Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?
>I think there is a misconception floating around that processing your >data with "anomalous turned on" will somehow degrade the quality of >"normal" intensity data. I can think of very few circumstances when I would NOT want anomalous data, yet for many data processing pipelines, it is the default not to give you the I+ and I- separately. Anomalous data are very useful for locating metal ions that you might not even have suspected to exist in your structure. Can I make a plea that all data processing packages/pipelines give you anomalous data by default? Can anyone think of a good reason why they shouldn't? James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of James Holton [jmhol...@lbl.gov] Sent: Wednesday, June 13, 2012 3:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique? I think there is a misconception floating around that processing your data with "anomalous turned on" will somehow degrade the quality of "normal" intensity data. I'm not exactly sure where this rumor comes from, but I imagine it has something to do with confusion about all the various "anomalous" options different scaling programs have. For example, some programs offer the option to treat all I+ and all I- as completely separate data sets, scaled and merged independently. I think this is called "scale anomalous" in SCALEPACK and "intensities anomalous" in SCALA. Neither of these is the default because such treatment is only helpful if the anomalous signal is absolutely huge (I have only seen this once). So, I imagine people who have never done experimental phasing (there are lots of them!) might read things like "Switching ANOMALOUS ON does affect the statistics and the outlier rejection" in the SCALA manual and decide that they had better turn off all those evil "anomalous" things. Then they tell their students to do the same, etc. -James Holton MAD Scientist On Tue, Jun 12, 2012 at 3:17 AM, Eleanor Dodson wrote: > Why would anyone ignore the anomalous data they had collected? It will always > help the phasing, and decide the hand for you.. > Eleanor > On 6 Jun 2012, at 03:55, Stefan Gajewski wrote: > >> Hey! >> >> I was just wondering, do you know of any recent (~10y) publication that >> presented a structure solution solely based on MIR? Without the use of any >> anomalous signal of some sort? >> >> When was the last time you saw a structure that was solved without the use >> of anomalous signal or homology model? Is there a way to look up the answer >> (e.g. filter settings in the RCSB) I am not aware of? >> >> Thanks, >> S. >> >> (Disclaimer: I am aware that isomorpous data is a valuable source of >> information)
Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?
Just to be clear, the CCP4 data processing programs (SCALA and its replacement AIMLESS) always give you I+ and I- in the output. The only difference between "anomalous on & off" is in the outlier rejection, since if you have a large anomalous signal you don't want to reject as "outliers" reflections with a good strong anomalous difference. AIMLESS now automatically detects whether there is a substantial anomalous signal and switches this option ON if there is (unless you specify the option explicitly). There are also different Rmeas etc values within I+/I- sets and over all data. In the scaling, as James points out, it is nearly always best to ignore the I+/I- distinction, unless you really have a huge anomalous signal (almost impossible for macromolecules), since you want to try to minimise anomalous differences to reduce systematic errors, so that what is left is more likely to be real signal. SCALA allows you the (unrecommended) option to separate I+ and I- in scaling, but I haven't programmed this in AIMLESS since I have never seen a case where it would be a good idea. As far as I know, in CCP4 you only lose I+ and I- if you explicitly remove them. Phil On 13 Jun 2012, at 08:03, Murray, James W wrote: >> I think there is a misconception floating around that processing your >> data with "anomalous turned on" will somehow degrade the quality of >> "normal" intensity data. > > I can think of very few circumstances when I would NOT want anomalous data, > yet for many data processing pipelines, it is the default not to give you the > I+ and I- separately. Anomalous data are very useful for locating metal ions > that you might not even have suspected to exist in your structure. Can I make > a plea that all data processing packages/pipelines give you anomalous data by > default? Can anyone think of a good reason why they shouldn't? > > James > > -- > Dr. James W. Murray > David Phillips Research Fellow > Division of Molecular Biosciences > Imperial College, LONDON > Tel: +44 (0)20 759 48895 > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of James Holton > [jmhol...