[ccp4bb] Difference map
Hi Folks I have a query regarding the difference map between the two structures ligand bound data (2.5A) and native (2.8A). I tried to calculate the fourier difference map between two data sets ligand bound- native. The protocol in CCP4 that i used is as: 1.merge the mtz file of native nad ligand bound using cad 2. scaling this combine file with scaleit program followed by map generation uisng fft. I got the map but i did not find the fu;ll map of the ligand,i can only see small density nera the ligand binding site at 5 sigma level. I have calculated omit map that cleraly showed the ligand. why is such discrepency in the two cases, is there is something missing from the calculation. kindly help me out. Thanks and regards Intekhab alam -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
Re: [ccp4bb] Difference map
Hi Intekhab Alam, an Fobs-Fobs' map usually works only if the two crystals are isomorphous, which means that there are neither large cell constant changes nor any other larger structural changes (like overall rotations, domain movements, other rearrangements) than the bound compound (ligand, heavy atom, ...). Scaleit produces a plot of Riso against resolution which ideally should resemble the scattering curve of the bound compound (~ like an atomic scattering curve for instance), plus a mild increase at high resolution due to the increasing noise component at higher resolution. If the curve starts at high Riso values and increases steeply, this would indicate anisomorphism, and there is probably no chance to detect your compound signal. All this is qualitative, but you could estimate the expected change with the Crick-Magdoff equation by replacing the heavy atoms with a sum over your compound atoms. The Crick-Magdoff equations has been recently discussed on this board: http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg10039.html Good luck, Dirk. Am 29.09.10 09:09, schrieb intekhab alam: Hi Folks I have a query regarding the difference map between the two structures ligand bound data (2.5A) and native (2.8A). I tried to calculate the fourier difference map between two data sets ligand bound- native. The protocol in CCP4 that i used is as: 1.merge the mtz file of native nad ligand bound using cad 2. scaling this combine file with scaleit program followed by map generation uisng fft. I got the map but i did not find the fu;ll map of the ligand,i can only see small density nera the ligand binding site at 5 sigma level. I have calculated omit map that cleraly showed the ligand. why is such discrepency in the two cases, is there is something missing from the calculation. kindly help me out. Thanks and regards Intekhab alam -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] Difference map
That seems the right procedure. I presume the two crystals have similar cell, etc? What is the Riso plot from scaleit look like - if it is 55% + then there is no isomorphism, but if it is 30% then the map should be reasonable. which phase did you use for the map calculation? Eleanor intekhab alam wrote: Hi Folks I have a query regarding the difference map between the two structures ligand bound data (2.5A) and native (2.8A). I tried to calculate the fourier difference map between two data sets ligand bound- native. The protocol in CCP4 that i used is as: 1.merge the mtz file of native nad ligand bound using cad 2. scaling this combine file with scaleit program followed by map generation uisng fft. I got the map but i did not find the fu;ll map of the ligand,i can only see small density nera the ligand binding site at 5 sigma level. I have calculated omit map that cleraly showed the ligand. why is such discrepency in the two cases, is there is something missing from the calculation. kindly help me out. Thanks and regards Intekhab alam
Re: [ccp4bb] Difference Map images
Tim, Not really. Fo's can be viewed as having 3 parts. 1) F from our modeled structure, 2) F from what we can't model. This can be bulk solvent, partial or multiple occupancies in low resolution structures, thermal anisotropy , etc. 3) F from random errors in measuring data. The first F is what we deal with. The third F we can minimize by collecting high quality data. The second F is usually crystal/space group dependent. It arises from crystallization conditions, crystal morphology and intermolecular contacts. Data from isomorphic crystals should have similar second F's so map (from first F's) is cleaner. Doug -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim Gruene Sent: Wednesday, June 17, 2009 4:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Difference Map images I know that sometimes Fo(complex)-Fo(apo) cannot be done because of nonisomorphism. We've had a lot of success with this with the dioxygenases because there is no large scale alteration in the active site. As for the technique itself, Brian Matthews drilled this into me when I was a postdoc in his lab. Wouldn't - in the case of non-isomorphsim - a molecular replacement with the apo-form come closest to a Fo(complex)-Fo(apo) map? Just a thought. Tim
