[ccp4bb] Off-topic: ligand enrichment
Dear guys, Sorry for the off-topic question. After I have solved my strucutre, I have found my target ligand bound at the potential binding site. Also, I have found that there are two more ligand molecules bound along the path from solvent to the binding site. I think this can enrich the ligand to binding site, enhancing the local concentration of the ligand, thus reducing the Km of the ligand. I am wondering if anybody can give some suggestions on how to solve this problem clearly. If there is any similar case, it will be better. Thank you in advance. Best wishes, Yingjie Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921117 Email: yjp...@sibs.ac.cn
Re: [ccp4bb] Twinning for P31
Hi, Peter, I have tried to carry out MR in P3. It turned out that P31 is the right one. After MR, I refined it using Phenix.refine with or without twin law. The result is as following: Twin law R Rfree Twin fraction in phenix.refine None 0.3869 0.4080 none -h,-k,l 0.2828 0.3584 0.16 h,-h-k,-l 0.2829 0.3575 0.50 -k,-h,-l 0.2833 0.3587 0.15 (The structure was refined against dataset @20.0~2.8 A) I am wondering why there is no difference under different twin laws. How can I determine the real twin law and the real twin fraction? Thanks very much. best regards, Yingjie On Thu, Oct 16, 2008 at 9:42 PM, Peter Zwart <[EMAIL PROTECTED]> wrote: > if possible try and solve it in P3(n) and take it from there. > > This data you show now might be 'overmerged'. > Also, the fact that the data is twinned doesn't really matter at this > point. The most important bit is to get the space group right, or at > least not too high so you can solve it. > When the spacegroup is too low, model information can be very helpfull. > > Peter > > > > > > 2008/10/16, Eleanor Dodson <[EMAIL PROTECTED]>: > > People dont read the CCP4 documentation on twinning! Grrr > > PG P3 can have 3 twinning operators; and these are: > > k,h,-l ( or symm equiv) - if this is a crystallographic operator the PG > > becomes P321 > > -h,-k,l (or symm equiv) - if this is a crystallographic operator the PG > > becomes P6 > > -k,-h,-l (or symm equiv if this is a crystallographic operator the PG > > becomes P31 2 > > > > The second moment test is not too badly affected if you get the PG wrong > ( > > some centric reflections are flagged as acentric, but these are usually a > > small % of the total) > > > > Neither is the l test, but this is easily disturbed by problems with > the > > data > > > > However the H-test, or the Britten test and some others look at > > correlations between possibly twinned intensities, and there if you have > the > > wrong point group, they can be very misleading.. > > > > From the information you have provided I would guess the PG is P321 but > I > > need the TRUNCATE plots to be happy about saying that; they give some > > feeling for data quality. > > Eleanor > > > > > > Yingjie Peng wrote: > > > > > Hi, > > > > > > I tried to processed it as P321. It seemed that it might be right. The > > > Rmerge increased > > > just a little. Then I used phenix.xtriage and sfcheck to check it. The > > > results are as following: > > > > > > phenix.xtriage: > > > > > > Twinning and intensity statistics summary (acentric data): > > > > > > Statistics independent of twin laws > > > - /^2 : 2.084 > > > - ^2/ : 0.827 > > > - <|E^2-1|> : 0.666 > > > - <|L|>, : 0.400, 0.227 > > > Multivariate Z score L-test: 9.082 > > > The multivariate Z score is a quality measure of the given > > > spread in intensities. Good to reasonable data are expected > > > to have a Z score lower than 3.5. > > > Large values can indicate twinning, but small values do not > > > necessarily exclude it. > > > > > > > > > Statistics depending on twin laws > > > > > - > > > | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | > > > > > - > > > | -h,-k,l | M | 0.461 | 0.102 | 0.065 | 0.022| > > > > > - > > > > > > Patterson analyses > > > - Largest peak height : 4.288 > > > (corresponding p value : 0.98768) > > > > > > > > > The largest off-origin peak in the Patterson function is 4.29% of the > > > height of the origin peak. No significant pseudotranslation is > detected. > > > > > > The results of the L-test indicate that the intensity statistics > > > are significantly different than is expected from good to reasonable, > > > untwinned data. > > > As there are twin laws possible given the crystal symmetry, twinning > could > > > be the
