[ccp4bb] Arp/Wrap problem while model building

[Rojan Shrestha]

Hello,

 

I am facing a problem in running "auto_tracing" in arp/wrap. At first I have
tried using command line mode however I missed R-free value. I have added
the "freelabin " FREE=FREE "" and have got an error 

 

"Refmac_5.5.0102: Error in label assignments in LABIN"

 

In another way, I used ccp4i, here also the error was appeared as "Cannot
extract ARP/wARP asymmetric unit limit."

 

I guess the root of both errors are same. Have somebody faced same problems
before?

 

Could you tell me that how this type of problem can be solved?

 

Regards,

 

Rojan

[ccp4bb] low resolution refinement

[Qixu Cai]

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!

Re: [ccp4bb] low resolution refinement

[Elizabeth McSweeney]

 Hi

 I highly recommend reading the two Brunger papers below: they will
explain the important factors to take note of when refining low-resolution
structures.� I found them very useful when I was learning about
low-resolution refinement.

 I would suggest using the deformable elastic network with simulated
annealing in CNS, and then follow that with refinement in PHENIX using
tight geometery restraints and doing TLS and grouped-B factor refinement.�
Your problem is that at low resolutions, the data to parameters ratio is
low, and you need to use a number of restraints along with good
bulk-solvent and anisotropic corections in order to keep your R-factor and
R-free values within 3-4%.� Perhaps you could send your PHENIX input file
and we could see what you did and maybe point what needs to be corrected.

 Hope that was helpful.� good luck!

 Schroder, Levitt, Brunger, Super-resolution biomolecular crystallography
with low-resolution data Nature. 2010 Apr 22;464(7292):1218-22Brunger, AT
et al.� X-ray structure determination at low resolution. Acta Cryst D.
2009 Feb;65(Pt 2):128-33

 On Sat 07/09/11 4:59 AM , 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!

 

Re: [ccp4bb] low resolution refinement

[Robbie Joosten]

Dear Qixu,

 

refamac 5.6 works well at these resolutions. You can add commands to your 
refinement in CCP4i by using the 'Run and view command script' (or something 
like that) option and just typing in the extra commands. Jelly-body has worked 
very well for me (although I use tigheter restraints than the default). Also 
local NCS works well (provided you have NCS). I never used reference 
structures, but I heared good things about it. Don't forget to use riding 
hydrogens, for some reason it is not the deafault. 
 Perhaps you should also switch of the automatic X-ray weighting in favour of 
optimizing the matrix weight yourself (start with 0.05 and compare refinements 
for higher and lower values).

 

HTH,

Robbie

 

 

> Date: Sat, 9 Jul 2011 16:59:29 +0800
> From: caiq...@gmail.com
> Subject: [ccp4bb] low resolution refinement
> To: CCP4BB@JISCMAIL.AC.UK
>
> 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!

[ccp4bb] Fwd: [ccp4bb] Beta test versions of Mosflm & iMosflm

[harry powell]

Dear all

an addendum from Phil Evans - his aimless & pointless stuff is on our  
anonymous ftp site at


ftp.mrc-lmb.cam.ac.uk

then login with user name "anonymous" or "ftp", then cd to /pub/pre.  
Some web browsers (especially more modern ones) will take you  
straight there if you use the URL ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/


Note that he doesn't have a Windows build...

Begin forwarded message:


From: Phil Evans 
Date: 8 July 2011 17:27:43 BDT
To: Harry Powell 
Subject: Re: [ccp4bb] Beta test versions of Mosflm & iMosflm

I note you recommend Aimless.

