[ccp4bb] Stable Refinement as Low(ish) resolution - Follow up

[Rhys Grinter]

Hi All,

Thanks for all the useful comments. Buster gave me quite stable refinement
and nice maps, so I've been working with that so far. I'll work through the
other suggestions however, and see what gives the best results.

Cheers,

Rhys

-- 
Dr Rhys Grinter
Sir Henry Wellcome Fellow
Monash University
+61 (0)3 9902 9213
+61 (0)403 896 767

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Tristan Croll]

*Ahem*

*ISOLDE

 
 
Tristan Croll
Research Fellow
Cambridge Institute for Medical Research
University of Cambridge CB2 0XY
 

 

> On 13 Jul 2017, at 08:02, Tristan Croll  wrote:
> 
> This is more-or-less exactly the task I'm building ISODE for. It's early 
> days, still quite rough around the edges and by no means complete, but if you 
> have a machine with a decent GPU running a modern Linux distro, then it's 
> easily available as a plugin to ChimeraX after installing the latest daily 
> build from https://www.rbvi.ucsf.edu/chimerax/download.html. Think of it as 
> an interactive real-space refinement environment, and you won't be far off. 
> This video is getting a little dated now, but should give you some idea of 
> what it can do: 
> https://drive.google.com/open?id=0B6uMjfjuw4k8aUdyTEJWeEJ1UnM. I'm currently 
> working on a longer, narrated demo/tutorial video showing all the latest 
> features (interactive position/rotamer/secondary structure assignment, a very 
> fun tool for shifting stretches in register, ...).
> 
> There are some caveats:
> - It doesn't yet do any building (adding or removing of atoms/residues)
> - It requires all residues to be complete including hydrogens
> - Currently only protein, nucleic acid and metal ions are supported (although 
> the metal ions need more work to be properly useful)
> 
> Let me know if it's of interest, and we can talk more offline.
> 
> Cheers,
> 
> Tristan
> 
>> On 2017-07-13 00:17, Rhys Grinter wrote:
>> Dear All,
>> I'm currently in the process of refining a low(ish) resolution
>> structure at 3.2 Ang, with a fair level of anisotropy. I processed the
>> data through the anisotropy server
>> (https://services.mbi.ucla.edu/anisoscale/ [1]), which elliptically
>> truncated the data to 4.0, 3.8 and 3.2 Ang. This really improved the
>> maps and allowed me to trace the majority of the chain and build most
>> side chains.
>> The R-factors are reasonable (0.29 work and 0.35 free respectively).
>> but I'm having trouble with over fitting in refinement as I continue
>> to refine. What parameters/restraints would the community generally
>> use when refining this kind of structure? Additionally Refmac doesn't
>> seem to read the structure factors from the anisotropy server output
>> file properly, giving vastly inflated R values and strange looking
>> maps.
>> Cheers,
>> Rhys
>> --
>> Dr Rhys Grinter
>> Sir Henry Wellcome Fellow
>> Monash University
>> +61 (0)3 9902 9213
>> +61 (0)403 896 767
>> Links:
>> --
>> [1] https://services.mbi.ucla.edu/anisoscale/

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Tristan Croll]

This is more-or-less exactly the task I'm building ISODE for. It's early 
days, still quite rough around the edges and by no means complete, but 
if you have a machine with a decent GPU running a modern Linux distro, 
then it's easily available as a plugin to ChimeraX after installing the 
latest daily build from 
https://www.rbvi.ucsf.edu/chimerax/download.html. Think of it as an 
interactive real-space refinement environment, and you won't be far off. 
This video is getting a little dated now, but should give you some idea 
of what it can do: 
https://drive.google.com/open?id=0B6uMjfjuw4k8aUdyTEJWeEJ1UnM. I'm 
currently working on a longer, narrated demo/tutorial video showing all 
the latest features (interactive position/rotamer/secondary structure 
assignment, a very fun tool for shifting stretches in register, ...).


There are some caveats:
- It doesn't yet do any building (adding or removing of atoms/residues)
- It requires all residues to be complete including hydrogens
- Currently only protein, nucleic acid and metal ions are supported 
(although the metal ions need more work to be properly useful)


Let me know if it's of interest, and we can talk more offline.

Cheers,

Tristan

On 2017-07-13 00:17, Rhys Grinter wrote:

Dear All,

I'm currently in the process of refining a low(ish) resolution
structure at 3.2 Ang, with a fair level of anisotropy. I processed the
data through the anisotropy server
(https://services.mbi.ucla.edu/anisoscale/ [1]), which elliptically
truncated the data to 4.0, 3.8 and 3.2 Ang. This really improved the
maps and allowed me to trace the majority of the chain and build most
side chains.

The R-factors are reasonable (0.29 work and 0.35 free respectively).
but I'm having trouble with over fitting in refinement as I continue
to refine. What parameters/restraints would the community generally
use when refining this kind of structure? Additionally Refmac doesn't
seem to read the structure factors from the anisotropy server output
file properly, giving vastly inflated R values and strange looking
maps.

