Re: [ccp4bb] Restrained refinement problem
Hi, did you check in the refmac logfile that the cif file created at the first step was actually read ? If it is, you will have to carefully check the cif file and you ligand structure : 1) is the ligand structure included in the PDB at the fisrt step complete ?? Maybe you included only a partial lignad because of weak density... when you add other atoms, they will be missing from the cif file. 2) are all the ligand atoms present in the cif file with correct names ? 3) are the information present in the cif file correct (connectivity, bond order, planar group, chiral center). This will not produce the error you have, but will certainly distort the geometry... hope this helps laurent Le 12/03/2012 06:21, Dipankar Manna a écrit : Dear All, As I am practicing new in the crystallography, I am facing some difficulties in refining the ligand bound structure. Protein I am working with has SG P212121, it’s a dimer. I fitted the ligand on the density with COOT--calculate--Model/Fit/Refine--Rotate/Translate Zone. Then I merged both (protein ligand) the pdb structure and saved the coordinate as .pdb file. By taking this pdb when I am running restrained refinement it was showing: -- -- Laurent Maveyraud laurent.maveyraud AT ipbs DOT fr Université Paul Sabatier / CNRS / I.P.B.S. UMR 5089 PICT -- Plateforme Intégrée de Criblage de Toulouse Département BiologieStructurale et Biophysique BP 64182 - 205 rte de Narbonne - 31077 TOULOUSE FRANCE Tél: +33 (0)561 175 435 Fax : +33 (0)561 175 994 --
Re: [ccp4bb] Restrained refinement problem
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Dipankar, according to the log-file, the restraints-file was not read in the second time you ran refmac5. It should otherwise be listet in the section underneath Information from CCP4Interface script where it is mentioned as LIBOUT instead of LIBIN. Can you confirm the you added the cif-file as LIB in in the ccp4i GUI and not Output lib? Regards, Tim On 03/12/2012 06:21 AM, Dipankar Manna wrote: Dear All, As I am practicing new in the crystallography, I am facing some difficulties in refining the ligand bound structure. Protein I am working with has SG P212121, it's a dimer. I fitted the ligand on the density with COOT--calculate--Model/Fit/Refine--Rotate/Translate Zone. Then I merged both (protein ligand) the pdb structure and saved the coordinate as .pdb file. By taking this pdb when I am running restrained refinement it was showing: Number of atoms:2508 Number of residues : 319 Number of chains : 3 I am reading library. Please wait. mon_lib.cif WARNING : link:SS is found dist = 2.026 ideal_dist= 2.031 ch:AA res: 15 CYS at:SG .-AA res: 29 CYS at:SG . WARNING : link:SS is found dist = 2.069 ideal_dist= 2.031 ch:AA res: 30 CYS at:SG .-AA res: 43 CYS at:SG . WARNING : link:SS is found dist = 2.031 ideal_dist= 2.031 ch:AA res: 33 CYS at:SG .-AA res: 52 CYS at:SG . . PDB_code: PDB_name: PDB_date:XX-XXX-9- ATTENTION: atom:CD LYS75 BB is missing in the structure ATTENTION: atom:CE LYS75 BB is missing in the structure ATTENTION: atom:NZ LYS75 BB is missing in the structure .. ERROR : atom :C1 LIG 900 CC is absent in the library ERROR : atom :O1 LIG 900 CC is absent in the library ERROR : atom :C2 LIG 900 CC is absent in the library ERROR : atom :C3 LIG 900 CC is absent in the library In the next cycle I put the .cif (newly generated) as LIB in, and rerun the job. Again it was showing some error like: Number of atoms:2508 Number of residues : 319 Number of chains : 3 I am reading library. Please wait. mon_lib.cif WARNING : residue: LIG 900 chain:CC atom: C28 is absent in coord_file atom: C26 is absent in coord_file atom: C24 is absent in coord_file atom: C1 is absent in lib description. atom: O1 is absent in lib description. atom: C2 is absent in lib description. ... WARNING : LIG : program can not match library description program will create complete description for:LIG * Plotfile: C:\Ccp4Temp\refmac5_temp1.01004_new_LIG_N1.ps WARNING : residue: LIG 900 chain:CC - rename LIG -- LIG_N1 WARNING : link:SS is found dist = 2.026 ideal_dist= 2.031 ch:AA res: 15 CYS at:SG .-AA res: 29 CYS at:SG . PDB_code: PDB_name: PDB_date:XX-XXX-9- ATTENTION: atom:CD LYS75 BB is missing in the structure ATTENTION: atom:CE LYS75 BB is missing in the structure ATTENTION: atom:NZ LYS75 BB is missing in the structure ATTENTION: atom:CD LYS78 BB is missing in the structure --- Important, Important, Important! Your coordinate file has a ligand which has either minimum or no description in the library A new ligand description has been added to H:/DATA/TR/020312_C5/TR_8_lib.cif Picture of the new ligand can be viewed using postscript file. See above Check description in this file and, if satisfied, use it as the input library Otherwise either edit bond orders manually or use CCP4i Sketcher to view and edit the ligand and create a library entry by running libcheck It is