It might be worth pointing out that if they are going to do this,  
they may want to install the ccp4i GUI, in which case they also  
need the updated gui for pointless, and maybe the updated exe


Relevant files in .../ftp/pub/pre/ are

Executables:
-rwxrw-r-- 1 pre pre 6858272 May 18 16:38 pointless-1.5.22.osx
-rwxr-xr-x 1 pre pre 6933140 May 18 16:41 pointless-1.5.22.linux
-rwxr-xr-x 1 pre pre 6757679 Jul  8 16:03 aimless-0.0.14.linux
-rwxr-xr-x 1 pre pre 7776865 Jul  8 16:03 aimless-0.0.14.linux64
-rwxrw-r-- 1 pre pre 6553648 Jul  8 17:23 aimless-0.0.14.osx

ccp4i tasks (Install New Task in ccp4i)
-rwxr--r-- 1 pre pre   11039 Jun 15 13:36  
pointless_ccp4i_1.3.tar.gz
-rwxr--r-- 1 pre pre   20412 Jul  8 16:03  
aimless_dev_ccp4i_0.8.tar.gz


Source code:
-rw-r--r-- 1 pre pre  411762 Jun 21 14:31 pointless-1.5.22.tar.gz
-rw-r--r-- 1 pre pre  447811 Jul  8 16:09 aimless-0.0.14.tar.gz

Documentation:
-rwxr--r-- 1 pre pre   54889 Jul  8 17:18 aimless.html
-rwxr--r-- 1 pre pre   54889 Jul  8 17:18 aimless.html


Phil

On 8 Jul 2011, at 16:57, Harry Powell wrote:


Dear all

New versions of ipmosflm (7.0.8) and the matched iMosflm (1.0.6)  
are now available for beta testing.


http://www.mrc-lmb.cam.ac.uk/harry/mosflm/betas/index.html

The changes in ipmosflm are of particular relevance to processing  
very weak Pilatus images (with background pixel values of zero),  
but will also have some beneficial effect for any fine sliced data  
and result in both more stable detector parameter refinement and  
improved intensity estimates. The new version is strongly  
recommended when trying to measure small anomalous differences.


The major change in iMosflm has eliminated the gradual slowing  
down of the rate of data processing when dealing with large  
datasets (>500 images) that is observed with iMosflm 1.0.5. Again,  
this is a particular issue with fine sliced Pilatus datasets.


We would welcome any feedback on the performance of the new release.


Harry
--
Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre,  
Hills Road, Cambridge, CB2 0QH


http://www.iucr.org/resources/commissions/crystallographic- 
computing/schools/mieres2011




Harry
--
Dr Harry Powell,
MRC Laboratory of Molecular Biology,
Hills Road,
Cambridge,
CB2 0QH

Re: [ccp4bb] Potential Space Group Issue

[harry powell]

Hi

I'd run the reflection file through pointless rather than rely on  
cell dimensions and/or systematic absences. This is a quick and easy  
test to do and more reliable. As Bert suggests, the only real way to  
know the symmetry is after successful structure solution and  
refinement (but even then you can be fooled...).


On 8 Jul 2011, at 21:07, Van Den Berg, Bert wrote:

I'd say its very likely to be orthorhombic. Refinement should tell  
you.its the best way to determine the space group anyway. Why  
do you doubt its orthorhombic? Is Vm reasonable?
It could be monoclinic and merohedrally winned with the beta angle  
very close to 90 degrees, but my money is on orthorhombic. if  
refinement fails I would try monoclinic plus/minus twinning. As for  
the operators, xx.triage will tell you and xx.refine will  
apply them for you during refinement;-)


Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji  
Edayathumangalam [r...@brandeis.edu]

Sent: Friday, July 08, 2011 3:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Potential Space Group Issue

Oops sorry for the slippery fingers. I meant h00, 0k0 and 00l in my  
original email and NOT "00h, 00k, 00l". Note the correction  
especially if you are a first-year graduate student trying to learn  
stuff from these emails :)


Raji



On Thu, Jul 7, 2011 at 11:30 PM, Raji Edayathumangalam  
mailto:r...@brandeis.edu>> wrote:

Hello Everyone,

I have a 3.1 Ang dataset for which I'd like to get to the bottom of  
what the correct space group is.


The current unit cell in p212121 is 98.123   101.095   211.201 
90.00090.00090.000
I fed the reflection data into Xtriage to look for twinning and  
pseudotranslational NCS and there is no indication for either issue  
in the Xtriage output. Also, all odd 00h, 00k, 00l reflections are  
systematically absent as they should be for p212121.