Cheers,

Rhys

--

Dr Rhys Grinter
Sir Henry Wellcome Fellow
Monash University
+61 (0)3 9902 9213
+61 (0)403 896 767

Links:
--
[1] https://services.mbi.ucla.edu/anisoscale/

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Robbie Joosten]

In addition: pdb-redo automatically makes (homology-based) hydrogen bond 
restraints for Refmac at this resolution. You could gibe those a try.

Cheers,
Robbie

Sent from my Windows 10 phone

From: Sudipta Bhattacharyya<mailto:sudiptabhattacharyya.iit...@gmail.com>
Sent: 13 July 2017 06:25
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Stable Refinement as Low(ish) resolution

Hi Rhys,

Along with what others have suggested, you can always try prosmart refinement. 
I found it really helpful in my cases.

Best of luck,
Sudipta.

Sudipta Bhattacharyya
Postdoctoral research fellow
University of Texas at Austin
Texas, USA.

On Jul 12, 2017 6:20 PM, "Rhys Grinter" 
<rhys.grin...@monash.edu<mailto:rhys.grin...@monash.edu>> wrote:
Dear All,

I'm currently in the process of refining a low(ish) resolution structure at 3.2 
Ang, with a fair level of anisotropy. I processed the data through the 
anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which 
elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really improved 
the maps and allowed me to trace the majority of the chain and build most side 
chains.

The R-factors are reasonable (0.29 work and 0.35 free respectively). but I'm 
having trouble with over fitting in refinement as I continue to refine. What 
parameters/restraints would the community generally use when refining this kind 
of structure? Additionally Refmac doesn't seem to read the structure factors 
from the anisotropy server output file properly, giving vastly inflated R 
values and strange looking maps.

Cheers,

Rhys

--
Dr Rhys Grinter
Sir Henry Wellcome Fellow
Monash University
+61 (0)3 9902 9213<tel:+61%203%209902%209213>
+61 (0)403 896 767<tel:+61%20403%20896%20767>

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Sudipta Bhattacharyya]

Hi Rhys,

Along with what others have suggested, you can always try prosmart
refinement. I found it really helpful in my cases.

Best of luck,
Sudipta.

Sudipta Bhattacharyya
Postdoctoral research fellow
University of Texas at Austin
Texas, USA.

On Jul 12, 2017 6:20 PM, "Rhys Grinter"  wrote:

> Dear All,
>
> I'm currently in the process of refining a low(ish) resolution structure
> at 3.2 Ang, with a fair level of anisotropy. I processed the data through
> the anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which
> elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really
> improved the maps and allowed me to trace the majority of the chain and
> build most side chains.
>
> The R-factors are reasonable (0.29 work and 0.35 free respectively). but
> I'm having trouble with over fitting in refinement as I continue to refine.
> What parameters/restraints would the community generally use when refining
> this kind of structure? Additionally Refmac doesn't seem to read the
> structure factors from the anisotropy server output file properly, giving
> vastly inflated R values and strange looking maps.
>
> Cheers,
>
> Rhys
>
> --
> Dr Rhys Grinter
> Sir Henry Wellcome Fellow
> Monash University
> +61 (0)3 9902 9213 <+61%203%209902%209213>
> +61 (0)403 896 767 <+61%20403%20896%20767>
>

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Jason Busby]

Pretty much what the others have said, but there is a low resolution refinement 
pipeline in CCP4 called LORESTR (LOw-REsolution STructure Refinement).  Several 
people I know have had good results with this.  It automates the process of 
finding homologues, generating restraints, trying different scaling parameters 
etc.

In CCP4i it is “Run Low Resolution Refinement” on the refinement page.  I don’t 
think it is in CCP4i2 yet.

Jason.

--
Dr Jason Busby
Laboratory of Structural Biology
School of Biological Sciences
The University of Auckland
Thomas Building 110
3A Symonds St
Private Bag 92019
Auckland Mail Centre
Auckland
New Zealand

ph: +64 9 3737599 ext 83888




On 13/07/2017, at 11:17 AM, Rhys Grinter 
> wrote:

Dear All,

I'm currently in the process of refining a low(ish) resolution structure at 3.2 
Ang, with a fair level of anisotropy. I processed the data through the 
anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which 
elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really improved 
the maps and allowed me to trace the majority of the chain and build most side 
chains.

The R-factors are reasonable (0.29 work and 0.35 free respectively). but I'm 
having trouble with over fitting in refinement as I continue to refine. What 
parameters/restraints would the community generally use when refining this kind 
of structure? Additionally Refmac doesn't seem to read the structure factors 
from the anisotropy server output file properly, giving vastly inflated R 
values and strange looking maps.

Cheers,

Rhys

--
Dr Rhys Grinter
Sir Henry Wellcome Fellow
Monash University
+61 (0)3 9902 9213
+61 (0)403 896 767

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Andreas Forster]

Dear Rhys,

I second Roger on the use of jelly-body refinement.  In addition, give
Buster a try.  It sometimes does magic at lowish resolution.

All best.