[ccp4bb] recombinant enterokinase
Dear all, Would anyone know of a source for recombinant enterokinase? Best, Elias
Re: [ccp4bb] recombinant enterokinase
http://www.neb.com/nebecomm/products/productP8070.asp Date: Mon, 12 Mar 2012 09:38:22 -0400 From: elias.fernan...@utk.edu Subject: [ccp4bb] recombinant enterokinase To: CCP4BB@JISCMAIL.AC.UK Dear all,Would anyone know of a source for recombinant enterokinase? Best,Elias
Re: [ccp4bb] recombinant enterokinase
Dear Elias, Here are three sources for recombinant enterokinase: http://www.genscript.com/protein/Z02199-Porcine_Enterokinase_Lyophilized.html http://www.prospecbio.com/Enterokinase/?gclid=CMuXvK6-4a4CFUQUKgodjGwlXQ http://www.neb.com/nebecomm/products/productP8070.asp Hope it helps! Take Care, Sean Seaver P212121 http://store.p212121.com/
Re: [ccp4bb] recombinant enterokinase
Hi All, I misspoke - what I'm looking for is a source for an expression system for recombinant enterokinase. Elias Subject: recombinant enterokinase Dear all, Would anyone know of a source for recombinant enterokinase? Best, Elias
Re: [ccp4bb] Help! weird thing
Hi - I agree with Garib that its likely a pseudo-translation issue. I also agree with that the advice he gives is correct, but ... ... since I am evidently less smart to follow all these steps, I like to use phenix.xtriage that will tell me if there is pseudo- translation or not, and will give a p-value for that being significant. Its at the end of the text output. I am not sure if Phaser deals these days with pseudo-translation - I guess Randy can tell us. If not, there is a very simple trick to make Phaser work with pseudo- translation, but since I threw the ball to Randy's court and he told me the trick a few years ago, I will let him explain only if needed ;-) Best, Tassos On Mar 11, 2012, at 12:55, Garib N Murshudov wrote: Hi Could you please check: 1) If there is psedotranslation. It could be done by using sfcheck, molrep, ctruncate or calculating patterson map and displaying using coot at 8-10 sigma level (it is my favourite method for analysis of pseudo translations), whole unit cell ( a bit bigger than whole unit cell). Then if you see large no origin peak (very likely along one of the axis, could be a). If yes then you have several options: using phaser - 1) reduce cell, find solution in smaller cell and then expand; 2) use molrep to solve. When there are two copies related with pseudo translation molrep can give you solution; 3) as far as I am aware latest version of phaser works with pseudo translation. If you have pseudtranslation you should be aware that even if you solve the structure starting R factors could be 70-80%. Then you may want to do 40 cycles of rigid body and 40-100 cycles of ljelly body 2) Check your space group in pdb and mtz file. They may not be consistent. I hope it helps. Garib On 11 Mar 2012, at 07:33, xiaoyazi2008 wrote: Hi All, I have an interesting thing to share. 2.3A dataset with good quality, P21 Partial model is available (~60% of the target protein). It seems that there are 4 copies in the ASU (Matthews_coef 2.6, 53%solvent) Molecular replacement gave two copies of the model (Z scores are R6.2, T6.2, R6.8, T13.4). The solution is very clear. It could not locate the rest two copies. However, a quick refmac5 refinement gave a very high R factor. The funny part is the symmetry operation in Coot. As shown in the JPEG figure, it looks like there should be another two copies (based on strong fo-fc green map), which locate in the empty space between models found by Phaser. Why is that Phaser could not find the remaining two copies even there are strong fo-fc density? Any suggestions... Thanks a lot! Zhihong weird thing.jpg Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
[ccp4bb] My protein precipitates at r.t and dissolves at 4 oC
Hi all, I have a homotetrameric coiled-coil domain sample with 45aa per each. While I store this sample at 4oC, the sample looks clear w/o any particles. But when I took out the sample to my bench at r.t, I can see there are precipitates (as stack of needle like particles) at the bottom of the tube after several hours. Interestingly, when I put it back into 4oC fridge, the precipitates disappeared and the solution turned into clear again. Does anyone have knowledge of such behavior of any protein? I appreciate any information related. Min-Kyu
Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC
Which kind of buffer you use? If it is Tris, then temperature change will cause pH change. Actually, this is a good way for crystallization. Kevin On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho min-kyu@live.com wrote: Hi all, I have a homotetrameric coiled-coil domain sample with 45aa per each. While I store this sample at 4oC, the sample looks clear w/o any particles. But when I took out the sample to my bench at r.t, I can see there are precipitates (as stack of needle like particles) at the bottom of the tube after several hours. Interestingly, when I put it back into 4oC fridge, the precipitates disappeared and the solution turned into clear again. Does anyone have knowledge of such behavior of any protein? I appreciate any information related. Min-Kyu
Re: [ccp4bb] Help! weird thing
Yes, the current version of Phaser will do the same test that xtriage carries out, and if it finds a sufficiently high non-origin Patterson peak, it will automatically characterise the translational NCS and use this for molecular replacement. This is working pretty well in our tests. In the near future you will be able to get the current version of Phaser as part of the upcoming CCP4 release, but at the moment the easiest way to get it is to download a recent version of Phenix. You should be able to run that through ccp4i by downloading and installing the updated GUI files from our website (and getting ccp4i to interpret the command phaser as phenix.phaser). Best wishes, Randy Read On 12 Mar 2012, at 16:06, Anastassis Perrakis wrote: Hi - I agree with Garib that its likely a pseudo-translation issue. I also agree with that the advice he gives is correct, but ... ... since I am evidently less smart to follow all these steps, I like to use phenix.xtriage that will tell me if there is pseudo-translation or not, and will give a p-value for that being significant. Its at the end of the text output. I am not sure if Phaser deals these days with pseudo-translation - I guess Randy can tell us. If not, there is a very simple trick to make Phaser work with pseudo-translation, but since I threw the ball to Randy's court and he told me the trick a few years ago, I will let him explain only if needed ;-) Best, Tassos On Mar 11, 2012, at 12:55, Garib N Murshudov wrote: Hi Could you please check: 1) If there is psedotranslation. It could be done by using sfcheck, molrep, ctruncate or calculating patterson map and displaying using coot at 8-10 sigma level (it is my favourite method for analysis of pseudo translations), whole unit cell ( a bit bigger than whole unit cell). Then if you see large no origin peak (very likely along one of the axis, could be a). If yes then you have several options: using phaser - 1) reduce cell, find solution in smaller cell and then expand; 2) use molrep to solve. When there are two copies related with pseudo translation molrep can give you solution; 3) as far as I am aware latest version of phaser works with pseudo translation. If you have pseudtranslation you should be aware that even if you solve the structure starting R factors could be 70-80%. Then you may want to do 40 cycles of rigid body and 40-100 cycles of ljelly body 2) Check your space group in pdb and mtz file. They may not be consistent. I hope it helps. Garib On 11 Mar 2012, at 07:33, xiaoyazi2008 wrote: Hi All, I have an interesting thing to share. 2.3A dataset with good quality, P21 Partial model is available (~60% of the target protein). It seems that there are 4 copies in the ASU (Matthews_coef 2.6, 53%solvent) Molecular replacement gave two copies of the model (Z scores are R6.2, T6.2, R6.8, T13.4). The solution is very clear. It could not locate the rest two copies. However, a quick refmac5 refinement gave a very high R factor. The funny part is the symmetry operation in Coot. As shown in the JPEG figure, it looks like there should be another two copies (based on strong fo-fc green map), which locate in the empty space between models found by Phaser. Why is that Phaser could not find the remaining two copies even there are strong fo-fc density? Any suggestions... Thanks a lot! Zhihong weird thing.jpg Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791 -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] Matthews coeff. from model
Dear CCP4bbers, Is there any tool to calculate the Matthews coefficient from a crystallographic model of RNA-protein complex? Thanking you. James.
