Re: [ccp4bb] protein oligomer
Dear: The protein was purified in 4 degree, and the expression level is low, so the aggregation is not by high concentration; the buffer pH is 7.5 which is not colse to the PI 8.6. It should be a dimer when function, but it was aggregated when negative staining. Maybe I could try to add arginine when purification, or do mutantions. anyone has website for prediction the mutation sites of protein? Thanks! Best, shijun -原始邮件- 发件人:"Nikolay Dobrev" 发送时间:2020-07-19 20:29:49 (星期日) 收件人: CCP4BB@JISCMAIL.AC.UK 抄送: 主题: Re: [ccp4bb] protein oligomer It really depends from the nature of the protein and if is oligomerizing/agregating/forming polymers if any of this is reversible. On the other side if you are working with one of the fibril forming protein it will require optimizaiton on its on as they will form naturally polymers. Do you observed different specises when you analyze your protein by SEC or if you are able to perfom DLS? Additional information regarding your protein will be really helpful for more detalied suggestions how to overcome your protein. Best, Nikolay Nikolay Dobrev Scientific Officer, Protein Expression and Purification Core Facility EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany T +49 6221 387 8633 | M +49 173 684 0532 twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia Visit www.embl.org/events for a complete list of all EMBL events. On 19/07/2020 14:15, S. Mohanty wrote: Keep the protein concentration low during purification steps along with using other anti-aggregation agent/s. Make sure that the pH at which you are purifying is not close to the pI of the protein. Until completely purified, all purification steps should be performed in a cold room if it is a soluble protein. Smita On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga wrote: I am not sure what you mean by polymer formation. Presuming that you have optimized your protein concentration, pH and salt concentration, you could try arginine as an anti-aggregation agent in your purification (I presume you do FPLC). Have a look at chaotropic agents used in protein purification, The answer is generally dependent on the protein/proteins you are trying to purify and is not necessarily straightforward. Kinds regards On Sun, Jul 19, 2020 at 12:08 PM 张士军 <21620150150...@stu.xmu.edu.cn> wrote: Dear all: Any ideas to decrease protein polymer formation? my protein was easy to form oligomers and precipitation when do purification,I have tried add glycerol and DTT,both didn't work. Does anyone has experience to avoid it happening. Thanks! Best Regards To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 -- Nikolay Dobrev Scientific Officer, Protein Expression and Purification Core Facility EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany T +49 6221 387 8633 | M +49 173 684 0532 twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia Visit www.embl.org/events for a complete list of all EMBL events. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] protein oligomer
Dear all: Any ideas to decrease protein polymer formation? my protein was easy to form oligomers and precipitation when do purification,I have tried add glycerol and DTT,both didn't work. Does anyone has experience to avoid it happening. Thanks! Best Regards To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] cannot open NOMAD-Ref server
Dear all Does anyone have used NOMAD-Ref(normal mode analysis, deformation and refinement) server (http://lorentz.immstr.pasteur.fr/nomad-ref.php)? I want to do refinement of my crystal structure, but it cannot be opened when I using "X-ray refinement", the link is "http://lorentz.immstr.pasteur.fr/xray/standard_submission.php;, it warn me "You don't have permission to access /xray/standard_submission.php on this server", does it mean I have no permission to access it, or my browser didn't allow it? anyone can try it for me to check whether it can be opened? Thanks Best Regards Shijun To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] muti-crystal averaging
Thanks all, I can merge them together with HKL2000 now, and I found it also can do "No merge original index" and merge different data sets with same unit cell by new version of HKL2000, which is better than old version -Original Messages- From:"Eleanor Dodson" Sent Time:2019-07-15 22:08:01 (Monday) To: "张士军" <21620150150...@stu.xmu.edu.cn> Cc: "CCP4BB@JISCMAIL.AC.UK" Subject: Re: [ccp4bb] muti-crystal averaging Do you mean these crystals have different unit cells? If so then DMMULTI is a useful technique.. Or do you mean you have collected isomorphous data from several crystals and want to merge that data? In that case BLEND will help you decide how isomorhous the data sets are.. Eleanor On Mon, 15 Jul 2019 at 13:12, 张士军 <21620150150...@stu.xmu.edu.cn> wrote: Dear all I want to average several sets of crystal data together, and I saw some paper used DMMULTI program in CCP4, but I didn't find this program in ccp4, but only find Blend in ccp4 which only used for unmerged reflection files. I wondering is there any programs could use HKL2000 processed *.sca file to do this. Thanks a lot!!! Best Regards Shijun To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] muti-crystal averaging
