Re: [ccp4bb] low-resolution data and SG
Dear SDY, It is impossible to deduct from data statistics alone the difference between e.g. P43 21 2 and P41 21 2. Also with weak data like you have, a lot of artifacts may arise due to (weak) ice rings, intensity from neighboring strong reflections getting into the integration boxes of weak reflections, spurious reflections due to contaminating salt microcrystals etc.etc.etc. What you need to do is to integrate your data in the basic point group: P4 or P422, depending how sure you are about the additional twofold, and then run Phaser with the SGALTERNATIVE ALL option, so it will check all possible space groups (P41 2 2, P41 21 2, P42 2 2, P42 21 2 etc.). I am pretty sure you will find that the space group which will come out then will not be the P43 21 2 you have assumed right now and that refinement in this space group will solve your problem. Since with your current solution, you will have most of your symmetries correct, you still can get Rfactors in the 30-40% range like you observe. If you wish, after you found the correct space group with Phaser, you could reprocess your data using this correct space group. Best regards, Herman Schreuder From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of SD Y Sent: Sunday, November 04, 2012 4:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] low-resolution data and SG Dear All, I have few basic questions for which I need help. I have a 3.4 A data and I have processed it to P4. !--[if !supportLists]--1. !--[endif]--I used pointless to find SG, it suggests P41 21 2. But I see two strong intensities in systematic absences Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 2 -0.7 0.3 -2.0 0 0 3 1.0 0.4 2.3 0 0 5 0.3 0.7 0.4 0 0 6 -0.7 0.9 -0.8 0 0 7 -0.4 0.9 -0.4 0 0 9 -0.2 0.9 -0.2 0 0 10 1.3 1.2 1.1 0 0 11 -0.8 2.1 -0.4 0 0 13 1.2 2.1 0.6 0 0 14 2.3 1.8 1.3 0 0 15 -1.0 1.9 -0.5 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 3 0 0 -0.1 0.2 -0.8 5 0 0 0.2 0.2 0.9 7 0 0 -0.3 0.2 -1.3 9 0 0 0.0 0.5 0.0 11 0 0 -0.2 0.6 -0.4 13 0 0 0.8 0.7 1.1 15 0 0 -1.2 0.6 -1.9 17 0 0 -0.3 0.8 -0.4 19 0 0 -1.4 0.6 -2.6 21 0 0 -2.2 1.2 -1.9 23 0 0 -0.8 1.3 -0.6 25 0 0 -1.2 1.1 -1.1 27 0 0 -0.9 1.6 -0.5 29 0 0 -0.4 1.7 -0.2 31 0 0 -7.1 1.3 -5.3 33 0 0 -2.4 2.1 -1.1 !--[if !supportLists]--2. !--[endif]--When I used phaser for MR, it gave weak solution in p43, so I scaled data in p43 21 2 (this also two intesities high like above in systamatic absences) and used for Phaser to get the following solution SINGLE solution SOLU SET RFZ=4.5 TFZ=9.4 PAK=0 LLG=105 TFZ==10.1 RF++ TFZ=17.7 PAK=0 LLG=282 TFZ==15.6 LLG=285 TFZ==12.4 SOLU SPAC P 43 21 2 SOLU 6DIM ENSE ensemble1 EULER 153.1 50.3 73.2 FRAC -0.11 0.03 -0.94 BFAC -2.65 SOLU 6DIM ENSE ensemble1 EULER 148.4 129.9 252.8 FRAC -0.32 -0.35 1.07 BFAC 4.01 Ensemble ensemble1 RMS variance(s): 1.13 !--[if !supportLists]--3. !--[endif]--I used this solution to further refine the model in refmac, using local ncs, with/without jelly, optimized weight/weight of 0.03, map sharpening with B=20 in several rounds. I noticed that R factor R factor stayed around 33% while R free keeps floating around 42%. I could see some density for missing loop in the model and I could build but the R work and R free moving apart. By reading, I understand that this is very common for low resolution data unless I use appropriate restraints. I am wondering if my space group is correct? I had understood that if it's right SG, high intensity reflections will not be found in systematic absences but I started doubting if I have understood correctly. This is my first low resolution data, I want use this
[ccp4bb] refmac5 problem
Dear all, What's the difference between no prior phase information, phase and FOM, Hendrickson-Lattman coefficients, and SAD data directly in the refmac5 GUI of CCP4i? I have a SAD dataset and solve the phase by phenix.autosol. Now I want to refine the structure by refmac5, which kind of input above I should choose? Another question is in the Refinement Parameters of refmac5. What's the meaning of Use experimental sigmas to weight Xray terms? If I use molecular replacement to solve the structure, shall I uncheck this item? Thanks very much for your help. Best wishes, Q. Cai
[ccp4bb]
Dear all, I am now determining a structure at 2.2 A resolution. The space group is C2, and there is only one molecular in an ASU. During the refinement, some weird electron density appears right on the two-fold axis. Does anyone have any idea what could this be? Thanks a lot. Best regards Shutong Xu
[ccp4bb] Weird electron density on the two-fold axis
Dear all, I am now determining a structure at 2.2 A resolution. The space group is C2, and there is only one molecular in an ASU. During the refinement, some weird electron density appears right on the two-fold axis. Does anyone have any idea what could this be? Thanks a lot. Best regards Shutong Xu
[ccp4bb] Copying R-free flags - possibly daft question.
