Thank you. I think I will then first try to optimize the weight and then increase the cycles. Herman
-----Ursprüngliche Nachricht----- Von: Tom Burnley - UKRI STFC <tom.burn...@stfc.ac.uk> Gesendet: Donnerstag, 30. Juli 2020 14:35 An: ccp4bb@jiscmail.ac.uk; Schreuder, Herman /DE <herman.schreu...@sanofi.com> Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement EXTERNAL : Real sender is tom.burn...@stfc.ac.uk Hi Herman, Just a couple of tips for Refmac... 1) increase number of cycles - we find some cases require a lot of cycles, more than you may be used from MX refinement. 2) optimise weight - this is important and at present the autoweighting option is not as reliable for EM refinement as MX. You could run these in parallel whilst exploring the other very nice tools suggested 😉! Cheers, Tom ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Schreuder, Herman /DE <herman.schreu...@sanofi.com> Sent: 30 July 2020 13:28:00 To: ccp4bb Subject: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement Hi everybody, Thank you very much for the many tips! I discovered that in Coot by clicking on the N- and C-terminus of a protein chain, one can indeed run a real-space refinement on the complete chain and the same is true for a rigid body fit. However, subsequent refinement with Refmac did not converge, so it looks like I still have to go manually through the chains. ☹ Still on my to do list are: - test Isolde - test Main - test the wiggle-fit option in Coot. Although the Refmac procedure consisting of converting the cryo-EM map into structure factors and running a "crystallographic" refinement on it seems to work, in my opinion the future will be a robust real-space refinement on the cryo-EM map itself, instead of a refinement on derived structure factors. However, this is my personal opinion. Best regards, Herman -----Ursprüngliche Nachricht----- Von: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> Im Auftrag von Tom Burnley - UKRI STFC Gesendet: Mittwoch, 29. Juli 2020 18:51 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk Hi Herman, There is lots of information on CCP-EM's modelling tools here: https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ccpem.ac.uk_training_icknield-5F2019_icknield-5F2019.php&d=DwIFAg&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=aaxzRy8JT0AM0_MqNvU2fr6IB4wIbkY8SsgvQkYcw_w&s=D5VOMK2Fz8AOV1PCvJpD9tm914KUxJlsRNRWXYPb7tI&e= This includes a tutorial on Refmac. As you note this is done in reciprocal space but the CCP-EM pipeline handles conversions from MRC to MTZ format (and runs Refmac via CCP4). All the best, Tom ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Boaz Shaanan <bshaa...@bgu.ac.il> Sent: 29 July 2020 16:31:46 To: ccp4bb Subject: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement Hi, Try searching ccp-em. Boaz Boaz Shaanan, Ph.D. Department of Life Sciences Ben Gurion University of the Negev Beer Sheva Israel On Jul 29, 2020 18:29, "Schreuder, Herman /DE" <herman.schreu...@sanofi.com> wrote: Coot does it, but if one wants to do it for a complete protein, it is a lot of clicking. On the other hand, I have not tried to just select the N- and C-terminus for real-space refinement to see what happens. I would prefer to have some script to do it. Best, Herman Von: Eleanor Dodson <eleanor.dod...@york.ac.uk> Gesendet: Mittwoch, 29. Juli 2020 17:23 An: Schreuder, Herman /DE <herman.schreu...@sanofi.com> Cc: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] Re: [ccp4bb] real real-space-refinement EXTERNAL : Real sender is eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk> Wont coot do that? Eleanor On Wed, 29 Jul 2020 at 16:20, Schreuder, Herman /DE <herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com>> wrote: Dear BB, I would like to do a real real-space-refinement of a protein against a cryo-EM map; not the mtz-based Refmac approach. A quick internet search produced a lot of Phenix hits, but little ccp4 hits. Does somebody know how to do this using ccp4 programs, or has someone a Coot script to do this? Thank you for your help! 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