Re: [ccp4bb] Refmac5 and Coot for refining cryo-EM structures
The main issue is that carboxyls seem to be invisible and Coot tries to fit them as though the map had them there Well, if the atoms are part of the model, then Coot will try to fit them to the "data." I routinely chop off GLU and ASP side-chain when modelling into cryo-EM maps (that's what the K key is for). That seems unnecessarily harsh. Remember that EM maps do not show electron density but electric potential, so undamaged negatively charged moieties should occur at negative density levels. There is an "Aha-Erlebnis" paper (at least, that's the effect it had on me :-) ) by Jimin Wang and Peter Moore (entitled "On the interpretation of electron microscopic maps of biological macromolecules") on this very topic in last month's EM-special of Protein Science, Vol 26, pp 122-129. --Gerard ** Gerard J. Kleywegt http://xray.bmc.uu.se/gerard mailto:ger...@xray.bmc.uu.se ** The opinions in this message are fictional. Any similarity to actual opinions, living or dead, is purely coincidental. ** Little known gastromathematical curiosity: let "z" be the radius and "a" the thickness of a pizza. Then the volume of that pizza is equal to pi*z*z*a ! **
Re: [ccp4bb] Refmac5 and Coot for refining cryo-EM structures
On 01/02/2017 17:11, Trevor Sewell wrote: I have a 3.4 A (enzyme – protein only) map that I have fitted manually using Coot and automatically with Rapper. It all looks very nice – I can fit all but 13 of the 330 residues. I have the following questions: Is there a way of having Coot know that it is an electron map and not an x-ray map? Yes, and/but it's not under user control. > The main issue is that carboxyls seem to be invisible and Coot tries to fit them as though the map had them there Well, if the atoms are part of the model, then Coot will try to fit them to the "data." I routinely chop off GLU and ASP side-chain when modelling into cryo-EM maps (that's what the K key is for). Coot refines differently to Refmac. What is a reasonable starting weight for vdwrestraints ? Is there a rule of thumb for choosing this? I don't typically adjust this. What make you think that you need to do so? Paul.
Re: [ccp4bb] Refmac5 and Coot for refining cryo-EM structures
Dear Trevor, I do not know if you are aware of it but there is a different version of refmac which is just made for Cryo-EM refinements. (http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/software.html) In there, Refmac reports an average FSC value which is way more useful than the R-factor. You want to have this value as high as possible (based on your resolution it might be around 0.79) But you always have to make sure to not do overfitting. You will have to test for overfitting as it is described and done for example in the most recent publications of Venki Ramakrishnan. Cheers Marcus Marcus Fislage, PhD Howard Hughes Medical Institute (HHMI) Columbia University Department of Biochemistry and Biophysics Lab of Joachim Frank New York, NY email address: mf2...@cumc.columbia.edu Phone: 212.305.9524 Fax: 212.305.9500 Marcus Fislage, PhD Howard Hughes Medical Institute (HHMI) Columbia University Department of Biochemistry and Biophysics Lab of Joachim Frank New York, NY email address: mf2...@cumc.columbia.edu Phone: 212.305.9524 Fax: 212.305.9500 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Trevor Sewell [trevor.sew...@uct.ac.za] Sent: Wednesday, February 01, 2017 12:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Refmac5 and Coot for refining cryo-EM structures I have a 3.4 A (enzyme – protein only) map that I have fitted manually using Coot and automatically with Rapper. It all looks very nice – I can fit all but 13 of the 330 residues. I have the following questions: Does refmac5 have a way of refining the scale of the map? – I did my best to calibrate the microscope – but it would be nice to have the scale as a refinable parameter. Is there a way of having Coot know that it is an electron map and not an x-ray map? The main issue is that carboxyls seem to be invisible and Coot tries to fit them as though the map had them there – which results in some pretty dopey stuff. The problem doesn’t seem to apply in refmac5 if you use the source EM as described in the instructions. What is a reasonable starting weight for vdwrestraints ? Is there a rule of thumb for choosing this? What are acceptable values for the Rfactor produced by Refmac5? The fit looks pretty good with an Rfactor of .39. What is the experience of the community in this regard? Many thanks Trevor Sewell Disclaimer - University of Cape Town This e-mail is subject to UCT policies and e-mail disclaimer published on our website at http://www.uct.ac.za/about/policies/emaildisclaimer/ or obtainable from +27 21 650 9111. If this e-mail is not related to the business of UCT, it is sent by the sender in an individual capacity. Please report security incidents or abuse via cs...@uct.ac.za
[ccp4bb] Refmac5 and Coot for refining cryo-EM structures
I have a 3.4 A (enzyme – protein only) map that I have fitted manually using Coot and automatically with Rapper. It all looks very nice – I can fit all but 13 of the 330 residues. I have the following questions: Does refmac5 have a way of refining the scale of the map? – I did my best to calibrate the microscope – but it would be nice to have the scale as a refinable parameter. Is there a way of having Coot know that it is an electron map and not an x-ray map? The main issue is that carboxyls seem to be invisible and Coot tries to fit them as though the map had them there – which results in some pretty dopey stuff. The problem doesn’t seem to apply in refmac5 if you use the source EM as described in the instructions. What is a reasonable starting weight for vdwrestraints ? Is there a rule of thumb for choosing this? What are acceptable values for the Rfactor produced by Refmac5? The fit looks pretty good with an Rfactor of .39. What is the experience of the community in this regard? Many thanks Trevor Sewell Disclaimer - University of Cape Town This e-mail is subject to UCT policies and e-mail disclaimer published on our website at http://www.uct.ac.za/about/policies/emaildisclaimer/ or obtainable from +27 21 650 9111. If this e-mail is not related to the business of UCT, it is sent by the sender in an individual capacity. Please report security incidents or abuse via cs...@uct.ac.za