Am I worrying about something unnecessarily? I have several protein-drug datasets, all in the same spacegroup, but wildly varying resolutions. I wish to use the same reflections for calculating R-free in all cases. Using xia2 with a reference dataset for both indexing and R-free seems to work fine, apart from the fact that the resulting mtz file, now contains R-free labels for reflections that have no observations… i.e. taken from the higher resolution reference dataset; see output below. I get essentially the same results using CAD to copy the R-free column between mtzfiles…. Is this actually a problem - or is it just my innate sense of tidiness that wants the resolution values to be the same? Many thanks, Antony. Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 36 0 100.00 14.2 14.2 57.66 2.02 H H 2 NONE 0 36 0 100.00 14.6 14.6 57.66 2.02 H K 3 NONE 0 38 0 100.00 14.8 14.8 57.66 2.02 H L 4 NONE0.019.0 132 99.18 9.52 9.52 57.40 2.02 I FreeR_flag 5 NONE -30.6 10855.5 11647 28.03 202.72 203.43 57.66 3.17 J IMEAN 6 NONE1.5 356.4 11647 28.0311.5211.52 57.66 3.17 Q SIGIMEAN 7 NONE7.8 1040.0 11647 28.03 109.05 109.05 57.66 3.17 F F 8 NONE1.423.8 11647 28.03 6.59 6.59 57.66 3.17 Q SIGF No. of reflections used in FILE STATISTICS16183 --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512
Re: [ccp4bb] Extra electron density
Dear Yogesh, Since your difference density is at the center of a hexamer, you might be looking at the 6-fold average density of one or two fatty acid molecules. Just try to fit one and generate to other 5 equivalent molecules and look if this makes sense. Otherwise, I agree with Mark that with hardly any 2mFo-dFC density, you may also be looking at an artifact. Best regards, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J van Raaij Sent: Monday, November 05, 2012 9:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Extra electron density It may just be noise. Notice there there appears to be no electron density, only difference density. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 5 Nov 2012, at 01:38, yogesh khandokar wrote: Dear All I am working on an enzyme which involved in fatty acid biosynthesis. We solved the crystal structure of it. Biological unit of this enzyme is Hexamer and showing extra electron density in the center of Hexamer. Wincoot software doesn't recognize this extra electron density as water molecules/ substrate. My question is:Is there any way to find the molecules responsible for extra electron density? Picture is attached with email. Thanks in advance for your comments. Regards -- Yogesh Khandokar PhD Student School of Biomedical Sciences Charles Sturt University Wagga Wagga, NSW, Australia http://www.csu.edu.au/faculty/science/biomed/ Doc1.doc
[ccp4bb]
It might be a 50% occupied protein side chain(s), since only one of the two equivalent protein side chains can occupy this position. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Crystal Xu Sent: Monday, November 05, 2012 11:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] I am sorry that I forget to attach the electron density map 2012/11/5 Crystal Xu crystalh...@gmail.com Dear all, I am now determining a structure at 2.2 A resolution. The space group is C2, and there is only one molecular in an ASU. During the refinement, some weird electron density appears right on the two-fold axis. Does anyone have any idea what could this be? Thanks a lot. Best regards Shutong Xu
Re: [ccp4bb] Weird electron density on the two-fold axis
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Shutong Xu, one often finds artefacts on special positions. As far as I understand, these are numerical 'instabilities' or artefacts rather than of chemical origin. Best, Tim On 11/05/2012 10:50 AM, Crystal Xu wrote: Dear all, I am now determining a structure at 2.2 A resolution. The space group is C2, and there is only one molecular in an ASU. During the refinement, some weird electron density appears right on the two-fold axis. Does anyone have any idea what could this be? Thanks a lot. Best regards Shutong Xu - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQl5NkUxlJ7aRr7hoRAkUtAKC/6mrt5LdcG9bjZm3rN6UfDzpotQCg7B20 WknHYPcUECMxk//k9zKjiqE= =WCPn -END PGP SIGNATURE-
Re: [ccp4bb] Copying R-free flags - possibly daft question.
