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 <[log in to unmask]> on behalf of Schreuder, Herman /DE <[log in to unmask]>
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 <[log in to unmask]> Im Auftrag von Tom Burnley - UKRI STFC
Gesendet: Mittwoch, 29. Juli 2020 18:51
An: [log in to unmask]
Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement
EXTERNAL : Real sender is [log in to unmask]
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 <[log in to unmask]> on behalf of Boaz Shaanan <[log in to unmask]>
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" <[log in to unmask]> 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 <[log in to unmask]>
Gesendet: Mittwoch, 29. Juli 2020 17:23
An: Schreuder, Herman /DE <[log in to unmask]>
Cc: [log in to unmask]
Betreff: [EXTERNAL] Re: [ccp4bb] real real-space-refinement
EXTERNAL : Real sender is [log in to unmask]<mailto:[log in to unmask]>
Wont coot do that?
Eleanor
On Wed, 29 Jul 2020 at 16:20, Schreuder, Herman /DE <[log in to unmask]<mailto:[log in to unmask]>> 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!
Herman
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