Re: [ccp4bb] How to reduce free-R factor
Hi Pankaj, if you are refining in phenix.refine: - is automatic ordered solvent (water) update is turned on; - you can try weight optimization: "optimize_wxc=true optimize_wxu=true"; - is NCS available. Use it if so. In fact, the Rworks seems fine, but the gap Rfree-Rwork seems large. Insignificant remark: TLS is a more adequate model for ADPs, and not the tool to reduce R-factors. Good luck! Pavel. On 12/3/09 9:07 PM, Pankaj Chauhan wrote: Hi all, In one of my 2.52 A structure, which is dimeric, the R-factor is 21.4 and free-R is 29.3. I have tried all ways to reduce the free-R including TLS (even by defining the number of TLS group per chain), but still factors are not getting reduced. Even i have tried some other space group, but no use. Even i have the same structure with some complex at 1.86 A in the same space group , but everything is fine in that. Any suggestion, Regards, -- Pankaj Chauhan Senior Research Fellow, Graduate student Crystallography Laboratory Division of Protein Science & Engg. IMTECH , Chandigarh, INDIA
[ccp4bb] How to reduce free-R factor
Hi all, In one of my 2.52 A structure, which is dimeric, the R-factor is 21.4 and free-R is 29.3. I have tried all ways to reduce the free-R including TLS (even by defining the number of TLS group per chain), but still factors are not getting reduced. Even i have tried some other space group, but no use. Even i have the same structure with some complex at 1.86 A in the same space group , but everything is fine in that. Any suggestion, Regards, -- Pankaj Chauhan Senior Research Fellow, Graduate student Crystallography Laboratory Division of Protein Science & Engg. IMTECH , Chandigarh, INDIA
Re: [ccp4bb] How to add a single water molecule
Frederic VELLIEUX wrote: Hi Rui, Use the control key (pressed down) to translate the map and get the center point on top of your positive density (using the mouse at the same time, left button I think). Do not hold cntrl any more to rotate the map by 90 degrees. Cntrl again to move the map etc etc until the center point is right in the middle of the density. Then you use the menu, add atom, select Water, make sure you insert into your current molecule. One water atom added, done. That is a fair enough way if you want to add one a single water (as per the question). If you want to add several, I suggest the following (add-key-binding "Add Water +" "W" (lambda () (blob-under-pointer-to-screen-centre) (place-typed-atom-at-pointer "Water") (refine-active-residue))) Then point at a water blob with the mouse and use Shift-W Paul.
Re: [ccp4bb] How to add a single water molecule
Thank you all for the quick reply. Kim and Ingrid both pointed me to the same direction!! Thanks a bunch :-) On Thu, Dec 3, 2009 at 2:37 PM, Frederic VELLIEUX < frederic.velli...@orange.fr> wrote: > Hi Rui, > > I am at home so this is all from memory. In coot. > > Use the control key (pressed down) to translate the map and get the center > point on top of your positive density (using the mouse at the same time, > left button I think). Do not hold cntrl any more to rotate the map by 90 > degrees. Cntrl again to move the map etc etc until the center point is right > in the middle of the density. > > Then you use the menu, add atom, select Water, make sure you insert into > your current molecule. One water atom added, done. > > Repeat the process until you're finished adding waters. Then save as > usual... > > HTH, > > Fred. > > > Message du 03/12/09 19:59 > > De : "rui" > > A : CCP4BB@JISCMAIL.AC.UK > > Copie à : > > Objet : [ccp4bb] How to add a single water molecule > > > > Hi, All, > > > > I tried to use phenix to add water molecules, and when I check those > waters > > in coot, I can easily delete them if they are not in right spot. However, > if > > I see some clear positive maps that seems like water, can I add manually > at > > that position? How to do this in coot?Or any other way? > > >
Re: [ccp4bb] How to add a single water molecule
Hi Rui, I am at home so this is all from memory. In coot. Use the control key (pressed down) to translate the map and get the center point on top of your positive density (using the mouse at the same time, left button I think). Do not hold cntrl any more to rotate the map by 90 degrees. Cntrl again to move the map etc etc until the center point is right in the middle of the density. Then you use the menu, add atom, select Water, make sure you insert into your current molecule. One water atom added, done. Repeat the process until you're finished adding waters. Then save as usual... HTH, Fred. > Message du 03/12/09 19:59 > De : "rui" > A : CCP4BB@JISCMAIL.AC.UK > Copie à : > Objet : [ccp4bb] How to add a single water molecule > > Hi, All, > > I tried to use phenix to add water molecules, and when I check those waters > in coot, I can easily delete them if they are not in right spot. However, if > I see some clear positive maps that seems like water, can I add manually at > that position? How to do this in coot?Or any other way? >
[ccp4bb] How to add a single water molecule
Hi, All, I tried to use phenix to add water molecules, and when I check those waters in coot, I can easily delete them if they are not in right spot. However, if I see some clear positive maps that seems like water, can I add manually at that position? How to do this in coot?Or any other way?