Yes, if your data does not extend to high resolution, your free reflections will not extend to high resolution either (e.g. are not observed). Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Antony Oliver Sent: Monday, November 05, 2012 10:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Copying R-free flags - possibly daft question. Am I worrying about something unnecessarily? I have several protein-drug datasets, all in the same spacegroup, but wildly varying resolutions. I wish to use the same reflections for calculating R-free in all cases. Using xia2 with a reference dataset for both indexing and R-free seems to work fine, apart from the fact that the resulting mtz file, now contains R-free labels for reflections that have no observations... i.e. taken from the higher resolution reference dataset; see output below. I get essentially the same results using CAD to copy the R-free column between mtzfiles Is this actually a problem - or is it just my innate sense of tidiness that wants the resolution values to be the same? Many thanks, Antony. Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. Low High label 1 ASC 0 36 0 100.00 14.2 14.2 57.66 2.02 H H 2 NONE 0 36 0 100.00 14.6 14.6 57.66 2.02 H K 3 NONE 0 38 0 100.00 14.8 14.8 57.66 2.02 H L 4 NONE0.019.0 132 99.18 9.52 9.52 57.40 2.02 I FreeR_flag 5 NONE -30.6 10855.5 11647 28.03 202.72 203.43 57.66 3.17 J IMEAN 6 NONE1.5 356.4 11647 28.0311.5211.52 57.66 3.17 Q SIGIMEAN 7 NONE7.8 1040.0 11647 28.03 109.05 109.05 57.66 3.17 F F 8 NONE1.423.8 11647 28.03 6.59 6.59 57.66 3.17 Q SIGF No. of reflections used in FILE STATISTICS16183 --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512
Re: [ccp4bb] Copying R-free flags - possibly daft question.
Hi Antony, Using xia2 with a reference dataset for both indexing and R-free seems to work fine, apart from the fact that the resulting mtz file, now contains R-free labels for reflections that have no observations… i.e. taken from the higher resolution reference dataset; see output below. I get essentially the same results using CAD to copy the R-free column between mtzfiles…. This is exactly what xia2 does, so no surprise there. Does anyone have a neater CAD script which does this? Alternatively I guess it would be the work of an hour or so using CCTBX Python to make it so… if no-one has a neat CAD script I will look at doing this. It is untidy, I don't really like untidy things however I suspect it will make no difference at all - the missing values will be flagged as such so should not mess anything up. Best wishes, Graeme
Re: [ccp4bb] Weird electron density on the two-fold axis
As only small part of the molecules is shown and it appears to be in poly-alanine representation, more interesting thing may happened apart of numerical instabilities of Fourier transform. These can be mutually exclusive conformations that brake local symmetry but preserve overall symmetry. The alts penetrate forbidden region of the symmetry, but only from one molecule. I have seen such things. BTW what program was used to calculate maps? FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 5, 2012, at 12:22 , Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Shutong Xu, one often finds artefacts on special positions. As far as I understand, these are numerical 'instabilities' or artefacts rather than of chemical origin. Best, Tim On 11/05/2012 10:50 AM, Crystal Xu wrote: Dear all, I am now determining a structure at 2.2 A resolution. The space group is C2, and there is only one molecular in an ASU. During the refinement, some weird electron density appears right on the two-fold axis. Does anyone have any idea what could this be? Thanks a lot. Best regards Shutong Xu - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQl5NkUxlJ7aRr7hoRAkUtAKC/6mrt5LdcG9bjZm3rN6UfDzpotQCg7B20 WknHYPcUECMxk//k9zKjiqE= =WCPn -END PGP SIGNATURE-
Re: [ccp4bb] low-resolution data and SG
There are several questions here. 1) What is the point Group - P4 or P422. I find the pointless statistics which check all symmetry operators singly useful. If the 2 fold kh-l gives about the same CC as the 4 fold operators k-hl -h -k l -k h l then you probably do have point group P422. ( But check the twinning graph to see if twinning is likely - twinning in this space group can mimic the 2 fold k,h,-l ) and if it is present you should work in point group P4. 2) What is the space group? The absences make it look like P42 21 2 or P 43 21 2 except for the rogues 00 19 and 00 20 which maybe are just that - rogues - see Jims suggestions..) But do you have a non-crystallographic translation? this can be found by a native patterson peak search, and is part of the truncate output. If there IS a peak at x,y,z=1/4 say then that might mislead you be producing weak reflections along 0 0 l axis. 3) Find the MR solution - and that is easiest done using the Phaser command test alterspacegroups.. Eleanor On 4 Nov 2012, at 15:03, SD Y wrote: Dear All, I have few basic questions for which I need help. I have a 3.4 A data and I have processed it to P4. 