@lbl.gov] > Sent: Wednesday, June 13, 2012 3:47 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an > obsolete technique? > > I think there is a misconception floating around that processing your > data with "anomalous turned on" will somehow degrade the quality of > "normal" intensity data. I'm not exactly sure where this rumor comes > from, but I imagine it has something to do with confusion about all > the various "anomalous" options different scaling programs have. For > example, some programs offer the option to treat all I+ and all I- as > completely separate data sets, scaled and merged independently. I > think this is called "scale anomalous" in SCALEPACK and "intensities > anomalous" in SCALA. Neither of these is the default because such > treatment is only helpful if the anomalous signal is absolutely huge > (I have only seen this once). So, I imagine people who have never > done experimental phasing (there are lots of them!) might read things > like "Switching ANOMALOUS ON does affect the statistics and the > outlier rejection" in the SCALA manual and decide that they had better > turn off all those evil "anomalous" things. Then they tell their > students to do the same, etc. > > -James Holton > MAD Scientist > > > On Tue, Jun 12, 2012 at 3:17 AM, Eleanor Dodson > wrote: >> Why would anyone ignore the anomalous data they had collected? It will >> always help the phasing, and decide the hand for you.. >> Eleanor >> On 6 Jun 2012, at 03:55, Stefan Gajewski wrote: >> >>> Hey! >>> >>> I was just wondering, do you know of any recent (~10y) publication that >>> presented a structure solution solely based on MIR? Without the use of any >>> anomalous signal of some sort? >>> >>> When was the last time you saw a structure that was solved without the use >>> of anomalous signal or homology model? Is there a way to look up the answer >>> (e.g. filter settings in the RCSB) I am not aware of? >>> >>> Thanks, >>> S. >>> >>> (Disclaimer: I am aware that isomorpous data is a valuable source of >>> information)
[ccp4bb] ICCBM14 Workshop & Conference, Hunstville, Alabama
We cordially invite you to participate in the 14th International Conference on the Crystallization of Biological Macromolecules (ICCBM14) on September 23-28, 2012. The conference will be held in Huntsville, the "/Rocket City/," in the beautiful state of Alabama. The meeting site will be at the Westin within the second largest research park in the U.S. nearby the NASA Marshall Space Flight Center, the Redstone Arsenal and the new HudsonAlpha Institute for Biotechnology. The theme of the meeting will be synthetic design, engineering and strategies for macromolecular crystallization. Focus will be on the practical and theoretical basis of macromolecular crystallization involving synthetic biology, protein complexes, nucleic acids, membrane proteins and convection-free crystallization platforms. A practical workshop will precede the main conference devoted to hands-on instruction on selected aspects of macromolecule production, synthetic biology engineering and crystal growth. Lodging will be available at the Westin conference center as well as other convenient and affordable housing nearby the University of Alabama in Huntsville and Research Park. Early registration deadline has been extended to July 14, 2012 Please visit the conference homepage at http://www.iccbm14.org We look forward to seeing you in September 2012! /Joseph Ng and Marc Pusey/, Co-chairs of ICCBM14
[ccp4bb] PHENIX: sequence files input problem
Dear all, Sorry for the non-CCP4 question. My crystal is a complex of two proteins. I use Se-Met SAD method to solve the structure, and only one of the proteins is Se-Met protein. When I use phenix.autobuild to auto-build the model, how do I input the sequences? in a file or two files? And is that necessary to specify the location of MSE? Thanks a lot for your help. Qixu Cai
Re: [ccp4bb] how to get phase of huge complex