Re: [ccp4bb] High R factors in Orthorhombic Space Group
Rwork = 0.26 and Rfree = 0.32 are not necessarily unacceptably high for a 2.3 A dataset in my opinion. Not all crystals are the same, some just have more disorder than others and not all of this disorder is modelable (if that is a word). Having said that: - is the 2.3A data cutoff perhaps a tad optimistic and is your structure really more like 2.5-2.7A? - have you correctly modelled solvent/ions etc.? - did you try TLS analysis and refinement? - is yours a protein/DNA complex? I understand they often have a higher R-factors, the specialists may contribute their opinions on the reason (disorder in DNA? restraints for nucleic acids being less good than for protein?). From the information you give, I think you can be confident about the P21212 spacegroup. Mark Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ researcherID: B-3678-2009 On 3 Dec 2009, at 18:49, Robert Radford wrote: Hello- I am working up a data that is giving me unacceptable high R factors. I am solving a data set that is good to ~ 2.3 Å using molecular replacement based on a previously crystalized variant. The maps look good with all the density fitting well to my model. I originally worked up the data using PHASER for the molecular replacement. It searched all alternative orthorhombic space groups and found a single solution in P2(1)2(1)2 but that only gave me final R factors of (Rwork = 0.26 and Rfree = 0.32) Next I went back and tried to work the data up in a different space group. Thus far I have tried P21 and P1. Both these space groups give me essential the same final statistics (~Rwork = 0.26 and Rfree = 0.32). Furthermore, the solution looks the same, the only change being the number for monomers in the asymmetric unit (1, 2, 4 for P21212, P21 and P1 respectively) The diagnostics don't indicate that the crystal is twined and the data set looks good (i.e. the spots are nice and well separated) The unit cell dimensions for the three cases as P1: a = 34.55 b = 46.95 c = 88.56, alpha = 89.94, beta = 90.03 gamma = 89.99 P21: a = 46.91 b = 34.52 c = 88.47, alpha = 90 , beta = 90.03 gamma = 90.0 P21212 a = 88.44 b = 34.51 c = 46.89, alpha = 90.0, beta = 90.00 gamma = 90.0 One possible complication is that the biological unit is dimeric with 2 fold symmetry. The 2 fold symmetry axis for the protein appears to be sitting on a 2-fold crystallographic axis. Any suggestions? Thank you in advance. Robert Robert Radford Graduate Researcher Dept. Of Chemistry UC San Diego
[ccp4bb] High R factors in Orthorhombic Space Group
Hello- I am working up a data that is giving me unacceptable high R factors. I am solving a data set that is good to ~ 2.3 Å using molecular replacement based on a previously crystalized variant. The maps look good with all the density fitting well to my model. I originally worked up the data using PHASER for the molecular replacement. It searched all alternative orthorhombic space groups and found a single solution in P2(1)2(1)2 but that only gave me final R factors of (Rwork = 0.26 and Rfree = 0.32) Next I went back and tried to work the data up in a different space group. Thus far I have tried P21 and P1. Both these space groups give me essential the same final statistics (~Rwork = 0.26 and Rfree = 0.32). Furthermore, the solution looks the same, the only change being the number for monomers in the asymmetric unit (1, 2, 4 for P21212, P21 and P1 respectively) The diagnostics don't indicate that the crystal is twined and the data set looks good (i.e. the spots are nice and well separated) The unit cell dimensions for the three cases as P1: a = 34.55 b = 46.95 c = 88.56, alpha = 89.94, beta = 90.03 gamma = 89.99 P21: a = 46.91 b = 34.52 c = 88.47, alpha = 90 , beta = 90.03 gamma = 90.0 P21212 a = 88.44 b = 34.51 c = 46.89, alpha = 90.0, beta = 90.00 gamma = 90.0 One possible complication is that the biological unit is dimeric with 2 fold symmetry. The 2 fold symmetry axis for the protein appears to be sitting on a 2-fold crystallographic axis. Any suggestions? Thank you in advance. Robert Robert Radford Graduate Researcher Dept. Of Chemistry UC San Diego