Re: [ccp4bb] Twinning for P31
Hi, I tried to processed it as P321. It seemed that it might be right. The Rmerge increased just a little. Then I used phenix.xtriage and sfcheck to check it. The results are as following: phenix.xtriage: Twinning and intensity statistics summary (acentric data): Statistics independent of twin laws - /^2 : 2.084 - ^2/ : 0.827 - <|E^2-1|> : 0.666 - <|L|>, : 0.400, 0.227 Multivariate Z score L-test: 9.082 The multivariate Z score is a quality measure of the given spread in intensities. Good to reasonable data are expected to have a Z score lower than 3.5. Large values can indicate twinning, but small values do not necessarily exclude it. Statistics depending on twin laws - | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | - | -h,-k,l | M | 0.461 | 0.102 | 0.065 | 0.022| - Patterson analyses - Largest peak height : 4.288 (corresponding p value : 0.98768) The largest off-origin peak in the Patterson function is 4.29% of the height of the origin peak. No significant pseudotranslation is detected. The results of the L-test indicate that the intensity statistics are significantly different than is expected from good to reasonable, untwinned data. As there are twin laws possible given the crystal symmetry, twinning could be the reason for the departure of the intensity statistics from normality. It might be worthwhile carrying out refinement with a twin specific target function. sfcheck: Pseudo-translation is not detected. Minimal estimated error : 0.0864 Perfect twinning test /^2 : 2.0191 Partial Twinning test: -h,-k,+l Polar angles:0.000.00 180.00 Alpha(twin fraction),Npair,Ior,Tol : 0.148 1188122 0.000 Then, what should I do? I did not deal with any twinning dataset. Any comments and suggestions will be greatly appreciated. Thanks! Yingjie On Thu, Oct 16, 2008 at 12:24 AM, Eleanor Dodson <[EMAIL PROTECTED]>wrote: > I should have said - most likely explanation is point group is reall P321 > > Eleanor > > > >> 2008/10/15, Eleanor Dodson <[EMAIL PROTECTED]>: >> >> >>> I cant follow this very well. >>> Try SFCHECK as well which will do the same tests and give a differently >>> formatted output.. >>> or TRUNCATE which gives you plots of these stats v resolution.. >>> >>> /^2 : 2.351 This is higher than the expected value of 2 for >>> untwinned data. (1.5 for perfectly twinned data) >>> However it can be distorted by non-crystallographic translation, but you >>> dont seem to have that.. >>> Or by experimental errors and you need to inspect it in resolution >>> ranges >>> to detect that - assuming your low res data is more accurate than the >>> high >>> res. >>> >>> Eleanor >>> >>> >>> >>> >>> >>> Yingjie Peng wrote: >>> >>> >>> >>>> Dear guys, >>>> >>>> I have collected a dataset with the sg as P31. I ran pehnix.xtriage to >>>> analyse the data with >>>> following result: >>>> >>>> Twinning and intensity statistics summary (acentric data): >>>> >>>> Statistics independent of twin laws >>>> - /^2 : 2.351 >>>> - ^2/ : 0.788 >>>> - <|E^2-1|> : 0.766 >>>> - <|L|>, : 0.446, 0.270 >>>> Multivariate Z score L-test: 3.358 >>>> The multivariate Z score is a quality measure of the given >>>> spread in intensities. Good to reasonable data are expected >>>> to have a Z score lower than 3.5. >>>> Large values can indicate twinning, but small values do not >>>> necessarily exclude it. >>>> >>>> >>>> Statistics depending on twin laws >>>> >>>> >>>> >>> -- >>> >>> >>>> | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | >>>> >>>> >>>> >>> -- >>> >>> >>>> | -h,-k,l | M | 0.460 | 0.043 | 0.039 | 0.022| >>>> | h,-h-k,-l | M | 0.054 | 0.423 | 0.459 | 0.478| >>>> | -k,-h,-l | M | 0.476 | 0.042 | 0.043 | 0.022| >>>> >>>>