However, my colleague who is also working on the same dataset  
recently reprocessed the data in P21. Here's the cell in p21:

98.010  100.940  210.470  90.00  90.04  90.00 p21

I am not sure if BETA=90.04 is significant enough to treat as p21  
(0.04% deviation of beta angle from ideal lattice for p212121). I  
don't think so but I could be wrong. Could someone please clarify?


Also, what kind of twinning and twinning operators can relate a  
p212121 cell to a p21 cell with almost identical unit cell  
parameters as that of the p212121 cell and leave all systematic  
absences intact?


Thanks much.
Raji


---
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University




--

--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University


Harry
--
Dr Harry Powell,
MRC Laboratory of Molecular Biology,
Hills Road,
Cambridge,
CB2 0QH

[ccp4bb] Post-doc position in Structural Biology of RNA decay components near Paris (France)

[Marc Graille]

Post-doctoral position in Structural biology of RNA decay components.

Location: Orsay, near Paris (45 minutes by regular trains), France



A 2,5 years post-doctoral position, funded by the ANR (Agence  
Nationale pour la Recherche), is available to study structural aspects  
of protein-protein and protein-RNA complexes involved in eukaryotic  
mRNA decay.


For this purpose, the successful candidate will combine structural  
biology methods (X-ray crystallography and small angle X-ray  
scattering) together with biophysical approaches (ITC, DSC, MALLS,  
fluorescence polarization, CD, …). All these technologies are  
currently available at the state-of the-art structural biology and  
biophysical platforms implemented in the host institute IBBMC  
(Institute for Biophysics and Molecular and Cellular Biochemistry,  
CNRS & University Paris-Sud), i.e. 3 nanodrop crystallization robots  
(Tecan, Cartesian and Mosquito), a Formulatrix crystal imager, an  
ITC200, an in-house SAXS apparatus, … . The lab is also well equipped  
for protein production in E. coli or eukaryotic systems (P. pastoris  
and baculovirus) and protein purification (several AKTA purification  
systems).


The project will be carried-out under the supervision of Dr Marc  
Graille at IBBMC (Orsay, Paris-Sud University, France) in close  
collaboration with Dr Dominique Durand (IBBMC) for SAXS studies and Dr  
Séraphin (IGBMC, Illkirch, France) for functional studies. For recent  
publications related to the subject by Dr Graille and co-workers, see  
van den Elzen et al; Nat. Struct. Mol. Biol, 2010; Henri J et al, J.  
Biol. Chem., 2010; Rispal D et al, RNA, 2011 & Liger D, et al, Nuc.  
Acids Res., 2011.


The IBBMC, an institute from CNRS and University Paris-Sud (one of the  
largest and most renowned French universities), is located in a green  
and timbered campus, 25km south from Paris. The lab is within 10  
minutes walk from the train station, with regular connections to Paris  
city centre (45 minutes). The IBBMC is 2km away from the recently  
built French synchrotron SOLEIL, where we have regular access to  
protein crystallography (Proxima-1) and SAXS (Swing) beamlines.


		The successful candidate will have a PhD in protein biochemistry,  
biophysics, or structural biology. He/she should express strong  
interest in working at the interface between physics and biology.  
Expertise in RNA production and purification will be very much  
appreciated.


Net annual salary: around 27,000€.

Starting from January 2012.

Duration: 30 months.

Applications (including CV, covering and reference letters) or  
information requests should be sent to Marc Graille (marc.grai...@u-psud.fr 
) and Dominique Durand (dominique.dur...@u-psud.fr) before 15th of  
September, 2011.




Dr Marc Graille, CR1-CNRS,
Equipe "Fonction et Architecture des Assemblages Macromoléculaires"
IBBMC, CNRS UMR8619, Bât. 430 Université Paris-Sud   91405 ORSAY
Tel: 33 (0)1 69 15 31 57 / Fax: 33 (0)1 69 85 37 15

Re: [ccp4bb] how to distinguish between phase separation, spherulite, and microcrystalline?

[Seema Nath]

I also got something shown in pic.3 and supposed to be some oil droplet,when I 
tried to break them I found that was actually a hemisphere type material 
attached to the coverslip like a 2D circle and sphere like shape in the hanging 
drop, a small hanging-drop within a large one.Ammonium sulfate was used as 
precipitant,even I put it to run SDS-PAGE and it was (may be a quasi-crystal 
of) the protein of interest.