Re: [ccp4bb] Difference Map images
Hello Doug, I understand your arguing, although I do not understand why Fo contains a contribution from the model(led structure). My idea was only supposed to be a backup in case the apo-form had not been measuredcor could not be used. I agree the Fo(complex)-Fo(apo) gives a much more realistic result than the MR-solution I suggested. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Thu, 18 Jun 2009, Doug Ohlendorf wrote: Tim, Not really. Fo's can be viewed as having 3 parts. 1) F from our modeled structure, 2) F from what we can't model. This can be bulk solvent, partial or multiple occupancies in low resolution structures, thermal anisotropy , etc. 3) F from random errors in measuring data. The first F is what we deal with. The third F we can minimize by collecting high quality data. The second F is usually crystal/space group dependent. It arises from crystallization conditions, crystal morphology and intermolecular contacts. Data from isomorphic crystals should have similar second F's so map (from first F's) is cleaner. Doug -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim Gruene Sent: Wednesday, June 17, 2009 4:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Difference Map images I know that sometimes Fo(complex)-Fo(apo) cannot be done because of nonisomorphism. We've had a lot of success with this with the dioxygenases because there is no large scale alteration in the active site. As for the technique itself, Brian Matthews drilled this into me when I was a postdoc in his lab. Wouldn't - in the case of non-isomorphsim - a molecular replacement with the apo-form come closest to a Fo(complex)-Fo(apo) map? Just a thought. Tim
[ccp4bb] Difference Map images
Hello, I was wondering what people used to generate difference map images of, say, a ligand in their structures? e.g. Figure 2a here http://journals.iucr.org/f/issues/2009/05/00/tt5012/tt5012.pdf Cheers, Andy
Re: [ccp4bb] Difference Map images
Andy, One important thing if your complex crystals are isomorphous with apo is to use nFo(complex) - Fo(apo). These maps give the maximum information as the uninterpretable 'stuff' in the Fo-Fc map is likely quite similar in both crystal forms so the difference signal should be cleaner. Douglas H. Ohlendorf Phone: 612-624-8436 Professor FAX: 612-624-5121 Dept. of Biochemistry, Molecular Biology Biophysics Twin Cities Campus, University of Minnesota Lab web site: http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of ANDY DODDS Sent: Wednesday, June 17, 2009 8:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Difference Map images Hello, I was wondering what people used to generate difference map images of, say, a ligand in their structures? e.g. Figure 2a here http://journals.iucr.org/f/issues/2009/05/00/tt5012/tt5012.pdf Cheers, Andy
Re: [ccp4bb] Difference Map images
Andy, on the practical point of view, you can create such images using Pymol and the command 'carve'. load composite_omit-ccp4.map, map-to-display, format=ccp4 isomesh mesh, map-to-display, 1.5, resi 45, carve=3 color blue, mesh this will display the map at 1.5 sigma, at 3 angstroms around the residue 45. it gives you nice clean maps and it's a nice trick to remove the noise in your density, with obvious ethic limitations... But if you carve 20A around your residue, you're safe. If i remember correctly, ligands create problems because of the HETAM in the PDb file but you can replace it with ATOM and it should work. the commands 'around' or 'expand' can achieve similar figures i believe. Check he pymol wiki for more infos. vincent ANDY DODDS wrote: Hello, I was wondering what people used to generate difference map images of, say, a ligand in their structures? e.g. Figure 2a here http://journals.iucr.org/f/issues/2009/05/00/tt5012/tt5012.pdf Cheers, Andy IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer.