1. I used pointless to find SG, it suggests P41 21 2. But I see two strong intensities in systematic absences Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 2 -0.7 0.3 -2.0 0 0 3 1.0 0.4 2.3 0 0 5 0.3 0.7 0.4 0 0 6 -0.7 0.9 -0.8 0 0 7 -0.4 0.9 -0.4 0 0 9 -0.2 0.9 -0.2 0 0 10 1.3 1.2 1.1 0 0 11 -0.8 2.1 -0.4 0 0 13 1.2 2.1 0.6 0 0 14 2.3 1.8 1.3 0 0 15 -1.0 1.9 -0.5 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 3 0 0 -0.1 0.2 -0.8 5 0 0 0.2 0.2 0.9 7 0 0 -0.3 0.2 -1.3 9 0 0 0.0 0.5 0.0 11 0 0 -0.2 0.6 -0.4 13 0 0 0.8 0.7 1.1 15 0 0 -1.2 0.6 -1.9 17 0 0 -0.3 0.8 -0.4 19 0 0 -1.4 0.6 -2.6 21 0 0 -2.2 1.2 -1.9 23 0 0 -0.8 1.3 -0.6 25 0 0 -1.2 1.1 -1.1 27 0 0 -0.9 1.6 -0.5 29 0 0 -0.4 1.7 -0.2 31 0 0 -7.1 1.3 -5.3 33 0 0 -2.4 2.1 -1.1 2. When I used phaser for MR, it gave weak solution in p43, so I scaled data in p43 21 2 (this also two intesities high like above in systamatic absences) and used for Phaser to get the following solution SINGLE solution SOLU SET RFZ=4.5 TFZ=9.4 PAK=0 LLG=105 TFZ==10.1 RF++ TFZ=17.7 PAK=0 LLG=282 TFZ==15.6 LLG=285 TFZ==12.4 SOLU SPAC P 43 21 2 SOLU 6DIM ENSE ensemble1 EULER 153.1 50.3 73.2 FRAC -0.11 0.03 -0.94 BFAC -2.65 SOLU 6DIM ENSE ensemble1 EULER 148.4 129.9 252.8 FRAC -0.32 -0.35 1.07 BFAC 4.01 Ensemble ensemble1 RMS variance(s): 1.13 3. I used this solution to further refine the model in refmac, using local ncs, with/without jelly, optimized weight/weight of 0.03, map sharpening with B=20 in several rounds. I noticed that R factor R factor stayed around 33% while R free keeps floating around 42%. I could see some density for missing loop in the model and I could build but the R work and R free moving apart. By reading, I understand that this is very common for low resolution data unless I use appropriate restraints. I am wondering if my space group is correct? I had understood that if it’s right SG, high intensity reflections will not be found in systematic absences but I started doubting if I have understood correctly. This is my first low resolution data, I want use this opportunity to learn refmac well. So could you please let me know if my doubt is right regarding SG and how do I troubleshoot. Thanks SDY
Re: [ccp4bb] refmac5 problem
Dear Cai On 5 Nov 2012, at 09:46, Qixu Cai wrote: Dear all, What's the difference between no prior phase information, phase and FOM, Hendrickson-Lattman coefficients, and SAD data directly in the refmac5 GUI of CCP4i? I would use SAD data directly if you have SAD data set. It just means that the program uses observed F+ and F- directly in refinement. This part of the program has been developed by Leiden group so you can direct your questions to them: Navraj S. Pannu r...@chem.leidenuniv.nl Pavol Skubak p.sku...@chem.leidenuniv.nl I am sure they will help you to get started. I have a SAD dataset and solve the phase by phenix.autosol. Now I want to refine the structure by refmac5, which kind of input above I should choose? Another question is in the Refinement Parameters of refmac5. What's the meaning of Use experimental sigmas to weight Xray terms? If I use molecular replacement to solve the structure, shall I uncheck this item? No. It is related with sigmas of observations and it is desirable to use them. Although there are some question marks how sigmas are estimated, they seemed to be better than nothing. regards Garib Thanks very much for your help. Best wishes, Q. Cai Dr Garib N Murshudov Group Leader, 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
Re: [ccp4bb] refmac5 problem
I don't know what exactly the phenix.autosol output is. If you are satisfied with the solution and have a mtz file containing F sigF HLA etc, then use Hendrickson Lattmann coefficients. If there is only Phase/Fom then use that. I am not sure how you would fare with SAD data directly.. But beware - do the HLs etc combine phases from the existing model and the anomalous signal - if so they may be biased towards the existing model, and make it harder to correct Eleanor On 5 Nov 2012, at 09:46, Qixu Cai wrote: Dear all, What's the difference between no prior phase information, phase and FOM, Hendrickson-Lattman coefficients, and SAD data directly in the refmac5 GUI of CCP4i? I have a SAD dataset and solve the phase by phenix.autosol. Now I want to refine the structure by refmac5, which kind of input above I should choose? Another question is in the Refinement Parameters of refmac5. What's the meaning of Use experimental sigmas to weight Xray terms? If I use molecular replacement to solve the structure, shall I uncheck this item? Thanks very much for your help. Best wishes, Q. Cai
Re: [ccp4bb] Copying R-free flags - possibly daft question.