Re: [ccp4bb] Arp/Wrap problem while model building

[Victor Lamzin]

Dear Rojan,

On 09/07/2011 09:56, Rojan Shrestha wrote:


Hello,

I am facing a problem in running “auto_tracing” in arp/wrap. At first 
I have tried using command line mode however I missed R-free value. I 
have added the “freelabin “ FREE=FREE ”” and have got an error


“Refmac_5.5.0102: Error in label assignments in LABIN”



If your free column in the MTZ file is called 'FREE', then on the 
command line simply use

freelabin FREE

In another way, I used ccp4i, here also the error was appeared as 
“Cannot extract ARP/wARP asymmetric unit limit.”




Most likely ARP/wARP is not sourced. Type 'echo $warpbin' and see 
whether the outcome is something like:

/arp_warp/arp_warp_7.1/bin/bin-i386-Darwin

Please get back if there are problems.

With best regards,
Victor

Re: [ccp4bb] low resolution refinement

[CAI Qixu]

Hi,

Thank you for your papers. I will read them carefully.





2011/7/9 Elizabeth McSweeney 

> Hi
>
> I highly recommend reading the two Brunger papers below: they will explain
> the important factors to take note of when refining low-resolution
> structures.  I found them very useful when I was learning about
> low-resolution refinement.
>
> I would suggest using the deformable elastic network with simulated
> annealing in CNS, and then follow that with refinement in PHENIX using tight
> geometery restraints and doing TLS and grouped-B factor refinement.  Your
> problem is that at low resolutions, the data to parameters ratio is low, and
> you need to use a number of restraints along with good bulk-solvent and
> anisotropic corections in order to keep your R-factor and R-free values
> within 3-4%.  Perhaps you could send your PHENIX input file and we could see
> what you did and maybe point what needs to be corrected.
>
> Hope that was helpful.  good luck!
>
> Schroder, Levitt, Brunger, Super-resolution biomolecular crystallography
> with low-resolution data Nature. 2010 Apr 22;464(7292):1218-22 Brunger, AT
> et al.  X-ray structure determination at low resolution. Acta Cryst D. 2009
> Feb;65(Pt 2):128-33
>
>
> On Sat 07/09/11 4:59 AM , 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!
>
>

Re: [ccp4bb] low resolution refinement

[CAI Qixu]

Hi,

Thank you for your suggestion.
Could you tell me what is "riding hydrogens"?
And it seems there is not "reference model" function in refmac5.6?



2011/7/9 Robbie Joosten 

> Dear Qixu,
>
>
>
> refamac 5.6 works well at these resolutions. You can add commands to your
> refinement in CCP4i by using the 'Run and view command script' (or something
> like that) option and just typing in the extra commands. Jelly-body has
> worked very well for me (although I use tigheter restraints than the
> default). Also local NCS works well (provided you have NCS). I never used
> reference structures, but I heared good things about it. Don't forget to use
> riding hydrogens, for some reason it is not the deafault.
>  Perhaps you should also switch of the automatic X-ray weighting in favour
> of optimizing the matrix weight yourself (start with 0.05 and compare
> refinements for higher and lower values).
>
>
>
> HTH,
>
> Robbie
>
>
>
>
> 
> > Date: Sat, 9 Jul 2011 16:59:29 +0800
> > From: caiq...@gmail.com
> > Subject: [ccp4bb] low resolution refinement
> > To: CCP4BB@JISCMAIL.AC.UK
>  >
> > 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!
>

Re: [ccp4bb] low resolution refinement

[Robbie Joosten]

Hi Qixu,

 

In CCP4i the option is in the refinement parameters: 

Use hydrogen atoms: ["build all hydrogens"] and [] output to coordinate file

 

What is does is build all hydrogens at the expected coordinates and constrain 
them in refinement (i.e. adding hydrogens does not add extra parameters to the 
model). The effect on explaining your experminetal data is typically small, but 
the hydrogens help with the VdW restraints. In effect they reduce the number of 
bumps and improve your torsion angles.