Dear all I want to average several sets of crystal data together, and I saw some paper used DMMULTI program in CCP4, but I didn't find this program in ccp4, but only find Blend in ccp4 which only used for unmerged reflection files. I wondering is there any programs could use HKL2000 processed *.sca file to do this. Thanks a lot!!! Best Regards Shijun To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] RAMACHANDRAN outlier of Thr
Dear all The problem is when I check RAMACHANDRAN outlier with coot, it shows zero, while it become one Thr outlier in phenix.vadition. so I am confusing about the standard of RAMACHANDRAN outlier in coot and phenix.validation, any difference between them? Thanks yours shijun To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] SO4 or PO4
Dear all Thanks for all your suggestions. PO4 is from my Ligand PI3P, but I cannot see the electron density of the PI3P tail, and the binding site is exactly there. While my crystal buffer salt is 50mM (NH4)2SO4, so I am afraid the SO4 occupied the PO4 position.BTW, I can see the other SO4 electron density in the other places, but is small than this one, and the diffraction resolution is 1.6A. Best Regards Shijun -Original Messages- From:"Roger Rowlett" Sent Time:2019-02-17 00:47:55 (Sunday) To: CCP4BB@JISCMAIL.AC.UK Cc: Subject: Re: [ccp4bb] SO4 or PO4 Two things to look at that could provide a clue: Examine the anomalous map for some density over the central atom. Sulfur will often, but not always have significant anomalous density depending on the wavelength and quality of data set. Phosphate is normally HPO4= or H2PO4-. Look for phosphate donor to acceptor hydrogen bonding contacts. Sulfate rarely has donor to acceptor hydrogen bonding contacts, as it is SO4= at any reasonable pH. Roger Rowlett On Sat, Feb 16, 2019, 4:06 AM 张士军 <21620150150...@stu.xmu.edu.cn wrote: Dear all I have got a crystal grown at the condition both have ion of SO4 and PO4, and the diffraction resolution is very well, but the problem is coming: how to tell which is which just from electron density? I think they are exactly same. Thanks a lot !!! Beat Regards Shijun To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] SO4 or PO4
Dear all I have got a crystal grown at the condition both have ion of SO4 and PO4, and the diffraction resolution is very well, but the problem is coming: how to tell which is which just from electron density? I think they are exactly same. Thanks a lot !!! Beat Regards Shijun To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] R-flag choose
Dear all I am confusing the choose of free R-flags recently. Rfactor means calculatted from reflection not used in refinement,so what's big the difference between different percentage of R-flags,like it's about 5% in ccp4 -refmac, while it is 10% in phenix-refinement,what's the difference between them and how they affect the Rwork and Rfree values when do refinement. Thanks a lot ! best regards shijun To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] disulfate bond ?
Hi Thanks for all your suggestions . It is out of our hope even though it is disulfide bond, so I wondering what's the biological meaning for this kind of disulfide bond. I used AcCOA and Chloramphenicol when I screen crystal, but I could only put a COA in the election density even that the election density is not good enough.Any good suggestion or experience? Best Regards shijun -原始邮件- 发件人:"Artem Evdokimov" 发送时间:2018-07-04 20:01:47 (星期三) 收件人: CCP4BB@JISCMAIL.AC.UK 抄送: 主题: Re: [ccp4bb] disulfate bond ? It seems that your CoA is one carbon out of reference. You have spotty difference density over the ligand. Shift it left by one carbon bond and redune to see if density fits better. Artem On Wed, Jul 4, 2018, 02:26 张士军 <21620150150...@stu.xmu.edu.cn> wrote: Hi all I got a structure which has COA in it, and the SH in the tail of COA is very close to the SH side chain of Cys in the structure. I don't know whether it is disulfate bond or not? I remember they should link together if they are disulfate bond,am I right? so what could this be? Thanks a lot!!! best regards shijun To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Rfree going up after refinement