[ccp4bb] Twinning for P31
Dear guys, I have collected a dataset with the sg as P31. I ran pehnix.xtriage to analyse the data with following result: Twinning and intensity statistics summary (acentric data): Statistics independent of twin laws - /^2 : 2.351 - ^2/ : 0.788 - <|E^2-1|> : 0.766 - <|L|>, : 0.446, 0.270 Multivariate Z score L-test: 3.358 The multivariate Z score is a quality measure of the given spread in intensities. Good to reasonable data are expected to have a Z score lower than 3.5. Large values can indicate twinning, but small values do not necessarily exclude it. Statistics depending on twin laws -- | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | -- | -h,-k,l | M | 0.460 | 0.043 | 0.039 | 0.022| | h,-h-k,-l | M | 0.054 | 0.423 | 0.459 | 0.478| | -k,-h,-l | M | 0.476 | 0.042 | 0.043 | 0.022| -- Patterson analyses - Largest peak height : 4.693 (corresponding p value : 0.95672) The largest off-origin peak in the Patterson function is 4.69% of the height of the origin peak. No significant pseudotranslation is detected. The results of the L-test indicate that the intensity statistics behave as expected. No twinning is suspected. Even though no twinning is suspected, it might be worthwhile carrying out a refinement using a dedicated twin target anyway, as twinned structures with low twin fractions are difficult to distinguish from non-twinned structures. The correlation between the intensities related by the twin law h,-h-k,-l with an estimated twin fraction of 0.42 % is most likely due to an NCS axis parallel to the twin axis. This can be verified by supplying calculated data as well. Is it perfect twinning or partial twinning? I am supposed to do MR with this dataset. What should I do next with this dataset? Thanks very much. Best regards, Yingjie Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921117 Email: [EMAIL PROTECTED]
Re: [ccp4bb] CCP4i can NOT run
Hi Boaz,
Thanks for your suggestion. I have tried as you suggested. In my case,
CCP4_MASTER
can NOT set to be /home/prog.
The ccp4i did not launched because it can not locate the ccp4i.tcl file.
On Tue, Sep 30, 2008 at 8:53 PM, Boaz Shaanan <[EMAIL PROTECTED]> wrote:
> Your setup in ccp4.setup seems to be incorrect.
> You should have:
> setenv CCP4_MASTER/home/prog
>
> and then you would have:
>
> echo $CCP4 /home/prog/ccp4-6.0.2
>
> I don´t think your problem has anything to do with tcl setup.
>
>Boaz
>
> - Original Message -
> From: Yingjie Peng <[EMAIL PROTECTED]>
> Date: Tuesday, September 30, 2008 15:45
> Subject: Re: [ccp4bb] CCP4i can NOT run
> To: CCP4BB@JISCMAIL.AC.UK
>
> > Hi Iain,
> >
> > Thanks for your reply. The path for tclsh is
> > "/usr/local/tcltkblt/bin/".There are six files: bltsh, bltwish,
> > tclsh, tclsh8.4, wish and wish8.4. The
> > tclsh
> > and wish are symbolic links for tclsh8.4 and wish8.4,
> > respectively. For the
> > permission reason you mentioned, I have also tried this change:
> > sudo chown yjpeng:yjpeng -R /usr/local/tcltkblt
> > It does NOT work!
> >
> > For better analysis, I paste my ccp4.setup file as following:
> > ### THIS SECTION MUST BE EDITED #
> >
> > # CCP4_MASTER is the location of the top-level directory containing
> > `ccp4-'
> > # This is usually the directory in which you ran the tar command
> > to unpack
> > the
> > # code, and is assumed to be shared between machines at a multi-
> > machinesite.
> >
> > setenv CCP4_MASTER/home/prog/ccp4-6.0.2
> > setenv CCP4
> > $CCP4_MASTER/ccp4-6.0.2
> >
> > # Check for existence of CCP4_MASTER
> > if (! -e $CCP4_MASTER) then
> > echo "* WARNING
> > **"echo "The directory
> > $CCP4_MASTER"echo "(assigned to CCP4_MASTER)
> > does not exist."