[ccp4bb] pdbcur wont remove hydrogens on the residues that have alternative conformations
Just want to point this out although it's pretty easy to remove these hydrogens manually... Or, maybe this only happened to me. Xun -- Department of Molecular and Structural Biochemistry North Carolina State University
Re: [ccp4bb] Matthews coeff. from model
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear James, I do not know such a tool, but you can use 140A^3/a.a. and 380A^3/base to calculate the solvent content by hand. Regards, Tim On 03/12/2012 06:35 PM, james09 pruza wrote: Dear CCP4bbers, Is there any tool to calculate the Matthews coefficient from a crystallographic model of RNA-protein complex? Thanking you. James. - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPXjthUxlJ7aRr7hoRAqPSAJ0Zr7H/Zt0w8TnaJvHsc5g5mZbZngCcCTEC inwbgapeZ+O0jfc20pMVS/M= =0cVz -END PGP SIGNATURE-
Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC
I am using KPi buffer at pH 5.5, 100mM KCl, 2mM beta-mercaptoethanol, 0.02% NaN3. Yes, I agree I should check CD melting curve to see temperature preference of my protein. Min-Kyu | -Original Message- | From: Kevin Jin [mailto:kevin...@gmail.com] | Sent: Monday, March 12, 2012 11:16 AM | To: Min-Kyu Cho | Cc: CCP4BB@jiscmail.ac.uk | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 | oC | | Which kind of buffer you use? If it is Tris, then temperature change will | cause pH change. | | Actually, this is a good way for crystallization. | | Kevin | | On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho min-kyu@live.com wrote: | Hi all, | | I have a homotetrameric coiled-coil domain sample with 45aa per each. | While I store this sample at 4oC, the sample looks clear w/o any | particles. But when I took out the sample to my bench at r.t, I can | see there are precipitates (as stack of needle like particles) at the | bottom of the tube after several hours. Interestingly, when I put it | back into 4oC fridge, the precipitates disappeared and the solution | turned into clear again. | | Does anyone have knowledge of such behavior of any protein? I | appreciate any information related. | | Min-Kyu
Re: [ccp4bb] Matthews coeff. from model
... or rwcontents ? -- Ian On 12 March 2012 17:35, james09 pruza james09x...@gmail.com wrote: Dear CCP4bbers, Is there any tool to calculate the Matthews coefficient from a crystallographic model of RNA-protein complex? Thanking you. James.
Re: [ccp4bb] mosflm and pilatus 2M
SSRL 12-2 uses a Pilatus 6M and can be processed in Mosflm or XDS without problems (have not tried HKL or D*Trek). Do you have the right beam center ? Jürgen On Mar 12, 2012, at 2:22 PM, Dean Derbyshire wrote: Has anyone successfully processed images from the new Pilatus detector? I can visualize them and auto index but not refine the cell or integrate! Oh I’m using version 7.0.7 Cheers Dean .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] mosflm and pilatus 2M
Hi Dean I'd get in touch with the Mosflm authors and ask them for help ;-) A copy of the mosflm.lp file (or the date-stamped version from iMosflm) would be useful in diagnosing the problem. On 12 Mar 2012, at 18:22, Dean Derbyshire wrote: Has anyone successfully processed images from the new Pilatus detector? I can visualize them and auto index but not refine the cell or integrate! Oh I’m using version 7.0.7 Cheers Dean Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC
Min-Kyu, This sounds like to be a hydrophobicity ~ temperature issue (as Kevin pointed out, pH gets involved too). If your protein is sensitive in this regards, it could form different oligomerization states at diff temp, associated with diff solubility. You may want to crystallize it at diff temperatures. Lijun On Mar 12, 2012, at 11:02 AM, Min-Kyu Cho wrote: Hi all, I have a homotetrameric coiled-coil domain sample with 45aa per each. While I store this sample at 4oC, the sample looks clear w/o any particles. But when I took out the sample to my bench at r.t, I can see there are precipitates (as stack of needle like particles) at the bottom of the tube after several hours. Interestingly, when I put it back into 4oC fridge, the precipitates disappeared and the solution turned into clear again. Does anyone have knowledge of such behavior of any protein? I appreciate any information related. Min-Kyu
Re: [ccp4bb] Matthews coeff. from model
I can't imagine the results would be very different for protein-DNA vs. protein-RNA. The reason protein-nucleic acids is an extra category in mattprob is largely due to poorer statistics resulting from limited sample size and hence no reliable resolution dependence can be computed. In addition the partial specific volumes for protein (0.74 cm3/g) and nucleic acids (0.50 cm3/g) are different, so an exact calculation needs to consider their ratio to obtain the correct psv estimate BR - Original Message - From: Tim Gruene t...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 12, 2012 11:07:29 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Matthews coeff. from model -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear James, I do not know such a tool, but you can use 140A^3/a.a. and 380A^3/base to calculate the solvent content by hand. Regards, Tim On 03/12/2012 06:35 PM, james09 pruza wrote: Dear CCP4bbers, Is there any tool to calculate the Matthews coefficient from a crystallographic model of RNA-protein complex? Thanking you. James. - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPXjthUxlJ7aRr7hoRAqPSAJ0Zr7H/Zt0w8TnaJvHsc5g5mZbZngCcCTEC inwbgapeZ+O0jfc20pMVS/M= =0cVz -END PGP SIGNATURE- -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] mosflm and pilatus 2M
