are so many problems with my structure, that i dont 
know where to begin. Could you try some refinement for me, because this is 
first structure that i need to solve as a student and i dont have too many 
experience with it.

Greetings,

Tom

Re: [ccp4bb] CCP4BB Digest - 26 Mar 2013 to 27 Mar 2013 - Special issue (#2013-90)

[Shivam Mishra]

Hi,
Please unsubscribe me from this list as the posts are not relevant to my 
research profile.
Kind Regards,
Shivam
On 28 Mar, 2013, at 12:32 AM, CCP4BB automatic digest system 
 wrote:

> There are 9 messages totaling 164114 lines in this issue.
> 
> Topics in this special issue:
> 
>  1. ./configure problem (2)
>  2. High number of clashes with high TFZ score (2)
>  3. off topic, BN PAGE
>  4. IUCr Travel Grants Available - (11th International Conference on
> Biological Synchrotron Radiation (BSR), Hamburg, Germany, 8-11 Sept 2013)
>  5. Rec- variant of E coli B (2)
>  6. refinement protein structure
> 
> --
> 
> Date:Tue, 26 Mar 2013 20:16:12 -0400
> From:A K 
> Subject: ./configure problem
> 
> Hi there,
> I am trying to install ccp4 on a scientific linux system. I've got stuck in
> the step where I need to configure the system. After sourcing ccp4.setup, I
> go to the main ccp4 folder and try ./configure help, but I get the error
> "bash: ./configure: No such file or directory". It looks as if the
> configure script has not been included in my tar file. The file I unzipped
> was called ccp4-6.3.0.1-arp-linux-x86_64.tar.gz. Any suggestion is highly
> appreciated.
> Alex
> 
> --
> 
> Date:Tue, 26 Mar 2013 21:55:38 -0400
> From:Zhijie Li 
> Subject: Re: ./configure problem
> 
> Hi Alex,
> 
> The CCP4 package you downloaded seems to be a prebuilt binary package for the 
> X86_64 architecture. You do not need to compile yourself - and you can't 
> without the source code. Running ./configure is for configuring the 
> compilation environment before running "make" to compile source codes, which 
> dose not apply to binary packages.
> 
> I guess you probably was following this page: 
> http://www.ccp4.ac.uk/dist/INSTALL.html. The information on that page is for 
> compiling the source code, not for the binary packages. The layout of that 
> page is a little confusing: it does mention how to download the binary 
> packages and started to say "then first you need to decide where you want to 
> put the software", but then it suddenly jumps to how to build the CCP4 from 
> source code. I think this might be something the authors want to fix.
> 
> For prebuilt binary packages, please take a look at the INSTALL file in the 
> unpacked CCP4 directory for installation instructions. The CCP4 binary 
> installation is very simple: run the BINARY.setup in the unpacked CCP4 
> directory. After it is done, append this line to /etc/bashrc (assuming your 
> default shell is bash):
> 
> source /path_to_ccp4/ccp4.setup-sh
> 
> Then open a new shell, type ccp4i, you should see the CCP4 user interface.
> 
> Zhijie
> 
> 
> 
> 
> 
> From: A K 
> Sent: Tuesday, March 26, 2013 8:16 PM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [ccp4bb] ./configure problem
> 
> 
> Hi there,
> I am trying to install ccp4 on a scientific linux system. I've got stuck in 
> the step where I need to configure the system. After sourcing ccp4.setup, I 
> go to the main ccp4 folder and try ./configure help, but I get the error 
> "bash: ./configure: No such file or directory". It looks as if the configure 
> script has not been included in my tar file. The file I unzipped was called 
> ccp4-6.3.0.1-arp-linux-x86_64.tar.gz. Any suggestion is highly appreciated.
> Alex
> 
> --
> 
> Date:Wed, 27 Mar 2013 17:32:46 +0900
> From:Rojan Shrestha 
> Subject: High number of clashes with high TFZ score
> 
> Hello,
> 
> 
> 
> I am trying phasing using homology models with Phaser. We obtained very high
> TFZ scores with many clashes. The number of clashes are around more than
> 100. The space group is P321. We tried two other space groups P3121 and
> P3221 but both gave same problem. Can you help on this problem?
> 
> 
> 
> Regards,
> 
> 
> 
> Rojan
> 
> --
> 
> Date:Wed, 27 Mar 2013 08:42:19 +
> From:Randy Read 
> Subject: Re: High number of clashes with high TFZ score
> 
> In such circumstances, one of the most common issues is that the space group 
> is incorrect, which is often because twinning gives higher apparent symmetry. 
>  If (for example) the true space group is P3, there will be solutions in P321 
> that place the molecules correctly around the 3-fold axis (so they give a 
> better than random score in the translation search), but the extra symmetry 
> causes clashes.  Have you looked at the results of twinning tests?
> 
> If that doesn't explain your problem, you could send me the log file from one 
> of the Phaser runs (probably offline) and I could see if there are any hints 
> of other explanations in the output.
> 
> Best wishes,
> 
> Randy Read
> 
> On 27 Mar 2013, at 08:32, Rojan Shrestha  wrote:
> 
>> Hello,
>> 
>> I am trying phasing using homology models with Phaser. We obtained very high 
>> TFZ scores with many clashes. The number of clashes are around more t