> > echo "The CCP4 programs will not run
> > correctly, and any"
> > echo "installation attempt will have errors
> > or will fail."
> > echo "* WARNING
> > **"endif
> >
> > # CCP4_SCR: a per-user directory for run-time-generated scratch
> > # files. A dedicated scratch filesystem is probably better than
> > (/usr)/tmp# BINSORT_SCR: a scratch directory for binsort's use;
> > normally same as
> > CCP4_SCR
> >
> > setenv CCP4_SCR /data/tmp
> > # check to see if this exists and if not try to make it
> > if (! -e $CCP4_SCR) mkdir $CCP4_SCR
> > if (! -e $CCP4_SCR) \
> > echo "Unable to make CCP4_SCR. CCP4 progs
> > will not run correctly."
> >
> > setenv BINSORT_SCR $CCP4_SCR
> >
> > ### CCP4i setup - you may need to edit CCP4I_TCLTK ###
> > # CCP4I_TOP - the top directory of the interface
> > setenv CCP4I_TOP ${CCP4}/ccp4i
> > # CCP4I_TCLTK - directory containing tclsh,wish and bltwish
> > executables# as used in
> $CCP4I_TOP/bin/ccp4i,ccp4ish,loggraph
> > # For 'standard' installations this is /usr/local/bin
> > # but note the SGI distributed version of Tcl/Tk is not
> > # appropriate version
> > setenv CCP4I_TCLTK /usr/local/tcltkblt/bin
> > # CCP4I_HELP - directory contain ccp4i help - default is
> > $CCP4I_TOP/helpsetenv CCP4I_HELP ${CCP4I_TOP}/help
>
> >
> > ### Optional - setting http_proxy environment
> > # The commented out 'setenv' line below may have to be declared
> > to download
> > # and edit protein sequences using the new "Import/Edit Protein
> > Sequences"# (This may also be required for remote job submission
> > in arp/warp.)
> > # task. If so, uncomment this line and replace the example proxy
> > URL with
> > your
> > # relevant URL
> >
> > #setenv HTTP_PROXY wwwblah.blah.ac.uk:/blah.blah
> >
> > ### NOVICE USERS STOP HERE #
> >
> > ### OPTIONS TO CUSTOMISE CCP4 #
> >
> > # By default, CCP4 directories are appended to the end of paths (PATH,
> > # LD_LIBRARY_PATH, and DYLD_LIBRARY_PATH). If ccp4_first_in_path
> > is set
> > # to 1, then they will be prepended to the beginning of paths.
> > # When deciding local policy, bear in mind the possible
> > existence of
> > # other CCP4 installations, and the possibility of non-CCP4 program
Re: [ccp4bb] CCP4i can NOT run
I have checked /etc/hosts, it is similar to what you say. I also tried to set it exactly the same as yours. But it did NOT work. Thanks! On Tue, Sep 30, 2008 at 10:24 PM, Ben Eisenbraun <[EMAIL PROTECTED]>wrote: > On Tue, Sep 30, 2008 at 10:48:27AM +0800, Yingjie Peng wrote: > > I have installed the latest version of CCP4i under Fedora 9 on my laptop > > HP Compaq 6515b (two processors). The system setup was done and the CCP4i > > interface can run normally. But if I submit a job, it displayes > > "STARTING" the job in the job window and will NOT actually run the job. > > Also I can NOT kill the job with its status as STARTING. > > I have seen similar behavior before. Is your /etc/hosts correct? The > localhost line should look like this: > > 127.0.0.1 localhost > > Some of the CCP4 utilities act as miniature servers and bind to localhost > to communicate between the GUI and the process. If this is actually the > problem, you should see output in the shell that started ccp4i similar > to this: > > ERROR running script can not connect to server port (RunNotification) > SERVER_HOST localhost SERVER_PORT 4441 > > -ben > > -- > Ben Eisenbraun > Structural Biology Grid Harvard Medical School > http://sbgrid.org http://hms.harvard.edu >
Re: [ccp4bb] CCP4i can NOT run