Re: [ccp4bb] Bad geometry for alt. conformation refined in Refmac5
Before you all go away and install the latest Intel compilers to fix this bug - don't bother! We just installed the latest version (ifort 11.3) and it has the same bug! We plan to file a bug report with Intel. In case anyone is interested, this is the code snippet which exhibits the buggy behaviour (this was pruned down from s/r CHK_ALT in Refmac): CHARACTER A(2)*2 DATA A/2*'.A'/ CALL CA(A) STOP 1 END SUBROUTINE CA(A) CHARACTER A(2)*2 DO 1 I=1,2 C With given data, the condition "A(I)(2:).NE.'A'" is always FALSE, C so we should never execute the 'STOP 1' here: 1 IF(A(I)(2:).NE.'A' .AND. A(I).NE.'..') RETURN C Should always drop through to here: STOP 2 END It fails using -O2, -O3 and -Os, i.e. it only gives the right answer using -O0 & -O1. I should say that this is the first time we've ever encountered a bug in the Intel compilers - I wish I could say the same for g77 & gfortran! Cheers -- Ian > -Original Message- > From: owner-ccp...@jiscmail.ac.uk > [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Ian Tickle > Sent: 28 November 2009 13:22 > To: Garib Murshudov; john.pas...@jefferson.edu > Cc: CCP4BB@jiscmail.ac.uk > Subject: RE: [ccp4bb] Bad geometry for alt. conformation > refined in Refmac5 > > I think I've finally squashed this particular bug which had been > annoying me for quite some time! It appears that Intel Fortran v11.0 > has an optimisation bug in s/r CHK_ALT (i.e. checking alternate atom > codes), & possibly also 10.x - I don't have those to test - > though v9.1 > & possibly earlier seem to be bug-free (at least this bug anyway!). > Possibly this is fixed in the current version of ifort (11.1), again I > don't have it to test, maybe someone who does can try it. > This affects > compilations using optimisation levels -O2, -Os and -O3, but > -O0 and -O1 > are unaffected. > > So my solution is to recompile with -O1. The CCP4 6.1.2 distribution > uses -O2 so you need either to fix the makefile, or download Garib's > source code and make sure the makefile you use has the > appropriate flag > settings. > > As was pointed out earlier, having to use the > gfortran-compiled code is > an issue, because at least for the OS I'm using (Centos 4.6), > gfortran-compiled code runs ~ 300% slower than the ifort code!! > > Hope this helps! > > -- Ian > > > -Original Message- > > From: owner-ccp...@jiscmail.ac.uk > > [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Garib Murshudov > > Sent: 25 November 2009 16:27 > > To: john.pas...@jefferson.edu > > Cc: CCP4BB@jiscmail.ac.uk > > Subject: Re: [ccp4bb] Bad geometry for alt. conformation > > refined in Refmac5 > > > > Could you please try the version from York: > > > > www.ysbl.york.ac.uk/refmac/latest_refmac.html > > > > I think probkem you mention is related with compilation or > > something. > > At least I cannot repat it on my computer > > > > regards > > Garib > > > > On 25 Nov 2009, at 16:18, John Pascal wrote: > > > > > Hello All, > > > > > > We are trying to refine ARG residues with two conformations in > > > Refmac5, and the > > > refined atom positions in the