Andreas



On Thu, Jul 13, 2017 at 1:17 AM, Rhys Grinter 
wrote:

> Dear All,
>
> I'm currently in the process of refining a low(ish) resolution structure
> at 3.2 Ang, with a fair level of anisotropy. I processed the data through
> the anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which
> elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really
> improved the maps and allowed me to trace the majority of the chain and
> build most side chains.
>
> The R-factors are reasonable (0.29 work and 0.35 free respectively). but
> I'm having trouble with over fitting in refinement as I continue to refine.
> What parameters/restraints would the community generally use when refining
> this kind of structure? Additionally Refmac doesn't seem to read the
> structure factors from the anisotropy server output file properly, giving
> vastly inflated R values and strange looking maps.
>
> Cheers,
>
> Rhys
>
> --
> Dr Rhys Grinter
> Sir Henry Wellcome Fellow
> Monash University
> +61 (0)3 9902 9213 <+61%203%209902%209213>
> +61 (0)403 896 767 <+61%20403%20896%20767>
>

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Roger Rowlett]

Besides empirically adjusting the weighting factor for X-ray data to
increase the geometric constraints, have you tried jelly-body refinement or
refinement with external constraints? The latter two methods can be helpful
when refining low resolution data.

Roger Rowlett

On Jul 12, 2017 7:33 PM, "Christine Gee"  wrote:

> Hi Rhys,
>
> In Refmac, you can change the weighting of the Xray terms vs the geometry
> restraints rather than use what is automatically assigned. Have you tried
> this? In Phenix you can use secondary structure restraints (probably you
> can do this in Refmac too, but I haven't tried) and try checking the box
> optimize stereochemistry/Xray weight. This will probably help if your over
> fitting is moving things into density but making the geometry sub optimal.
>
> Regards
> Christine
>
> On Wed, Jul 12, 2017 at 4:17 PM, Rhys Grinter 
> wrote:
>
>> Dear All,
>>
>> I'm currently in the process of refining a low(ish) resolution structure
>> at 3.2 Ang, with a fair level of anisotropy. I processed the data through
>> the anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which
>> elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really
>> improved the maps and allowed me to trace the majority of the chain and
>> build most side chains.
>>
>> The R-factors are reasonable (0.29 work and 0.35 free respectively). but
>> I'm having trouble with over fitting in refinement as I continue to refine.
>> What parameters/restraints would the community generally use when refining
>> this kind of structure? Additionally Refmac doesn't seem to read the
>> structure factors from the anisotropy server output file properly, giving
>> vastly inflated R values and strange looking maps.
>>
>> Cheers,
>>
>> Rhys
>>
>> --
>> Dr Rhys Grinter
>> Sir Henry Wellcome Fellow
>> Monash University
>> +61 (0)3 9902 9213 <+61%203%209902%209213>
>> +61 (0)403 896 767 <+61%20403%20896%20767>
>>
>
>

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Christine Gee]

Hi Rhys,

In Refmac, you can change the weighting of the Xray terms vs the geometry
restraints rather than use what is automatically assigned. Have you tried
this? In Phenix you can use secondary structure restraints (probably you
can do this in Refmac too, but I haven't tried) and try checking the box
optimize stereochemistry/Xray weight. This will probably help if your over
fitting is moving things into density but making the geometry sub optimal.

Regards
Christine

On Wed, Jul 12, 2017 at 4:17 PM, Rhys Grinter 
wrote:

> Dear All,
>
> I'm currently in the process of refining a low(ish) resolution structure
> at 3.2 Ang, with a fair level of anisotropy. I processed the data through
> the anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which
> elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really
> improved the maps and allowed me to trace the majority of the chain and
> build most side chains.
>
> The R-factors are reasonable (0.29 work and 0.35 free respectively). but
> I'm having trouble with over fitting in refinement as I continue to refine.
> What parameters/restraints would the community generally use when refining
> this kind of structure? Additionally Refmac doesn't seem to read the
> structure factors from the anisotropy server output file properly, giving
> vastly inflated R values and strange looking maps.
>
> Cheers,
>
> Rhys
>
> --
> Dr Rhys Grinter
> Sir Henry Wellcome Fellow
> Monash University
> +61 (0)3 9902 9213 <+61%203%209902%209213>
> +61 (0)403 896 767 <+61%20403%20896%20767>
>

[ccp4bb] Stable Refinement as Low(ish) resolution

[Rhys Grinter]

Dear All,

I'm currently in the process of refining a low(ish) resolution structure at
3.2 Ang, with a fair level of anisotropy. I processed the data through the
anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which
elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really
improved the maps and allowed me to trace the majority of the chain and
build most side chains.

The R-factors are reasonable (0.29 work and 0.35 free respectively). but
I'm having trouble with over fitting in refinement as I continue to refine.
What parameters/restraints would the community generally use when refining
this kind of structure? Additionally Refmac doesn't seem to read the
structure factors from the anisotropy server output file properly, giving
vastly inflated R values and strange looking maps.

Cheers,

Rhys

-- 
Dr Rhys Grinter
Sir Henry Wellcome Fellow
Monash University
+61 (0)3 9902 9213
+61 (0)403 896 767