Lisa, As others have said, using careful data collection and the modern program suites available (SHARP, Phenix, etc.), a 300 KD complex with 111 Se-Met residues should be quite solvable. But you didn't state is what is in the asymmetric unit (the important figure): one complex with 111 Se-Met residues or 111 Se-Met residues and > 300KD in the ASU. We recently solved a structure by SAD with >750 KD of protein and 114 Se-Met residues in the ASU. The anomalous signal at 3.1 A was strong enough to find 109 Se-Met residues and trace about 70% of the chain after the first round of phasing. While we had the potential of NCS to work with, conformational changes between the different monomers meant that NCS methods could not be used in the initial phasing. Trying also James' suggestion of differential labeling and/or including MIRAS methods (Hg, Pt, Sm, etc.) with Se-Met protein should increase your chances. With modern beamlines, you can tune to the most optimal wavelengths for data collection. Good hunting, 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 Jun 12, 2012, at 10:46 PM, LISA wrote: > Hi all, > > My work is to solve huge complex containing 4 different proteins and total > molecular weight is about 300 KD. I can purify the complex by co-expression > them in E.coli. This complex contains 8 protein A, 2 protein B and 1 protein > C and D. protein B and protein C have homology structures deposited in PDB > database. No homology structure available for protein A and D, which > contribute 60% of the whole molecular weight for the complex. > > Now I am trying to find a way to solve the phase of this complex. I am > thinking of use sad or mad with se-Met. There total 111 Met residues in > this complex. Is it possible to solve this complex by se-Met? Does someone > have experience to solve huge complex structure with se-met? It is also very > welcome for all the suggestion. Thank you. > > All the best, > > Lisa
[ccp4bb] Recompile fsearch with increased memory
Hi, I am trying to run fsearch to do molecular replacement from a SAXS envelope, but it gives an error: FSEARCH: *out of allocated memory* I assume this is because of my large unit cell: 133.9180 147.8250 274.7790 90. 90. 90. Is it possible to increase the amount of memory available to fsearch? I have tried increasing MAX3D in the fsearch source, but could not get it to link on my system (ld: symbol(s) not found). I'm running ccp4 6.2.0 on Mac OS X 10.6.8 Any help would be appreciated. Thanks, Jason. -- Jason Busby PhD Student Laboratory of Structural Biology School of Biological Sciences University of Auckland Thomas Building 110 3a Symonds St Private Bag 92019 Auckland 1142 New Zealand ph: +64 9 3737599 ext 84155 fx: +64 9 3737414
Re: [ccp4bb] PHENIX: sequence files input problem
Hi Qixu Cai, first off -- as a reminder, there is Phenix mailing list to post Phenix-related questions: http://www.phenix-online.org/ Your first question: yes, give it one sequence file containing all. Your second question: see here http://phenix-online.org/documentation/autobuild_gui.htm Pavel On Wed, Jun 13, 2012 at 5:14 AM, Qixu Cai wrote: > Dear all, > > Sorry for the non-CCP4 question. > > My crystal is a complex of two proteins. I use Se-Met SAD method to solve > the structure, and only one of the proteins is Se-Met protein. > > When I use phenix.autobuild to auto-build the model, how do I input the > sequences? in a file or two files? > > And is that necessary to specify the location of MSE? > > Thanks a lot for your help. > > Qixu Cai >
[ccp4bb] Off-Topic
Dear Friends, Does anyone know how to control the polymerization of G-actin to F-actin? I need F-actin with <100 DP (degree of polymerization). Any suggestion is highly appreciated. Jahan
Re: [ccp4bb] Recompile fsearch with increased memory
Jason, It seems like you're on the right track - it's usually not a problem to increase array sizes and rebuild. How are you building (aka "cd $CCP4/src; make fsearch" or something else)? If you're calling the fortran compiler (or ld) directly, it looks like there's an a problem with the library options or search paths. Pete Jason Busby wrote: Hi, I am trying to run fsearch to do molecular replacement from a SAXS envelope, but it gives an error: FSEARCH: *out of allocated memory* I assume this is because of my large unit cell: 133.9180 147.8250 274.7790 90. 90. 90. Is it possible to increase the amount of memory available to fsearch? I have tried increasing MAX3D in the fsearch source, but could not get it to link on my system (ld: symbol(s) not found). I'm running ccp4 6.2.0 on Mac OS X 10.6.8 Any help would be appreciated. Thanks, Jason. -- Jason Busby PhD Student Laboratory of Structural Biology School of Biological Sciences University of Auckland Thomas Building 110 3a Symonds St Private Bag 92019 Auckland 1142 New Zealand ph: +64 9 3737599 ext 84155 fx: +64 9 3737414