output PDB file are all over the > > > place, as if the > > > geometry restraints are not well defined. > > > > > > We've tried several different formats for the input file, > based on > > > previous > > > postings to the bulletin board and the PDB standard (two > examples > > > below), but > > > the result is always the same. > > > > > > We are using Refmac5 in CCP4 Suite 6.1.2, GUI 2.0.5 on Mac OSX. > > > > > > We'd appreciate any suggestions. Thanks. -John > > > > > > Examples of ARG format: > > > > > > 1) > > > ATOM 1472 N ARG A 1 -5.737 26.887 38.372 1.00 > > > 29.53 CN > > > ATOM 1473 CA ARG A 1 -5.445 25.560 37.882 1.00 > > > 30.24 CC > > > ATOM 1474 CB ARG A 1 -5.314 24.548 39.036 1.00 > > > 30.63 CC > > > ATOM 1475 CG ARG A 1 -5.426 23.052 38.627 1.00 > > > 34.81 CC > > > ATOM 1476 CD AARG A 1 -4.827 22.075 39.644 0.50 > > > 37.09 CC > > > ATOM 1477 CD BARG A 1 -4.301 22.419 39.279 0.50 > > > 37.09 CC > > > ATOM 1478 NE AARG A 1 -3.430 21.777 39.304 0.50 > > > 42.71 CN > > > ATOM 1479 NE BARG A 1 -4.482 21.902 40.627 0.50 > > > 42.71 CN > > > ATOM 1480 CZ AARG A 1 -2.998 20.868 38.402 0.50 > > > 44.91 CC > > > ATOM 1481 CZ BARG A 1 -3.648 22.142 41.638 0.50 > > > 44.91 CC > > > ATOM 1482 NH1AARG A 1 -3.841 20.117 37.678 0.50 > > > 45.20 CN > > > ATOM 1483 NH1BARG A 1 -2.584 22.912 41.464 0.50 > > > 45.20 CN > > > ATOM 1484 NH2AARG A 1 -1.688 20.715 38.210 0.50 > > > 44.99 CN > > > ATOM 1485 NH2BARG A 1 -3.878 21.619 42.831 0.
Re: [ccp4bb] Refining residues as rigid bodies
Discussing with Fred Vellieux offlist I suggested the following: would it be
useful to have a sort of 'simulated annealing' of the B-factor values? - in
refinement I often try resetting them to higher values (Moleman has a function
for this I think) and then see which ones refine back down nicely - but it
would be good to have some sort of randomization method - that would be a test
of how much the values are inherited from the modelling early on - say from the
MR probe model for example.
We could do multiple B-factor refinements with different starting kicks to the
values and look for the best final Rfree or some measure of map quality?
Martyn
Martyn F. Symmons
Cambridge
'Chan fhiosrach mur feòraich.'
Gaelic proverb - Nothing asked, nothing learned.
From: Pavel Afonine
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, 2 December, 2009 22:52:45
Subject: Re: [ccp4bb] Refining residues as rigid bodies
Hi,
you can do similar thing (that is resulting in similar outcome) in
phenix.refine by increasing the weight on ADP restraints term. Example:
increase "wu" or decrease "wxu_scale". Although I believe a regular
refinement of individual isotropic ADPs should normally work just fine
at 3A resolution in phenix.refine.
Pavel.
On 12/1/09 11:18 PM, Frederic VELLIEUX wrote:
If the problem is B-factor refinement, you can do that easily at low resolution
with CNS. You just give tight restraints.
>
>You modify the file bindividual.inp
>
>This section:
>{* target sigma values for restrained B-factor refinement *}
>
>{* mainchain bonds *}
>{===>} bsig_main=1.5;
>{* mainchain angles *}
>{===>} asig_main=2.0;
>
>{* sidechain bonds *}
>{===>} bsig_side=2.0;
>{* sidechain angles *}
>{===>} asig_side=2.5;
>
>I cannot remember in which direction the values have to go. I think up. I have
>done this with a very low resolution structures (4.5 A?) a few years ago, you
>get smoothly varying B values, very tightly restrained. I do not think we
>published that structure, we obtained a higher resolution structure later (I
>don't think the referees would have been very happy seeing B factor refinement
>at low resolution). But it worked.