Re: [ccp4bb] Difference Map images
Wow, you can read too! Impressive. Indeed, it seems Larson et al did use Pymol, as have I. The question to the board was what they use, so that I might experiment with other methods. Any other helpful suggestions, please don't hesitate Jon. Thanks to all for their responses. A 2009/6/17 Jon Wright wri...@esrf.fr: There seems to be a clue in the text? Models were displayed and figures were produced with PyMOL (Delano, 2002), which you can read at the end of section 2 Materials and Methods. ANDY DODDS wrote: Hello, I was wondering what people used to generate difference map images of, say, a ligand in their structures? e.g. Figure 2a here http://journals.iucr.org/f/issues/2009/05/00/tt5012/tt5012.pdf Cheers, Andy
Re: [ccp4bb] Difference Map images
Hi Douglas Do you have some references with examples of this technique? In my experience this is a difficult experiment to perform routinely except in a few special cases. The first problem is that soaking the ligand can easily induce significant cell dimension changes, which if large enough causes non-isomorphism errors to wipe out the advantage of the similarity of apo complex crystal. One may be able to get around this of course by soaking the apo crystal in the same concentration of DMSO (or whatever solvent you use) as the complex was soaked in, but even then if the ligand is a tight binder it can induce conformational changes that again cause significant non-isomorphism errors. Then even if you can get around these problems, you have the problem that freezing the crystals usually causes differential cell dimension changes, possibly due to differing concentrations of organic solvent, but more likely it's simply that it's almost impossible to control the rate of freezing reproducibly. Is there a trick to avoid this? - of course you could simply not freeze, but then this would limit it to strongly diffracting crystals where you could afford to attenuate the beam to reduce radiation damage to an acceptable level. Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Doug Ohlendorf Sent: 17 June 2009 16:41 To: CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Difference Map images Andy, One important thing if your complex crystals are isomorphous with apo is to use nFo(complex) - Fo(apo). These maps give the maximum information as the uninterpretable 'stuff' in the Fo-Fc map is likely quite similar in both crystal forms so the difference signal should be cleaner. Douglas H. Ohlendorf Phone: 612-624-8436 Professor FAX: 612-624-5121 Dept. of Biochemistry, Molecular Biology Biophysics Twin Cities Campus, University of Minnesota Lab web site: http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of ANDY DODDS Sent: Wednesday, June 17, 2009 8:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Difference Map images Hello, I was wondering what people used to generate difference map images of, say, a ligand in their structures? e.g. Figure 2a here http://journals.iucr.org/f/issues/2009/05/00/tt5012/tt5012.pdf Cheers, Andy Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] Difference Map images
Ian, I know that sometimes Fo(complex)-Fo(apo) cannot be done because of nonisomorphism. We've had a lot of success with this with the dioxygenases because there is no large scale alteration in the active site. As for the technique itself, Brian Matthews drilled this into me when I was a postdoc in his lab. All the best, Doug Douglas H. Ohlendorf Phone: 612-624-8436 Professor FAX: 612-624-5121 Dept. of Biochemistry, Molecular Biology Biophysics Twin Cities Campus, University of Minnesota Lab web site: http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html -Original Message- From: Ian Tickle [mailto:i.tic...@astex-therapeutics.com] Sent: Wednesday, June 17, 2009 12:08 PM To: Doug Ohlendorf Cc: CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Difference Map images Hi Douglas Do you have some references with examples of this technique? In my experience this is a difficult experiment to perform routinely except in a few special cases. The first problem is that soaking the ligand can easily induce significant cell dimension changes, which if large enough causes non-isomorphism errors to wipe out the advantage of the similarity of apo complex crystal. One may be able to get around this of course by soaking the apo crystal in the same concentration of DMSO (or whatever solvent you use) as the complex was soaked in, but even then if the ligand is a tight binder it can induce conformational changes that again cause significant non-isomorphism errors. Then even if you can get around these problems, you have the problem that freezing the crystals usually causes differential cell dimension changes, possibly due to differing concentrations of organic solvent, but more likely it's simply that it's almost impossible to control the rate of freezing reproducibly. Is there a trick to avoid this? - of course you could simply not freeze, but then this would limit it to strongly diffracting crystals where you could afford to attenuate the beam to reduce radiation damage to an acceptable level. Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Doug Ohlendorf Sent: 17 June 2009 16:41 To: CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Difference Map images Andy, One important thing if your complex crystals are isomorphous with apo is to use nFo(complex) - Fo(apo). These maps give the maximum information as the uninterpretable 'stuff' in the Fo-Fc map is likely quite similar in both crystal forms so the difference signal should be cleaner. Douglas H. Ohlendorf Phone: 612-624-8436 Professor FAX: 612-624-5121 Dept. of Biochemistry, Molecular Biology Biophysics Twin Cities Campus, University of Minnesota Lab web site: http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of ANDY DODDS Sent: Wednesday, June 17, 2009 8:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Difference Map images Hello, I was wondering what people used to generate difference map images of, say, a ligand in their structures? e.g. Figure 2a here http://journals.iucr.org/f/issues/2009/05/00/tt5012/tt5012.pdf Cheers, Andy Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] Difference Map images
I know that sometimes Fo(complex)-Fo(apo) cannot be done because of nonisomorphism. We've had a lot of success with this with the dioxygenases because there is no large scale alteration in the active site. As for the technique itself, Brian Matthews drilled this into me when I was a postdoc in his lab. Wouldn't - in the case of non-isomorphsim - a molecular replacement with the apo-form come closest to a Fo(complex)-Fo(apo) map? Just a thought. Tim
[ccp4bb] Difference Map in COOT - Possible lignad but clash with structure?