Hi again, it's the 2M detector I'm having problems with. Got different data from a 6M and yep that works a dream. ? :0) From: Harry [ha...@mrc-lmb.cam.ac.uk] Sent: 12 March 2012 20:30 To: Dean Derbyshire Cc: Harry; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] mosflm and pilatus 2M Hi Dean I'd get in touch with the Mosflm authors and ask them for help ;-) A copy of the mosflm.lp file (or the date-stamped version from iMosflm) would be useful in diagnosing the problem. On 12 Mar 2012, at 18:22, Dean Derbyshire wrote: Has anyone successfully processed images from the new Pilatus detector? I can visualize them and auto index but not refine the cell or integrate! Oh I’m using version 7.0.7 Cheers Dean Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] Matthews coeff. from model
Is there a difference in the psv between DNA and RNA? I assumed (possibly incorrectly) that they would be very close if not exactly the same? Is mattprob more applicable to protein-DNA complexes than Protein-RNA complexes? Thanks for the insight, Mike - Original Message - From: Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com To: Michael Thompson mi...@chem.ucla.edu, CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 12, 2012 12:48:42 PM GMT -08:00 US/Canada Pacific Subject: RE: [ccp4bb] Matthews coeff. from model I can't imagine the results would be very different for protein-DNA vs. protein-RNA. The reason protein-nucleic acids is an extra category in mattprob is largely due to poorer statistics resulting from limited sample size and hence no reliable resolution dependence can be computed. In addition the partial specific volumes for protein (0.74 cm3/g) and nucleic acids (0.50 cm3/g) are different, so an exact calculation needs to consider their ratio to obtain the correct psv estimate BR - Original Message - From: Tim Gruene t...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 12, 2012 11:07:29 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Matthews coeff. from model -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear James, I do not know such a tool, but you can use 140A^3/a.a. and 380A^3/base to calculate the solvent content by hand. Regards, Tim On 03/12/2012 06:35 PM, james09 pruza wrote: Dear CCP4bbers, Is there any tool to calculate the Matthews coefficient from a crystallographic model of RNA-protein complex? Thanking you. James. - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPXjthUxlJ7aRr7hoRAqPSAJ0Zr7H/Zt0w8TnaJvHsc5g5mZbZngCcCTEC inwbgapeZ+O0jfc20pMVS/M= =0cVz -END PGP SIGNATURE- -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
[ccp4bb] Envelope Phasing.
Hi CCP4bb, I would like to ask about envelope phasing - specifically with SAXS data. There are papers (1) and tutorials (2) describing how this might be done, but I have also found comments on the ccp4bb, such as this one (http://www.proteincrystallography.org/ccp4bb/message11690.html) which are somewhat less optimistic. I get the impression from my reading around that SAXS envelope phasing is somewhat difficult to do unless you have some NCS you can use to help the phase extension process. Does anybody have any opinions/evidence/examples/anecdotes/tips about how SAXS envelope phasing can be done successfully? Cheers, Dave (1) - eg - http://scripts.iucr.org/cgi-bin/paper?dz5081 (2) - eg - http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk Webs : http://flavors.me/xtaldave Twitter: @xtaldave Skype: DocDCB
Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC
Could be one of those weird behaviors displayed by detergents where cloud point anomalously changes with temperature... Artem On Mar 12, 2012 1:11 PM, Min-Kyu Cho min-kyu@live.com wrote: I am using KPi buffer at pH 5.5, 100mM KCl, 2mM beta-mercaptoethanol, 0.02% NaN3. Yes, I agree I should check CD melting curve to see temperature preference of my protein. Min-Kyu | -Original Message- | From: Kevin Jin [mailto:kevin...@gmail.com] | Sent: Monday, March 12, 2012 11:16 AM | To: Min-Kyu Cho | Cc: CCP4BB@jiscmail.ac.uk | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 | oC | | Which kind of buffer you use? If it is Tris, then temperature change will | cause pH change. | | Actually, this is a good way for crystallization. | | Kevin | | On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho min-kyu@live.com wrote: | Hi all, | | I have a homotetrameric coiled-coil domain sample with 45aa per each. | While I store this sample at 4oC, the sample looks clear w/o any | particles. But when I took out the sample to my bench at r.t, I can | see there are precipitates (as stack of needle like particles) at the | bottom of the tube after several hours. Interestingly, when I put it | back into 4oC fridge, the precipitates disappeared and the solution | turned into clear again. | | Does anyone have knowledge of such behavior of any protein? I | appreciate any information related. | | Min-Kyu
Re: [ccp4bb] Envelope Phasing.
