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
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