Dear all I have got my se-phase structure with ccp4-CRANK2-SAD, and its Rfree is 0.26 when CRANK2 output, but the Rfree is higher to 0.36 when I refine it with refmac or phenix-refinement. does it mean I got a wrong phase, or I didn't got the right parameters when refinement.BTW, the resolution is 2.1A.THANKS A LOT Best Regards shijun
[ccp4bb] phenix refinement about cis-proline
Dear all I am refining a structure which has cis-Pro and trans-Pro, the tans-Pro is gone when I set the "threshold degrees for cis-peptide " from default 45 to 65, but still has cis-Pro. While no significant change when I set it to 15. My question is how to set in phenix refinement to clear the Pro residues in cis- or trans- conformations. Best Regards shijun
[ccp4bb] BUCCANEER label choose
Dear all I am confusing how to choose or enter the labels when I do density modification and autobuilding with ccp4 parrot and buccaneer or ARP/wARP, there are three different options like "use Free R-flag ","use map coefficient" and "use PHI/FOM instead of HL coefficient" in the figure I attached. What they are mean, which one I should choose when I just want to do density modification after SAD or MR,and how to do when autobuilding? Even though when I choose"use map coefficient", it appeared HLA,HLB,HLC,HLD,and when I choose "use PHI/FOM instead of HL coefficient", it appeared PHI,FOM, how to enter these labels? Thanks a lot !!! Best Regards Shijun
[ccp4bb] Dials
Dear all When I run dials.index it result me "Sorry: No suitable lattice could be found", but the other software can give me C2 space group. How could I DO with this situation Best Regards Shijun
[ccp4bb] cannot read h5 data file
Dear all I just collected some data with Eiger16M whose file format is *.h5. But I cannot read it with HKL2000, because I don't have the Eiger16M detector license now, while, I have *.cbf detector license. So I transfered the *.h5 to *.cbf files, but the problem is XDSGUI still cannot read them,and HKL2000 can read it, can search peak, but cannot index it with warning me ''no enough peaks to index the data'',when I check the peaks are much enough to index, whearas dials can read it, but the cell parameters are not the same with before. Anyone can tell me what's wrong with it? Thanks a lot !!! Best Regards Shijun
[ccp4bb]
Hi all
It always fail when I run ccp4-AMPLE, and log file says
Cannot find executable theseus in PATH. Please give the path to theseus
***
* Information from CCP4Interface script
***
The program run with command:
/usr/local/programs/ccp4/ccp4-6.4.0/bin/ccp4-python -u
/usr/local/programs/ccp4/ccp4-6.4.0/bin/ample -mtz
/home/xingqiang/ZSJ/kif5b/430-566/A12P422output.mtz -fasta
/home/xingqiang/ZSJ/kif5b/430.seq -nmodels 500 -name MVD0 -run_dir
/home/xingqiang/ZSJ/kif5b/430-566 -nproc 4 -make_models True -rosetta_dir
/usr/local/programs/rosetta -frags_3mers
/home/xingqiang/ZSJ/kif5b/aat000_03_05.200_v1_3 -frags_9mers
/home/xingqiang/ZSJ/kif5b/aat000_09_05.200_v1_3 -make_frags False -F F -SIGF
SIGF -FREE FreeR_flag -early_terminate True -use_shelxe True -use_arpwarp
False
has failed with error message
Traceback (most recent call last):
File "/usr/local/programs/ccp4/ccp4-6.4.0/bin/ample", line 505, in
amopt.d['theseus_exe'] = ample_util.check_for_exe('theseus',
amopt.d['theseus_exe'])
File "/usr/local/programs/ccp4/ccp4-6.4.0/share/ample/python/ample_util.py",
line 93, in check_for_exe
raise RuntimeError,msg
RuntimeError: Cannot find executable theseus in PATH. Please give the path to
theseus
***
#CCP4I TERMINATION STATUS 0 "Traceback (most recent call last): File
"/usr/local/programs/ccp4/ccp4-6.4.0/bin/ample", line 505, in
amopt.d['theseus_exe'] = ample_util.check_for_exe('theseus',
amopt.d['theseus_exe']) File
"/usr/local/programs/ccp4/ccp4-6.4.0/share/ample/python/ample_util.py", line
93, in check_for_exe raise RuntimeError,msg RuntimeError: Cannot find
executable theseus in PATH. Please give the path to theseus"
#CCP4I TERMINATION TIME 31 Jul 2017 19:49:31
#CCP4I MESSAGE Task failed
Anyone know what's wrong with it? Does it mean I don't install Rosseta
successfully? Thanks
best regards
shijun
[ccp4bb] high Rfree
Hi everyone I have got a anti-parallel coiled-coil structure in a short fragment recently, then I want to solve a longer fragment structure with phenix-MR using this short fragment structure as a model.The result is not good because of the Rwork and Rfree is high.So I think the longer fragment will be parallel coiled-coil which is different with the shorter one. I am wondering whether there are any other methods to handle this phenomenon besides heavy atom phasing? Thanks a lot!!!