Re: [ccp4bb] Rec- variant of E coli B

[Marko Hyvonen]

Hi Mark,

BLR(DE3) should be what you are after.

http://www.emdmillipore.com/life-science-research/blrde3-competent-cells/EMD_BIO-69053/p_yYub.s1Ol18AAAEjORl9.zLX

hth, Marko

On Wed, 27 Mar 2013, Mark J van Raaij wrote:


Dear All,
we were wondering if knows of an Escherichia coli B strain that is Rec 
deficient (Rec-).
We want to compare certain properties of E coli B and K12, and for the 
experiment it would be best to use a Rec- variant.
We have TOP10, which is a K12 derivative that is Rec-, but derivatives of B 
like BL21 are Rec+.
Have a happy Easter,
Mark

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij




 _

 Marko Hyvonen
 Department of Biochemistry, University of Cambridge
 ma...@cryst.bioc.cam.ac.uk
 http://www-cryst.bioc.cam.ac.uk/groups/hyvonen
 tel:+44-(0)1223-766 044
 mobile: +44-(0)7796-174 877
 fax:+44-(0)1223-766 002
 --

[ccp4bb] Rec- variant of E coli B

[Mark J van Raaij]

Dear All,
we were wondering if knows of an Escherichia coli B strain that is Rec 
deficient (Rec-).
We want to compare certain properties of E coli B and K12, and for the 
experiment it would be best to use a Rec- variant.
We have TOP10, which is a K12 derivative that is Rec-, but derivatives of B 
like BL21 are Rec+.
Have a happy Easter,
Mark

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij

[ccp4bb] IUCr Travel Grants Available - (11th International Conference on Biological Synchrotron Radiation (BSR), Hamburg, Germany, 8-11 Sept 2013)

[margret]

*IUCr Travel Grants Available!! *
*APPLY NOW!!*

*11th International Conference on Biological Synchrotron Radiation (BSR)*
*Hamburg, Germany*
*8th - 11th September 2013*

We are pleased to announce that we have a number of travel awards 
available for young scientists, kindly provided by the International 
Union of Crystallography (IUCr). Up to 450 Euros will be awarded to 
outstanding students for supporting travel costs. Applicants must be 
graduate students, post-graduate students or post doctoral fellows under 
the age of 30 (exceptionally 35). If you are eligible and wish to apply, 
please indicate this when you register for the conference. Successful 
applicants will be notified in the summer.


The 11th International conference on Biological Synchrotron Radiation 
(BSR) aims to bring together scientists involved in the methodical 
developments on synchrotron and laser sources with a broad community of 
biologists with an ambition to make the best use of the most advanced 
infrastructures in structural biology.


Topics include:

Biological small angle X-ray scattering

Macromolecular X-ray crystallography

Biological X-ray imaging and spectroscopy

Biological sample preparation

Free electron laser applications

Synchrotron instrumentation

Industrial applications

Structural biology hybrid methods

Ultimate storage rings


For more information and to register, please go to: www.bsr2013.org 




Looking forward to seeing you in Hamburg!

Margret Fischer on behalf of

Matthias Wilmanns,  Dmitri Svergun (EMBL Hamburg)

[ccp4bb] off topic, BN PAGE

[Careina Edgooms]

Hi

Has anyone found the coomassie in a BN PAGE to be interfering with the 
oligomeric structure of their protein? If so, how did you deal with this?
Thanks

Careina

Re: [ccp4bb] High number of clashes with high TFZ score

[Randy Read]

In such circumstances, one of the most common issues is that the space group is 
incorrect, which is often because twinning gives higher apparent symmetry.  If 
(for example) the true space group is P3, there will be solutions in P321 that 
place the molecules correctly around the 3-fold axis (so they give a better 
than random score in the translation search), but the extra symmetry causes 
clashes.  Have you looked at the results of twinning tests?

If that doesn't explain your problem, you could send me the log file from one 
of the Phaser runs (probably offline) and I could see if there are any hints of 
other explanations in the output.

Best wishes,

Randy Read

On 27 Mar 2013, at 08:32, Rojan Shrestha  wrote:

> Hello,
>  
> I am trying phasing using homology models with Phaser. We obtained very high 
> TFZ scores with many clashes. The number of clashes are around more than 100. 
> The space group is P321. We tried two other space groups P3121 and P3221 but 
> both gave same problem. Can you help on this problem?
>  
> Regards,
>  
> Rojan

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

[ccp4bb] High number of clashes with high TFZ score

[Rojan Shrestha]

Hello,

 

I am trying phasing using homology models with Phaser. We obtained very high
TFZ scores with many clashes. The number of clashes are around more than
100. The space group is P321. We tried two other space groups P3121 and
P3221 but both gave same problem. Can you help on this problem?

 

Regards,

 

Rojan