Hi David - I think you have four hurdles to overcome: 1. How good is a SAXS envelope 2. How will you place it in the right place 3. How will you extend from ~15-20 A to around 4 A 4. How will you extend from 4 A to beyond Steps 2 and 4 might be the easiest - albeit far from trivial. Point 1 can be argued... However, I have not seen anyone cracking 3 in a convincing manner... (in the absence of serious NCS) I hope the next emails prove me wrong though! A. On 12 Mar 2012, at 21:10, David Briggs wrote: Hi CCP4bb, I would like to ask about envelope phasing - specifically with SAXS data. There are papers (1) and tutorials (2) describing how this might be done, but I have also found comments on the ccp4bb, such as this one (http://www.proteincrystallography.org/ccp4bb/message11690.html) which are somewhat less optimistic. I get the impression from my reading around that SAXS envelope phasing is somewhat difficult to do unless you have some NCS you can use to help the phase extension process. Does anybody have any opinions/evidence/examples/anecdotes/tips about how SAXS envelope phasing can be done successfully? Cheers, Dave (1) - eg - http://scripts.iucr.org/cgi-bin/paper?dz5081 (2) - eg - http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk Webs : http://flavors.me/xtaldave Twitter: @xtaldave Skype: DocDCB
Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC
I remember I saw the similar problem caused by beta-mercaptoethanol. Kevin On Mon, Mar 12, 2012 at 1:29 PM, Artem Evdokimov artem.evdoki...@gmail.com wrote: Could be one of those weird behaviors displayed by detergents where cloud point anomalously changes with temperature... Artem On Mar 12, 2012 1:11 PM, Min-Kyu Cho min-kyu@live.com wrote: I am using KPi buffer at pH 5.5, 100mM KCl, 2mM beta-mercaptoethanol, 0.02% NaN3. Yes, I agree I should check CD melting curve to see temperature preference of my protein. Min-Kyu | -Original Message- | From: Kevin Jin [mailto:kevin...@gmail.com] | Sent: Monday, March 12, 2012 11:16 AM | To: Min-Kyu Cho | Cc: CCP4BB@jiscmail.ac.uk | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 | oC | | Which kind of buffer you use? If it is Tris, then temperature change will | cause pH change. | | Actually, this is a good way for crystallization. | | Kevin | | On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho min-kyu@live.com wrote: | Hi all, | | I have a homotetrameric coiled-coil domain sample with 45aa per each. | While I store this sample at 4oC, the sample looks clear w/o any | particles. But when I took out the sample to my bench at r.t, I can | see there are precipitates (as stack of needle like particles) at the | bottom of the tube after several hours. Interestingly, when I put it | back into 4oC fridge, the precipitates disappeared and the solution | turned into clear again. | | Does anyone have knowledge of such behavior of any protein? I | appreciate any information related. | | Min-Kyu
Re: [ccp4bb] Matthews coeff. from model
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 The number 380A^3/base I gave results from a decent number of nucleic acids-only PDB files I once analysed with the 'volume' program from ccp4. I cannot think of an application where knowing the solvent content must be known to more than 1%, so I bet this number is fine for any such purposes. I got the number 140A^3/a.a. from George Sheldrick who told me where he got this number from, but I do not remember for sure - it could be Kevin Cowtan, but George may update my memory in order to pay proper credit. Cheers, Tim On 03/12/2012 08:48 PM, Bernhard Rupp (Hofkristallrat a.D.) wrote: I can't imagine the results would be very different for protein-DNA vs. protein-RNA. The reason protein-nucleic acids is an extra category in mattprob is largely due to poorer statistics resulting from limited sample size and hence no reliable resolution dependence can be computed. In addition the partial specific volumes for protein (0.74 cm3/g) and nucleic acids (0.50 cm3/g) are different, so an exact calculation needs to consider their ratio to obtain the correct psv estimate BR - Original Message - From: Tim Gruene t...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 12, 2012 11:07:29 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Matthews coeff. from model Dear James, I do not know such a tool, but you can use 140A^3/a.a. and 380A^3/base to calculate the solvent content by hand. Regards, Tim On 03/12/2012 06:35 PM, james09 pruza wrote: Dear CCP4bbers, Is there any tool to calculate the Matthews coefficient from a crystallographic model of RNA-protein complex? Thanking you. James. - -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPXl8gUxlJ7aRr7hoRAnKIAKC9BiLuWTo8j/NOUkKee1FlcOSULACggsPw uFJo7bCPaZ2mJ/LQcsxkDKU= =Wzko -END PGP SIGNATURE-
Re: [ccp4bb] Envelope Phasing.