Hello, I am fairly new to this lark so please forgive me if this question is unclear, but it is really puzzling me. I have used phaser to generate a molecular replacement structure of my target (which has 100% identity to my template) and this particular crystal I had soaked with a ligand. I have a density in my difference map which resembles my ligand. The thing is, it overlaps the density map of my structure and if I were to place the ligand in that map, then there would be a steric clash. Obviously I would expect the ligand to be near to the structure, but not overlapping it? I am uncertain how to proceed? Any helpful suggestions please? Thanks in advance Brenda
Re: [ccp4bb] Difference Map in COOT - Possible lignad but clash with structure?
Covalent modification?? Bias in 2fo-fc map? Can you show us a pic of offending density? Dave On 28/12/2007, Brenda Patterson [EMAIL PROTECTED] wrote: Hello, I am fairly new to this lark so please forgive me if this question is unclear, but it is really puzzling me. I have used phaser to generate a molecular replacement structure of my target (which has 100% identity to my template) and this particular crystal I had soaked with a ligand. I have a density in my difference map which resembles my ligand. The thing is, it overlaps the density map of my structure and if I were to place the ligand in that map, then there would be a steric clash. Obviously I would expect the ligand to be near to the structure, but not overlapping it? I am uncertain how to proceed? Any helpful suggestions please? Thanks in advance Brenda -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
Re: [ccp4bb] Difference Map in COOT - Possible lignad but clash with structure?
What's the resolution? At low res it is possible to miss a subtle movement of e.g. some sidechains which are being replaced by the ligand. What quality parameters did the MR have? Can you omit the entire ligand binding site, refine, and re-generate the map - what does it look like after that? A picture would help! Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Brenda Patterson Sent: Friday, December 28, 2007 10:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Difference Map in COOT - Possible lignad but clash with structure? Hello, I am fairly new to this lark so please forgive me if this question is unclear, but it is really puzzling me. I have used phaser to generate a molecular replacement structure of my target (which has 100% identity to my template) and this particular crystal I had soaked with a ligand. I have a density in my difference map which resembles my ligand. The thing is, it overlaps the density map of my structure and if I were to place the ligand in that map, then there would be a steric clash. Obviously I would expect the ligand to be near to the structure, but not overlapping it? I am uncertain how to proceed? Any helpful suggestions please? Thanks in advance Brenda
Re: [ccp4bb] Difference Map in COOT - Possible lignad but clash with structure?
There is another possibility that your ligand is not fully occupied, if the binding site of protein endures an apparent change when bound by ligand. Lijun On Dec 28, 2007, at 7:55 AM, Brenda Patterson wrote: Hello, I am fairly new to this lark so please forgive me if this question is unclear, but it is really puzzling me. I have used phaser to generate a molecular replacement structure of my target (which has 100% identity to my template) and this particular crystal I had soaked with a ligand. I have a density in my difference map which resembles my ligand. The thing is, it overlaps the density map of my structure and if I were to place the ligand in that map, then there would be a steric clash. Obviously I would expect the ligand to be near to the structure, but not overlapping it? I am uncertain how to proceed? Any helpful suggestions please? Thanks in advance Brenda Lijun Liu, PhD Institute of Molecular Biology HHMI Department of Physics University of Oregon Eugene, OR 97403 541-346-4080
Re: [ccp4bb] Difference Map in COOT - Possible lignad but clash with structure?
On Fri, 28 Dec 2007 15:55:39 + Brenda Patterson [EMAIL PROTECTED] wrote: Hello, I am fairly new to this lark No problem. With a name like Patterson, your future is guaranteed (unless of course you see everything in the world with intensity but no phase). I have a density in my difference map which resembles my ligand. The thing is, it overlaps the density map of my structure and if I were to place the ligand in that map, then there would be a steric clash. ... how to proceed? The best thing to do at this point is to refine your structure while omitting the residues involved in the potential steric clash. The ligand may bind by induced fit -- i.e., changing the conformation at the binding site from that of the original structure. When you do that, the density that comes back will be less biased, and the answer to how it fits together may be apparent at that point.