Just give CAD the resolution cut off of the new data set.. Eleanor From the CAD documentation.. RESOLUTION [ RESOLUTION OVERALL dmin dmax ] | [RESOLUTION FILE_NUMBER i dmin dmax ] Use either: RESOLUTION OVERALL dmin dmax for overall resolution limits, or: RESOLUTION FILE_NUMBER i dmin dmax to set input limit for FILE_NUMBER i. dmax, dmin are the resolution limits for the data to be included, i.e. data are included for which (1/dmax)**2 = 4 sin**2theta/lambda**2 =(1/dmin)**2 NOTE: Defaults are 0.1 and 1000.0 Angstrom. On 5 Nov 2012, at 09:53, Antony Oliver wrote: Am I worrying about something unnecessarily? I have several protein-drug datasets, all in the same spacegroup, but wildly varying resolutions. I wish to use the same reflections for calculating R-free in all cases. Using xia2 with a reference dataset for both indexing and R-free seems to work fine, apart from the fact that the resulting mtz file, now contains R-free labels for reflections that have no observations… i.e. taken from the higher resolution reference dataset; see output below. I get essentially the same results using CAD to copy the R-free column between mtzfiles…. Is this actually a problem - or is it just my innate sense of tidiness that wants the resolution values to be the same? Many thanks, Antony. Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 36 0 100.00 14.2 14.2 57.66 2.02 H H 2 NONE 0 36 0 100.00 14.6 14.6 57.66 2.02 H K 3 NONE 0 38 0 100.00 14.8 14.8 57.66 2.02 H L 4 NONE0.019.0 132 99.18 9.52 9.52 57.40 2.02 I FreeR_flag 5 NONE -30.6 10855.5 11647 28.03 202.72 203.43 57.66 3.17 J IMEAN 6 NONE1.5 356.4 11647 28.0311.5211.52 57.66 3.17 Q SIGIMEAN 7 NONE7.8 1040.0 11647 28.03 109.05 109.05 57.66 3.17 F F 8 NONE1.423.8 11647 28.03 6.59 6.59 57.66 3.17 Q SIGF No. of reflections used in FILE STATISTICS16183 --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512
[ccp4bb]
Any spurious blob on a symmetry axis is multiplied up of course. And when you finish building side chains it may reduce in size. But waters etc can sit on special positions - you just enter them with occ = 0.5 and refine as usual.. Eleanor On 5 Nov 2012, at 09:49, Crystal Xu wrote: Dear all, I am now determining a structure at 2.2 A resolution. The space group is C2, and there is only one molecular in an ASU. During the refinement, some weird electron density appears right on the two-fold axis. Does anyone have any idea what could this be? Thanks a lot. Best regards Shutong Xu
Re: [ccp4bb] Copying R-free flags - possibly daft question.
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 If you want to remove the indices which are not observed, you can use sftools (this has been suggested to me on this board, sorry I do not remember by whom). sftools eof read yourfile.mtz select only column IMEAN present write temp1.mtz END eof mv temp1.mtz yourfile.mtz This way mtzdmp reports the resolution of your data, not of the indices, and I find this a clean solution. Cheers, Tim On 11/05/2012 10:53 AM, Antony Oliver wrote: Am I worrying about something unnecessarily? I have several protein-drug datasets, all in the same spacegroup, but wildly varying resolutions. I wish to use the same reflections for calculating R-free in all cases. Using xia2 with a reference dataset for both indexing and R-free seems to work fine, apart from the fact that the resulting mtz file, now contains R-free labels for reflections that have no observations… i.e. taken from the higher resolution reference dataset; see output below. I get essentially the same results using CAD to copy the R-free column between mtzfiles…. Is this actually a problem - or is it just my innate sense of tidiness that wants the resolution values to be the same? Many thanks, Antony. Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 36 0 100.00 14.2 14.2 57.66 2.02 H H 2 NONE 0 36 0 100.00 14.6 14.6 57.66 2.02 H K 3 NONE 0 38 0 100.00 14.8 14.8 57.66 2.02 H L 4 NONE0.019.0 132 99.18 9.52 9.52 57.40 2.02 I FreeR_flag 5 NONE -30.6 10855.5 11647 28.03 202.72 203.43 57.66 3.17 J IMEAN 6 NONE1.5 356.4 11647 28.0311.5211.52 57.66 3.17 Q SIGIMEAN 7 NONE7.8 1040.0 11647 28.03 109.05 109.05 57.66 3.17 F F 8 NONE1.423.8 11647 28.03 6.59 6.59 57.66 3.17 Q SIGF No. of reflections used in FILE STATISTICS16183 --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQl6Q+UxlJ7aRr7hoRAnJxAKDJka3GNBDOePq29MyfsCUGTXhjggCffy4J QIlkkxo4iV3i9G4xtB+hCd0= =Mku1 -END PGP SIGNATURE-
Re: [ccp4bb] Copying R-free flags - possibly daft question.