Point 3: Use the saxs model for preliminary phasing of your HA derivative Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Mar 12, 2012, at 16:30, Anastassis Perrakis a.perra...@nki.nl wrote: Hi David - I think you have four hurdles to overcome: 1. How good is a SAXS envelope 2. How will you place it in the right place 3. How will you extend from ~15-20 A to around 4 A 4. How will you extend from 4 A to beyond Steps 2 and 4 might be the easiest - albeit far from trivial. Point 1 can be argued... However, I have not seen anyone cracking 3 in a convincing manner... (in the absence of serious NCS) I hope the next emails prove me wrong though! A. On 12 Mar 2012, at 21:10, David Briggs wrote: Hi CCP4bb, I would like to ask about envelope phasing - specifically with SAXS data. There are papers (1) and tutorials (2) describing how this might be done, but I have also found comments on the ccp4bb, such as this one (http://www.proteincrystallography.org/ccp4bb/message11690.html) which are somewhat less optimistic. I get the impression from my reading around that SAXS envelope phasing is somewhat difficult to do unless you have some NCS you can use to help the phase extension process. Does anybody have any opinions/evidence/examples/anecdotes/tips about how SAXS envelope phasing can be done successfully? Cheers, Dave (1) - eg - http://scripts.iucr.org/cgi-bin/paper?dz5081 (2) - eg - http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk Webs : http://flavors.me/xtaldave Twitter: @xtaldave Skype: DocDCB
Re: [ccp4bb] Matthews coeff. from model
My memory is not as good as it used to be, but I think that I indeed got the number of 140 cubic Angstroms per amino-acid from Kevin. It has worked fine ever since, and is how hkl2map calculates the solvent content for proteins. George On 12.03.2012 21:40, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 The number 380A^3/base I gave results from a decent number of nucleic acids-only PDB files I once analysed with the 'volume' program from ccp4. I cannot think of an application where knowing the solvent content must be known to more than 1%, so I bet this number is fine for any such purposes. I got the number 140A^3/a.a. from George Sheldrick who told me where he got this number from, but I do not remember for sure - it could be Kevin Cowtan, but George may update my memory in order to pay proper credit. Cheers, Tim On 03/12/2012 08:48 PM, Bernhard Rupp (Hofkristallrat a.D.) wrote: I can't imagine the results would be very different for protein-DNA vs. protein-RNA. The reason protein-nucleic acids is an extra category in mattprob is largely due to poorer statistics resulting from limited sample size and hence no reliable resolution dependence can be computed. In addition the partial specific volumes for protein (0.74 cm3/g) and nucleic acids (0.50 cm3/g) are different, so an exact calculation needs to consider their ratio to obtain the correct psv estimate BR - Original Message - From: Tim Gruenet...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 12, 2012 11:07:29 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Matthews coeff. from model Dear James, I do not know such a tool, but you can use 140A^3/a.a. and 380A^3/base to calculate the solvent content by hand. Regards, Tim On 03/12/2012 06:35 PM, james09 pruza wrote: Dear CCP4bbers, Is there any tool to calculate the Matthews coefficient from a crystallographic model of RNA-protein complex? Thanking you. James. - -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPXl8gUxlJ7aRr7hoRAnKIAKC9BiLuWTo8j/NOUkKee1FlcOSULACggsPw uFJo7bCPaZ2mJ/LQcsxkDKU= =Wzko -END PGP SIGNATURE-
Re: [ccp4bb] Envelope Phasing.