 

You can use a reference structure to generate external restraints:

http://www.ysbl.york.ac.uk/~garib/refmac/data/refmac_news.html#External

I hope someone else on the BB can explain how. I think it is also explained in 
the talk and tutorials of the Refmac website.

 

HTH,

Robbie

  

> From: caiq...@gmail.com 
> Date: Sun, 10 Jul 2011 00:44:25 +0800 
> Subject: Re: [ccp4bb] low resolution refinement 
> To: robbie_joos...@hotmail.com 
> CC: CCP4BB@jiscmail.ac.uk 
> 
> Hi, 
> 
> Thank you for your suggestion. 
> Could you tell me what is "riding hydrogens"? 
> And it seems there is not "reference model" function in refmac5.6? 
> 
> 
> 
> 2011/7/9 Robbie Joosten 
> mailto:robbie_joos...@hotmail.com>> 
> Dear Qixu, 
> 
> 
> 
> refamac 5.6 works well at these resolutions. You can add commands to 
> your refinement in CCP4i by using the 'Run and view command script' (or 
> something like that) option and just typing in the extra commands. 
> Jelly-body has worked very well for me (although I use tigheter 
> restraints than the default). Also local NCS works well (provided you 
> have NCS). I never used reference structures, but I heared good things 
> about it. Don't forget to use riding hydrogens, for some reason it is 
> not the deafault. 
> Perhaps you should also switch of the automatic X-ray weighting in 
> favour of optimizing the matrix weight yourself (start with 0.05 and 
> compare refinements for higher and lower values). 
> 
> 
> 
> HTH, 
> 
> Robbie 
> 
> 
> 
> 
>  
> > Date: Sat, 9 Jul 2011 16:59:29 +0800 
> > From: caiq...@gmail.com 
> > Subject: [ccp4bb] low resolution refinement 
> > To: CCP4BB@JISCMAIL.AC.UK 
> > 
> > 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! 
> 

[ccp4bb] PhD position in Protein Crystallography and Biochemistry at University Kiel (Germany)

[Scheidig, Axel]

I would like to announce a PhD position in Protein crystallography and 
Biochemistry, on a project entitled "Structure/function studies of 
Toll-like receptor related proteins”. The project is associated with the 
centre of excellence “Inflammation at Interfaces”. The main goal of this 
PhD project is to structurally and functionally characterize on the 
molecular level of host-pathogen interactions.


More information can be found at
http://www.strubio.uni-kiel.de  go to “Open Positions”

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Axel


Prof. Dr. Axel J. Scheidig
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Re: [ccp4bb] low resolution refinement

[Rob Nicholls]

Hi,
External structure atomic bond distance restraints for use in refmac are 
generated using ProSMART. 
The webpage is here: http://www.ysbl.york.ac.uk/mxstat/Rob/index.html 

Email me if you want a pre-release version. 
Regards,
Rob



On 9 Jul 2011, at 18:00, Robbie Joosten wrote:

> Hi Qixu,
> 
> 
> 
> In CCP4i the option is in the refinement parameters: 
> 
> Use hydrogen atoms: ["build all hydrogens"] and [] output to coordinate file
> 
> 
> 
> What is does is build all hydrogens at the expected coordinates and constrain 
> them in refinement (i.e. adding hydrogens does not add extra parameters to 
> the model). The effect on explaining your experminetal data is typically 
> small, but the hydrogens help with the VdW restraints. In effect they reduce 
> the number of bumps and improve your torsion angles.
> 
> 
> 
> You can use a reference structure to generate external restraints:
> 
> http://www.ysbl.york.ac.uk/~garib/refmac/data/refmac_news.html#External
> 
> I hope someone else on the BB can explain how. I think it is also explained 
> in the talk and tutorials of the Refmac website.
> 
> 
> 
> HTH,
> 
> Robbie
> 
> 
> 
>> From: caiq...@gmail.com 
>> Date: Sun, 10 Jul 2011 00:44:25 +0800 
>> Subject: Re: [ccp4bb] low resolution refinement 
>> To: robbie_joos...@hotmail.com 
>> CC: CCP4BB@jiscmail.ac.uk 
>> 
>> Hi, 
>> 
>> Thank you for your suggestion. 
>> Could you tell me what is "riding hydrogens"? 
>> And it seems there is not "reference model" function in refmac5.6? 
>> 
>> 
>> 
>> 2011/7/9 Robbie Joosten 
>> mailto:robbie_joos...@hotmail.com>> 
>> Dear Qixu, 
>> 
>> 
>> 
>> refamac 5.6 works well at these resolutions. You can add commands to 
>> your refinement in CCP4i by using the 'Run and view command script' (or 
>> something like that) option and just typing in the extra commands. 
>> Jelly-body has worked very well for me (although I use tigheter 
>> restraints than the default). Also local NCS works well (provided you 
>> have NCS). I never used reference structures, but I heared good things 
>> about it. Don't forget to use riding hydrogens, for some reason it is 
>> not the deafault. 
>> Perhaps you should also switch of the automatic X-ray weighting in 
>> favour of optimizing the matrix weight yourself (start with 0.05 and 
>> compare refinements for higher and lower values). 
>> 
>> 
>> 
>> HTH, 
>> 
>> Robbie 
>> 
>> 
>> 
>> 
>>  
>>> Date: Sat, 9 Jul 2011 16:59:29 +0800 
>>> From: caiq...@gmail.com 
>>> Subject: [ccp4bb] low resolution refinement 
>>> To: CCP4BB@JISCMAIL.AC.UK 
>>> 
>>> 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! 
>>

Re: [ccp4bb] low resolution refinement

[Steiner, Roberto]

Dear CAI Qixu

Refmac does an excellent job at low resolution. Many of the features available 
in version 5.6.x are described in a very recent Refmac paper

Murshudov et al. REFMAC5 for the refinement of macromolecular crystal 
structures.
Acta Crystallogr D Biol Crystallogr (2011) vol. 67 (Pt 4) pp. 355-67

Guidelines for Refmac use (including new functions) can be found at 
http://www.ysbl.york.ac.uk/~garib/refmac/data/refmac_news.html

For example:
Jelly body restraints are controlled by

ridge distance sigma  # Default 0.1
ridge distance dmax   # Default 4.2
ridge atoms 
ridge bvalue 

keywords. You will find info on this under Harmonic distance restraints (Ridge 
regression)

External restraints (distance) are controlled by a syntax like that below:

exte dist first chain A residue   78 atom  O   second chain A residue   82 atom 
 N   value 3.000 sigma 0.05

More information at under External restraints. As Rob pointed out ProSMART can 
generate the above type of restraints for you.
Phenix can also provide a list of external distance restraints which can be 
interpreted by Refmac
phenix.secondary_structure_restraints model.pdb format=refmac


Best wishes
Roberto



On 9 Jul 2011, at 17:44, CAI Qixu wrote:

Hi,

Thank you for your suggestion.
Could you tell me what is "riding hydrogens"?
And it seems there is not "reference model" function in refmac5.6?



2011/7/9 Robbie Joosten 
mailto:robbie_joos...@hotmail.com>>
Dear Qixu,



refamac 5.6 works well at these resolutions. You can add commands to your 
refinement in CCP4i by using the 'Run and view command script' (or something 
like that) option and just typing in the extra commands. Jelly-body has worked 
very well for me (although I use tigheter restraints than the default). Also 
local NCS works well (provided you have NCS). I never used reference 
structures, but I heared good things about it. Don't forget to use riding 
hydrogens, for some reason it is not the deafault.
 Perhaps you should also switch of the automatic X-ray weighting in favour of 
optimizing the matrix weight yourself (start with 0.05 and compare refinements 
for higher and lower values).



HTH,

Robbie





> Date: Sat, 9 Jul 2011 16:59:29 +0800
> From: caiq...@gmail.com
> Subject: [ccp4bb] low resolution refinement
> To: CCP4BB@JISCMAIL.AC.UK
>
> 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!


Roberto Steiner, PhD
Randall Division of Cell and Molecular Biophysics Group Leader
King's College London

Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL, London, UK
Tel 0044-20-78488216
Fax 0044-20-78486435
roberto.stei...@kcl.ac.uk