Re: [ccp4bb] Off-Topic
Matthew Vetting Enzyme Function Initiative Structural Biology Albert Einstein College of Medicine - Reply message - From: "Jahan Alikhajeh" To: Subject: [ccp4bb] Off-Topic Date: Wed, Jun 13, 2012 5:05 pm Dear Friends, Does anyone know how to control the polymerization of G-actin to F-actin? I need F-actin with <100 DP (degree of polymerization). Any suggestion is highly appreciated. Jahan
Re: [ccp4bb] PHENIX: sequence files input problem
Matthew Vetting Enzyme Function Initiative Structural Biology Albert Einstein College of Medicine - Reply message - From: "Pavel Afonine" To: Subject: [ccp4bb] PHENIX: sequence files input problem Date: Wed, Jun 13, 2012 4:51 pm Hi Qixu Cai, first off -- as a reminder, there is Phenix mailing list to post Phenix-related questions:http://www.phenix-online.org/ Your first question: yes, give it one sequence file containing all.Your second question: see herehttp://phenix-online.org/documentation/autobuild_gui.htm Pavel On Wed, Jun 13, 2012 at 5:14 AM, Qixu Cai wrote: Dear all, Sorry for the non-CCP4 question. My crystal is a complex of two proteins. I use Se-Met SAD method to solve the structure, and only one of the proteins is Se-Met protein. When I use phenix.autobuild to auto-build the model, how do I input the sequences? in a file or two files? And is that necessary to specify the location of MSE? Thanks a lot for your help. Qixu Cai
Re: [ccp4bb] Recompile fsearch with increased memory
Another option might be to reduce the resolution of the search. With SAXS you don't have much resolution in your search model anyway. On 06/13/12 16:33, Jason Busby wrote: Hi, I am trying to run fsearch to do molecular replacement from a SAXS envelope, but it gives an error: FSEARCH: *out of allocated memory* I assume this is because of my large unit cell: 133.9180 147.8250 274.7790 90. 90. 90. Is it possible to increase the amount of memory available to fsearch? I have tried increasing MAX3D in the fsearch source, but could not get it to link on my system (ld: symbol(s) not found). I'm running ccp4 6.2.0 on Mac OS X 10.6.8 Any help would be appreciated. Thanks, Jason. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Recompile fsearch with increased memory
Ah thanks, I've managed to get it sorted out. In case anyone else has this issue, I downloaded the CCP4 source code, increased MAX3D in fsearch.f, and recompiled. I first built CCP4 (./configure && make) and then fsearch explicitly (cd src && make fsearch). Thanks, Jason. -- Jason Busby PhD Student Laboratory of Structural Biology School of Biological Sciences University of Auckland Thomas Building 110 3a Symonds St Private Bag 92019 Auckland 1142 New Zealand ph: +64 9 3737599 ext 84155 fx: +64 9 3737414 On 14/06/2012, at 9:17 AM, Pete Meyer wrote: > Jason, > > It seems like you're on the right track - it's usually not a problem to > increase array sizes and rebuild. > > How are you building (aka "cd $CCP4/src; make fsearch" or something else)? > If you're calling the fortran compiler (or ld) directly, it looks like > there's an a problem with the library options or search paths. > > Pete > > Jason Busby wrote: >> Hi, >> I am trying to run fsearch to do molecular replacement from a SAXS envelope, >> but it gives an error: FSEARCH: *out of allocated memory* >> I assume this is because of my large unit cell: 133.9180 147.8250 >> 274.7790 90. 90. 90. >> Is it possible to increase the amount of memory available to fsearch? I >> have tried increasing MAX3D in the fsearch source, but could not get it to >> link on my system (ld: symbol(s) not found). >> I'm running ccp4 6.2.0 on Mac OS X 10.6.8 >> Any help would be appreciated. >> Thanks, >> Jason. >> -- >> Jason Busby >> PhD Student >> Laboratory of Structural Biology >> School of Biological Sciences >> University of Auckland >> Thomas Building 110 >> 3a Symonds St >> Private Bag 92019 >> Auckland 1142 >> New Zealand >> ph: +64 9 3737599 ext 84155 >> fx: +64 9 3737414 >