Thanks Eleanor - guess I should have plumbed the depths of the CAD manual a little further. Works perfectly. Antony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On Nov 5, 2012, at 11:24 AM, Eleanor Dodson wrote: Just give CAD the resolution cut off of the new data set.. Eleanor From the CAD documentation.. RESOLUTION [ RESOLUTION OVERALL dmin dmax ] | [RESOLUTION FILE_NUMBER i dmin dmax ] Use either: RESOLUTION OVERALL dmin dmax for overall resolution limits, or: RESOLUTION FILE_NUMBER i dmin dmax to set input limit for FILE_NUMBER i. dmax, dmin are the resolution limits for the data to be included, i.e. data are included for which (1/dmax)**2 = 4 sin**2theta/lambda**2 =(1/dmin)**2 NOTE: Defaults are 0.1 and 1000.0 Angstrom. On 5 Nov 2012, at 09:53, Antony Oliver wrote: Am I worrying about something unnecessarily? I have several protein-drug datasets, all in the same spacegroup, but wildly varying resolutions. I wish to use the same reflections for calculating R-free in all cases. Using xia2 with a reference dataset for both indexing and R-free seems to work fine, apart from the fact that the resulting mtz file, now contains R-free labels for reflections that have no observations… i.e. taken from the higher resolution reference dataset; see output below. I get essentially the same results using CAD to copy the R-free column between mtzfiles…. Is this actually a problem - or is it just my innate sense of tidiness that wants the resolution values to be the same? Many thanks, Antony. Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 36 0 100.00 14.2 14.2 57.66 2.02 H H 2 NONE 0 36 0 100.00 14.6 14.6 57.66 2.02 H K 3 NONE 0 38 0 100.00 14.8 14.8 57.66 2.02 H L 4 NONE0.019.0 132 99.18 9.52 9.52 57.40 2.02 I FreeR_flag 5 NONE -30.6 10855.5 11647 28.03 202.72 203.43 57.66 3.17 J IMEAN 6 NONE1.5 356.4 11647 28.0311.5211.52 57.66 3.17 Q SIGIMEAN 7 NONE7.8 1040.0 11647 28.03 109.05 109.05 57.66 3.17 F F 8 NONE1.423.8 11647 28.03 6.59 6.59 57.66 3.17 Q SIGF No. of reflections used in FILE STATISTICS16183 --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512
Re: [ccp4bb] refmac5 problem
I am not sure how you would fare with SAD data directly.. If you would like a comparison, check out the refmac paper. http://journals.iucr.org/d/issues/2011/04/00/ba5152/index.html See Figure 1 - this shows model building with ARP/wARP on over 100 data sets using the different Refmac functions that you asked about. The MLHL function is using phase and fom or Hendrickson-Lattman coefficients The Rice function is no prior phase information and the SAD function is using SAD data directly. The figure shows fraction of the model built automatically. If you see lot of red squares on the top left of the diagonal (which you do), this just means the SAD function outperforms the 'Rice' function and you see more Red squares then Blue circles, so SAD performs better than MLHL. You can get more information on individual functions from references cited in the Refmac paper. The top right shows data sets between 80-100% built by all functions: thus, if your model/map is good enough, any function can build it. For SAD data sets that we have collected here, we always use the SAD function even at the end of the refinement and found (unsurprisingly) a lower difference between R-free and R then other functions. As Garib mentioned, Pavol Skubak and/or I are happy to answer any questions. Best wishes, Raj But beware - do the HLs etc combine phases from the existing model and the anomalous signal - if so they may be biased towards the existing model, and make it harder to correct Eleanor On 5 Nov 2012, at 09:46, Qixu Cai wrote: Dear all, What's the difference between no prior phase information, phase and FOM, Hendrickson-Lattman coefficients, and SAD data directly in the refmac5 GUI of CCP4i? I have a SAD dataset and solve the phase by phenix.autosol. Now I want to refine the structure by refmac5, which kind of input above I should choose? Another question is in the Refinement Parameters of refmac5. What's the meaning of Use experimental sigmas to weight Xray terms? If I use molecular replacement to solve the structure, shall I uncheck this item? Thanks very much for your help. Best wishes, Q. Cai
[ccp4bb]
It might be a 50% occupied protein side chain(s), since only one of the two equivalent protein side chains can occupy this position. Best Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Crystal Xu Sent: Monday, November 05, 2012 11:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] I am sorry that I forget to attach the electron density map 2012/11/5 Crystal Xu crystalh...@gmail.com Dear all, I am now determining a structure at 2.2 A resolution. The space group is C2, and there is only one molecular in an ASU. During the refinement, some weird electron density appears right on the two-fold axis. Does anyone have any idea what could this be? Thanks a lot. Best regards Shutong Xu
Re: [ccp4bb] Extra electron density