Hi Francis, As you may have guessed - my question is stems from my recent acquisition of some native data for a protein that I have lots of SAXS data for. I suspect that more conventional MR is unlikely to work in this case as I only have a half-decent phasing model for ~30% of the protein (2 copies in the ASU, so 2x15%). I figured that seeing as I had all this SAXS data (and I have many datasets collected at multiple synchrotrons that all give very consistent results), I might as well have a pop at phasing with it. I have an Fsearch run running at the moment using Flms generated from the Crysol program (ATSAS package) - no output as yet - does this program only output results to the log file at the end of the run? I have about 6 weeks until my next available synchrotron time, where derivatives will be screened, better resolution native data will hopefully be obtained, etc. In the meantime... Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk Webs : http://flavors.me/xtaldave Twitter: @xtaldave Skype: DocDCB On 12 March 2012 22:09, Francis E Reyes francis.re...@colorado.edu wrote: Hi David I'm the original poster you mention in your email, and I'm sad to report I never really got anywhere with the initial inquiry outside of James Holton's original response. The good news is that I was able to solve the structure I mentioned at 4A. (albeit through other means). From my own literature research I can vaguely remember that bridging the SAXS resolution to the X-ray resolution required measuring some really low angle reflections in the x-ray data (100-20A). And of course it's the phase extension piece that James refers to. (20A to 4A is a hell of a jump). However, even John Tainer has suggested that SAXS envelopes can be used to locate heavy atoms in isomorphous or anomalous difference data, though this has yet to be shown in practice. Depending on the heavy atom this can certainly help with the phase extension. Was your question academic, or are you, like I was, stuck in low resolution land with no phases? (I can certainly assist with the latter). F On Mar 12, 2012, at 2:10 PM, David Briggs wrote: Hi CCP4bb, I would like to ask about envelope phasing - specifically with SAXS data. There are papers (1) and tutorials (2) describing how this might be done, but I have also found comments on the ccp4bb, such as this one (http://www.proteincrystallography.org/ccp4bb/message11690.html) which are somewhat less optimistic. I get the impression from my reading around that SAXS envelope phasing is somewhat difficult to do unless you have some NCS you can use to help the phase extension process. Does anybody have any opinions/evidence/examples/anecdotes/tips about how SAXS envelope phasing can be done successfully? Cheers, Dave (1) - eg - http://scripts.iucr.org/cgi-bin/paper?dz5081 (2) - eg - http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk Webs : http://flavors.me/xtaldave Twitter: @xtaldave Skype: DocDCB - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC
Hi Kevin, Could you tell me more detail about beta-mercaptoethanol problem? Min-Kyu | -Original Message- | From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of | Kevin Jin | Sent: Monday, March 12, 2012 3:32 PM | To: CCP4BB@JISCMAIL.AC.UK | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 | oC | | I remember I saw the similar problem caused by beta-mercaptoethanol. | | | Kevin | | | On Mon, Mar 12, 2012 at 1:29 PM, Artem Evdokimov | artem.evdoki...@gmail.com wrote: | Could be one of those weird behaviors displayed by detergents where | cloud point anomalously changes with temperature... | | Artem | | On Mar 12, 2012 1:11 PM, Min-Kyu Cho min-kyu@live.com wrote: | | I am using KPi buffer at pH 5.5, 100mM KCl, 2mM beta-mercaptoethanol, | 0.02% NaN3. | | Yes, I agree I should check CD melting curve to see temperature | preference of my protein. | | Min-Kyu | | | -Original Message- | | From: Kevin Jin [mailto:kevin...@gmail.com] | | Sent: Monday, March 12, 2012 11:16 AM | | To: Min-Kyu Cho | | Cc: CCP4BB@jiscmail.ac.uk | | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves | at 4 | | oC | | | | Which kind of buffer you use? If it is Tris, then temperature | change will | | cause pH change. | | | | Actually, this is a good way for crystallization. | | | | Kevin | | | | On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho | min-kyu@live.com | wrote: | | Hi all, | | | | I have a homotetrameric coiled-coil domain sample with 45aa per | each. | | While I store this sample at 4oC, the sample looks clear w/o any | | particles. But when I took out the sample to my bench at r.t, I | can | | see there are precipitates (as stack of needle like particles) | at the | | bottom of the tube after several hours. Interestingly, when I | put it | | back into 4oC fridge, the precipitates disappeared and the | solution | | turned into clear again. | | | | Does anyone have knowledge of such behavior of any protein? I | | appreciate any information related. | | | | Min-Kyu
[ccp4bb] RE : [ccp4bb] Envelope Phasing.
Dear David, I would remind that the molecular-replacement positionning of molecular envelopes and some methodological features (in comparison with a conventional MR) of this have been discussed in : Urzhumtsev Podjarny (1995). On the Solution of the Molecular Replacement Problem at Very Low Resolution: Application to Large Complexes. Acta Cryst. D51, 888-895. Concerning the practical results, at my knowlegde, this was the first step in the ribosome phasing by Nenad Ban in 1999 (while the envelope was from EM and not from SAXS, but this shall not make a difference). Best regards, Sacha Urzhumtsev De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Date d'envoi : lundi 12 mars 2012 21:10 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Envelope Phasing. Hi CCP4bb, I would like to ask about envelope phasing - specifically with SAXS data. There are papers (1) and tutorials (2) describing how this might be done, but I have also found comments on the ccp4bb, such as this one (http://www.proteincrystallography.org/ccp4bb/message11690.html) which are somewhat less optimistic. I get the impression from my reading around that SAXS envelope phasing is somewhat difficult to do unless you have some NCS you can use to help the phase extension process. Does anybody have any opinions/evidence/examples/anecdotes/tips about how SAXS envelope phasing can be done successfully? Cheers, Dave (1) - eg - http://scripts.iucr.org/cgi-bin/paper?dz5081 (2) - eg - http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk Webs : http://flavors.me/xtaldave Twitter: @xtaldave Skype: DocDCB