Re: [ccp4bb] SAD phasing
Dear Kay
You mean after convert .phs to .mtz ,use the .mtz file refine the initial
structure model? By the way , what's the difference between this density map
with the density map modified after phaser-EP density modification? Thanks a
lot!!!
Best Regard
Shijun
> -原始邮件-
> 发件人: "Kay Diederichs" <kay.diederi...@uni-konstanz.de>
> 发送时间: 2016年12月28日 星期三
> 收件人: CCP4BB@JISCMAIL.AC.UK
> 抄送:
> 主题: Re: [ccp4bb] SAD phasing
>
> Dear Shijun,
>
> hkl2map is a very nice graphical user interface that makes it easy to use the
> SHELX programs; I've used it successfully around 3A. You find documentation
> and download information for SHELX C/D/E and hklmap in the CCP4 community
> wiki, at
> http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/SHELX_C/D/E
>
> hkl2map also writes a script, xxx_phs2mtz.csh
>
> #!/bin/csh -f
> #
> # Shell script for converting phs to mtz-format
> # This script was written by HKL2MAP 0.4.c-beta
> # f2mtz keeps order of columns
> # cad picks up things in a different order and then writes E1...E4
> #
> # To convert from phs to mtz type:
> #./sad_phs2mtz.csh .phs
> #
> set fname = $1:r
> f2mtz hklin ${fname}.phs hklout t_tmp.mtz > t_f2mtz.log < CELL 79.100 79.100 38.3 90 90 90
> SYMM P212121
> LABOUT H K L FP FOM PHIB SIGFP
> CTYPOUT H H H F W P Q
> END
> #
> cad hklin1 t_tmp.mtz hklout $fname.mtz > t_cad.log < LABIN FILE 1 E1=FP E2=SIGFP E3=PHIB E4=FOM
> LABOUT E1=FP E2=SIGFP E3=PHIB E4=FOM
> eof-cad
>
>
> that helps with the conversion of the .phs file to a .mtz file. The latter
> can then be used in the buccaneer/refmac autobuild/refine pipeline provided
> by ccp4i, to complete the structure.
> Please note that for the final refinement stages (which involve manual model
> adjustment), one should switch from this SHELXE-provided file to a .mtz file
> written by XDSCONV or (C)TRUNCATE because SHELXE does not use the
> French procedure for converting intensities to amplitudes.
>
> Hope that helps,
>
> Kay
>
> On Wed, 28 Dec 2016 14:46:03 +0800, 张士军 <21620150150...@stu.xmu.edu.cn> wrote:
>
> >Hello everyone
> >
> > I am learning phasing SAD data now ,and I got some files(like i.phs , .pdb
> > ,fa.res, fa.pdb) when I using SHELXC/D/E,and I know fa.pdb and fa.res
> > (which contain heavy atom information) are used for the further solution
> > searching ,and .phs file can read by coot. my question are :what is the
> > .phs used for(or which step it can be used ?) ,just for check my structure
> > ? Can I convert this .phs file into .mtz ,and used it for refinement or
> > model building after I found structure solution ? And only the heavy atom
> > site file (fa.pdb)is used when I searching my structure solution using
> > software? Can you guys give me some software suggestions about solution
> > searching after SHELXC/D/E when the data resolution is around 3A? Thanks a
> > lot
> >
> >Best Regard
> >
> >Shijun
[ccp4bb] SAD phasing
Hello everyone I am learning phasing SAD data now ,and I got some files(like i.phs , .pdb ,fa.res, fa.pdb) when I using SHELXC/D/E,and I know fa.pdb and fa.res (which contain heavy atom information) are used for the further solution searching ,and .phs file can read by coot. my question are :what is the .phs used for(or which step it can be used ?) ,just for check my structure ? Can I convert this .phs file into .mtz ,and used it for refinement or model building after I found structure solution ? And only the heavy atom site file (fa.pdb)is used when I searching my structure solution using software? Can you guys give me some software suggestions about solution searching after SHELXC/D/E when the data resolution is around 3A? Thanks a lot Best Regard Shijun
[ccp4bb] merge different crystal data
Hi ccp4 guys I have a crystal whose spacegroup is I4,and the cell dimension are a=b=83，while ,the C axis is 315.SO the diffraction data is very bad when the crystal started to rotate to some degree ,and it is difficult to process the data .So I wondering there any strategy to resolve this kind of situation ,or any software to merge different crystal data together to process or phasing ? thanks a lot for your suggestions !!! Best Regards SHIJUN