You know what? Scratch what I said. As Mark says, there isn't any 2Fo-Fc density, which is suspicious. However, I would still model what may fit in it, with appropriate occupancy, based on the symmetry. To be doubly sure. I have, in the past, used ProDRGhttp://davapc1.bioch.dundee.ac.uk/prodrg/submit.htmlfor obtaining pdf, topology and parameter files for ligands. ARP/wARP can be used for a first fit of ligand into density. You may find appropriate fatty acid pdb-top-param files already in the HIC-UP http://xray.bmc.uu.se/hicup/ database. I would do a quick search for a fatty acid of an appropriate length. If it is truly spurious density as I suspect it is, you'll see negative density lighting up your screen despite using appropriately low occupancy. Good luck! Sangeetha. On Sun, Nov 4, 2012 at 8:17 PM, Sangeetha Vedula sangeetha...@gmail.comwrote: Could it be a fatty acid? It looks like it has a (hydrophobic?) tail with a (charged?) head. Partially occupied perhaps, as they're so close together. On Sun, Nov 4, 2012 at 7:38 PM, yogesh khandokar yogesh.khando...@gmail.com wrote: Dear All I am working on an enzyme which involved in fatty acid biosynthesis. We solved the crystal structure of it. Biological unit of this enzyme is Hexamer and showing extra electron density in the center of Hexamer. Wincoot software doesn't recognize this extra electron density as water molecules/ substrate. My question is:Is there any way to find the molecules responsible for extra electron density? Picture is attached with email. Thanks in advance for your comments. Regards -- Yogesh Khandokar PhD Student School of Biomedical Sciences Charles Sturt University Wagga Wagga, NSW, Australia http://www.csu.edu.au/faculty/science/biomed/
Re: [ccp4bb] protein cleavage
Dear Rana, I think you need to clear up some confusion about this experiment. MBP fusions suffer from a number of drawbacks depending on what you are doing. First, did you use the MPB domain to purify the fusion protein (with an amylose column)? If so, you also purified native MBP from the E. coli as well (and there is a good amount in the periplasm). Therefore you should expect to see MBP before and after TEV cleavage, regardless of whether you have a fusion or not. Second, did you see the MBP fusion on SDS PAGE, particularly on Westerns with anti-MBP? Depending on your answers, we can troubleshoot your situation. The MBP fusion vectors we have made incorporate an N-terminal His tag, followed by MBP and TEV, so we can purify the fusion either by Ni-chelation or amylose column chromatography (or both). Also we have experienced cases where, despite our best efforts, MBP fusion either is a truncated expression fragment (mostly MBP) or has a relatively inaccessible TEV site. For example, does DHBx dimerize? This could block access to the TEV site. But first, does the MBP-DHBx fusion exist and did you purify the fusion with an amylose column? Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Nov 4, 2012, at 10:24 AM, rana ibd wrote: Dear CCP4 I am having a problem with cleaving my fusion protein and I would be grateful if you advice me regarding this situation, I have an MBP-DHBx fusion protein and I am trying to cleave it using TEV protease, I have tried different ratios and different temperatures with different incubation time but still it will not cleave, all I observe on the gel is the bands of the fusion protein which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa and no sign of the DHBx which is 17kDa,I have also checked the sequence if there was any problem but I could not find anything unusual the sequence was fine , so if you have any suggestions regarding this situation I will be thankful Best Regards Rana
Re: [ccp4bb] Termination of Shelxc/d/e
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear, from the lack of any shelxc-related output in the log-file my first guess is you don't have shelxc installed on your system - do you? It is not part of ccp4 and must be installed separately. (http://shelx.uni-ac.gwdg.de/SHELX/index.html#Obtain%20SHELX) Regards, Tim On 11/05/2012 03:53 PM, Shanti Pal Gangwar wrote: Dear All I have installed ccp4 version 6.3.0. I am facing problem during shelxc/d/e run as it terminates with termination status shelxc failed to generate native hkl file Please suggest me how to rectify above mentioned problem. For your convenience I have attached the log file of this run. Thanks is advance. - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQl9ROUxlJ7aRr7hoRAmymAJ4ltmNhA9afo0IBx+4wZirhwApS/gCffynA 7VL0JKR+glPDH9xuZl8XDdE= =ujYt -END PGP SIGNATURE-
Re: [ccp4bb] Copying R-free flags - possibly daft question.
Thanks indeed Eleanor, when I get a moment I will add this too to the xia2 cad script! Best wishes, Graeme On 5 November 2012 11:37, Antony Oliver antony.oli...@sussex.ac.uk wrote: Thanks Eleanor - guess I should have plumbed the depths of the CAD manual a little further. Works perfectly. Antony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On Nov 5, 2012, at 11:24 AM, Eleanor Dodson wrote: Just give CAD the resolution cut off of the new data set.. Eleanor From the CAD documentation.. RESOLUTION [ RESOLUTION OVERALL dmin dmax ] | [RESOLUTION FILE_NUMBER i dmin dmax ] Use either: RESOLUTION OVERALL dmin dmax for overall resolution limits, or: RESOLUTION FILE_NUMBER i dmin dmax to set input limit for FILE_NUMBER i. dmax, dmin are the resolution limits for the data to be included, i.e. data are included for which (1/dmax)**2 = 4 sin**2theta/lambda**2 =(1/dmin)**2 NOTE: Defaults are 0.1 and 1000.0 Angstrom. On 5 Nov 2012, at 09:53, Antony Oliver wrote: Am I worrying about something unnecessarily? I have several protein-drug datasets, all in the same spacegroup, but wildly varying resolutions. I wish to use the same reflections for calculating R-free in all cases. Using xia2 with a reference dataset for both indexing and R-free seems to work fine, apart from the fact that the resulting mtz file, now contains R-free labels for reflections that have no observations… i.e. taken from the higher resolution reference dataset; see output below. I get essentially the same results using CAD to copy the R-free column between mtzfiles…. Is this actually a problem - or is it just my innate sense of tidiness that wants the resolution values to be the same? Many thanks, Antony. Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 36 0 100.00 14.2 14.2 57.66 2.02 H H 2 NONE 0 36 0 100.00 14.6 14.6 57.66 2.02 H K 3 NONE 0 38 0 100.00 14.8 14.8 57.66 2.02 H L 4 NONE0.019.0 132 99.18 9.52 9.52 57.40 2.02 I FreeR_flag 5 NONE -30.6 10855.5 11647 28.03 202.72 203.43 57.66 3.17 J IMEAN 6 NONE1.5 356.4 11647 28.0311.5211.52 57.66 3.17 Q SIGIMEAN 7 NONE7.8 1040.0 11647 28.03 109.05 109.05 57.66 3.17 F F 8 NONE1.423.8 11647 28.03 6.59 6.59 57.66 3.17 Q SIGF No. of reflections used in FILE STATISTICS16183 --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512