>
>Fred.
>
>
>Message du 01/12/09 23:51
>>De : "Jason C Porta"
>>A : CCP4BB@JISCMAIL.AC.UK
>>Copie à :
>>Objet : Re: [ccp4bb] Refining residues as rigid bodies
>>
>>
>>Basically my reasoning for doing this is a low data-to-parameter ratio, which
>>makes B-factor refinement unfeasible. So far I have had nice results with
>>breaking the complex into rigid subdomains. So i was basically just thinking
>>of a way I could refine the structure best, without using too many parameters.
>>
>I see now how this can be done in Phenix. I will give it a try.
>
>Thanks for all of your suggestions!
>
Re: [ccp4bb] Solubilization buffer
Hi Megha, You could make your buffer solutions at room temperature, so that the 8M urea dissolves completely. During solubilization of the protein, keep the tube on a shaker or agitate the solution using a magnetic strirrer. This will prevent any crystallization of the urea. In any case, urea crystallizes very slowly, even at 4 degrees C, as already mentioned . Another issue is the solubilization time. You've mentioned that you do it only for 1 hour. In the proteins I have worked with, I have had to solubilize overnight to get a reasonable yield. Of course this could be different for each protein. Hope this helps Ganesh On Wed, 2 Dec 2009 20:16:09 -0800, megha goyal wrote: Hi all, We use 8M urea solubilization buffer for our protein in inclusion bodies and recommended temperature is 10-15º C. but in 8M conc the urea does not dissolve and is in crystalline form only, will it have any effect on solubilzation efficiency. Our solubilization time is 1 Hr and after that we centrifuge and use the supernatant for refolding via dialysis. however the pellet after centrifugation of solubilzation show presence of our protein on sds page analysis. what should we do so that the process of solubilization is complete and our protein is not lost in pellet. thanks in anticipation. meg
Re: [ccp4bb] Solubilization buffer
Hi Meg I would highly recommend giving 6M guanidinium chloride a go in parallel, to compare the solubilization efficiency of the two approaches. We have had a couple of cases in the lab where homologous proteins showed strong preference either to urea OR guanidinium chloride when it came to solubilization of their inclusion bodies for refolding protocols. best wishes Savvas Savvas Savvides http://www.lprobe.ugent.be/xray.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Joerg Standfuss Sent: Thursday, December 03, 2009 8:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solubilization buffer Dear Meg, There is a database of refolding conditions from which you could get some inspiration. http://refold.med.monash.edu.au/search.php The Urea conditions I have seen there mostly use 37C. for solubilization/refolding and nearly all contain a reducing agent. Have a look. There are also a great number of other additives people use to improve things. Joerg > Hi all, > > We use 8M urea solubilization buffer for our protein in inclusion bodies > and > recommended temperature is 10-15º C. but in 8M conc the urea does not > dissolve and is in crystalline form only, will it have any effect on > solubilzation efficiency. Our solubilization time is 1 Hr and after that > we > centrifuge and use the supernatant for refolding via dialysis. however the > pellet after centrifugation of solubilzation show presence of our protein > on > sds page analysis. what should we do so that the process of solubilization > is complete and our protein is not lost in pellet. > > thanks in anticipation. > > meg > E-mail message checked by Spyware Doctor (6.1.0.447) Database version: 6.13830 http://www.pctools.com/en/spyware-doctor-antivirus/
[ccp4bb] UV detection of crystals/survey
Hello, I would be interested in people experiences and opinions about what types of fluorescence microscope setups have worked adequately to distinguish small protein crystals from background/ autofluorescence etc. ...general comments? (regular dichroic mirros etc dont allow Trp peak to pass through for instance etc and quartz becomes qute expensive i think?) i am looking for the cheapest but effective system (ie i want to see 10 um size things) regular sterefluor. microscope with filters + fiber UV light from the side? anybody has found something like that to work? thank you very much for comments, Tommi Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