ave to make a symbolic link to the executable, in the > same directory, as below: > > ln -s tclsh8.4 tclsh (or the other way round, check)..also make sure you > have read (and possibly execute) permission for this directory and the files > within. > > HTH, > Iain > > Yingjie Peng wrote: > >> Dear friends, >> I have installed the latest version of CCP4i under Fedora 9 on my laptop >> HP Compaq 6515b (two processors). The system >> setup was done and the CCP4i interface can run normally. But if I submit a >> job, it displayes "STARTING" the job in the job window >> and will NOT actually run the job. Also I can NOT kill the job with its >> status as STARTING. >> I have checked my Tcl/tk is version 8.4 and the CCP4I_TCLTK environment >> variable is pointed to the right directory containing >> tclsh, bltwish and other files. >> Is there any suggestion on how to solve this problem? Thank you very much >> in advance. >> Best wishes, >> Yingjie >> Yingjie PENG, Ph.D. student >> Structural Biology Group >> Shanghai Institute of Biochemistry and Cell Biology (SIBCB) >> Shanghai Institute of Biological Sciences (SIBS) >> Chinese Academy of Sciences (CAS) >> 320 Yue Yang Road, Shanghai 200031 >> P. R. China >> 86-21-54921117 >> Email: [EMAIL PROTECTED] >> >> >> >> No virus found in this incoming message. >> Checked by AVG - http://www.avg.com Version: 8.0.173 / Virus Database: >> 270.7.5/1698 - Release Date: 9/29/2008 7:25 PM >> >> >> >
[ccp4bb] CCP4i can NOT run
Dear friends, I have installed the latest version of CCP4i under Fedora 9 on my laptop HP Compaq 6515b (two processors). The system setup was done and the CCP4i interface can run normally. But if I submit a job, it displayes "STARTING" the job in the job window and will NOT actually run the job. Also I can NOT kill the job with its status as STARTING. I have checked my Tcl/tk is version 8.4 and the CCP4I_TCLTK environment variable is pointed to the right directory containing tclsh, bltwish and other files. Is there any suggestion on how to solve this problem? Thank you very much in advance. Best wishes, Yingjie Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921117 Email: [EMAIL PROTECTED]
[ccp4bb] Degradation and phasing
Dear friends, I have two problems with my proteins. For protein A, it was purified using the Nickel-affinity purification method. If the sample was placed at 4 degrees centigrade for 24 hrs, it didn't change obviously. But if it was concentrated by centrifuge at 4 degrees for further gel-filtration purification, it degraded very heavily during the concentrating process (~4hr), resulting there is nearly NO band corresponding to the full-length protein B nor any band corresponding to fragment of protein B. So I think protein B may undergo nonspecific degradation into very small fragments. I have used PMSF, Leupeptin, Pepstantin, and Aprotinin as protease inhibitor mixture to preventing the degradation, but it didn't work. For protein B, it can be purified to the purity of >95%, and can be concentrated to >20 mg/ml (although a little slowly). When setup hanging-drop crystallization trials, it was found phased heavily under most conditions. I have tried different buffer systems (Tris-HCl or NaH2PO4), with or without cofactor or substrate analogue. But none of these worked. The protein still phased very heavily. Would somebody share their experience on preventing protein degradation during purification or preventing phasing during crystallization. Any comments or suggestion are welcome. Thanks in advance. Yingjie Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921117 Email: [EMAIL PROTECTED]
[ccp4bb] Problem with the DLS instrument
Dear colleagues, We have encountered a problem with our dynamic light scattering instrument DynaPro from Protein Solutions (New Jersey) and would like to solicit your suggestions and advices. The instrument was purchased about six years ago. In the past years, the intensity (Cnt/s) has decreased dramatically to about 200 Cnt/s which practically makes the instrument unusable. We want to repair the instrument but could not find any representative of the company in China. I am wondering if someone could provide us advices on what we should do to get our instrument back to work. Many thanks in advance. Yingjie Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921117 Email: [EMAIL PROTECTED]
[ccp4bb] Binding pocket volume
Dear all, Is there anyone who can tell me how to calculate the volume of substrate binding pocket in a protein structure? I want to use it to the quantify the conformational change caused by induced fitting. Thanks very much. Yingjie Peng Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921217 Email: [EMAIL PROTECTED]