Re: [ccp4bb] Termination of Shelxc/d/e
Dear Sir Tim from the lack of any shelxc-related output in the log-file my first guess is you don't have shelxc installed on your system - do you? It is not part of ccp4 and must be installed separately. I have already have installed shelxc/d/e on my computer and its also shoeing in CCp4 program list.On intallation its shwoing successful installation but during Run it is failed. Thanks Dear Sir John Dear Shanti, Basic steps:- Print, sign and send the license form to Prof Sheldrick. He will then instruct you how to download the exe files. Take note of whether you have a 64 bit computer ie for the correct exe files. Place them into a folder eg in the ccp4 binaries program folder. In the ccp4 admin section of the GUI explicitly add the path to the program exe files, as above. Exit the GUI and re enter it. The program icon for Shelx C/D/E will now not be greyed out. The program inputs are obvious. So far I got C and D to work fine, but not E. Phaser EP also allows you to run C and D. The advantage of running them independently are the excellent graphs eg of delta anom/sigma delta anom versus resolution. I have downloaded Shelx exe files and tried to download on 64 Bit computer. during installtion it showed succesfull installtion but during run it fails. please suggest. Thanks On 5 November 2012 20:29, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear, from the lack of any shelxc-related output in the log-file my first guess is you don't have shelxc installed on your system - do you? It is not part of ccp4 and must be installed separately. (http://shelx.uni-ac.gwdg.de/SHELX/index.html#Obtain%20SHELX) Regards, Tim On 11/05/2012 03:53 PM, Shanti Pal Gangwar wrote: Dear All I have installed ccp4 version 6.3.0. I am facing problem during shelxc/d/e run as it terminates with termination status shelxc failed to generate native hkl file Please suggest me how to rectify above mentioned problem. For your convenience I have attached the log file of this run. Thanks is advance. - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQl9ROUxlJ7aRr7hoRAmymAJ4ltmNhA9afo0IBx+4wZirhwApS/gCffynA 7VL0JKR+glPDH9xuZl8XDdE= =ujYt -END PGP SIGNATURE- -- regards Shanti Pal Gangwar School of Life Sciences Jawaharlal Nehru University New Delhi-110067 India Email:gangwar...@gmail.com
Re: [ccp4bb] Termination of Shelxc/d/e
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Shanti, to track the problem I suggest you download the tutorial and data from my homepage at http://shelx.uni-ac.gwdg.de/~tg/teaching/anl-ccp4/index.php and run shelxc outside ccp4. If that works, one can further try to isolate the problem. Is there any other output other than the log-file you sent, e.g. to the terminal? Regards, Tim On 11/06/2012 07:10 AM, Shanti Pal Gangwar wrote: Dear Sir Tim from the lack of any shelxc-related output in the log-file my first guess is you don't have shelxc installed on your system - do you? It is not part of ccp4 and must be installed separately. I have already have installed shelxc/d/e on my computer and its also shoeing in CCp4 program list.On intallation its shwoing successful installation but during Run it is failed. Thanks Dear Sir John Dear Shanti, Basic steps:- Print, sign and send the license form to Prof Sheldrick. He will then instruct you how to download the exe files. Take note of whether you have a 64 bit computer ie for the correct exe files. Place them into a folder eg in the ccp4 binaries program folder. In the ccp4 admin section of the GUI explicitly add the path to the program exe files, as above. Exit the GUI and re enter it. The program icon for Shelx C/D/E will now not be greyed out. The program inputs are obvious. So far I got C and D to work fine, but not E. Phaser EP also allows you to run C and D. The advantage of running them independently are the excellent graphs eg of delta anom/sigma delta anom versus resolution. I have downloaded Shelx exe files and tried to download on 64 Bit computer. during installtion it showed succesfull installtion but during run it fails. please suggest. Thanks On 5 November 2012 20:29, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear, from the lack of any shelxc-related output in the log-file my first guess is you don't have shelxc installed on your system - do you? It is not part of ccp4 and must be installed separately. (http://shelx.uni-ac.gwdg.de/SHELX/index.html#Obtain%20SHELX) Regards, Tim On 11/05/2012 03:53 PM, Shanti Pal Gangwar wrote: Dear All I have installed ccp4 version 6.3.0. I am facing problem during shelxc/d/e run as it terminates with termination status shelxc failed to generate native hkl file Please suggest me how to rectify above mentioned problem. For your convenience I have attached the log file of this run. Thanks is advance. - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQmMBLUxlJ7aRr7hoRAo++AJ0T5jJzAIpBMhMlDetkj/N+WhJcSQCg8y9l efxq+yt+RWEkwuAqOogcC7o= =rcI9 -END PGP SIGNATURE-
