[phenixbb] high R-value with resolve_cryo_em
Pavel Afonine
pafonine at lbl.gov
Fri Jun 10 13:06:26 PDT 2022
Hi Tom and Daniel,
I'm confused about what R factor we are talking about here? You say
"This R value describes the mismatch between measured and map-based
structure factor amplitudes. ", then questions I'd ask are:
- what program was used to calculate it?
- Was all scaling between model and data generated Fs done properly? Not
treating solvent region (or whatever that is in cryo-em) alone can
result in large R factors.
- Was model refined properly (coordinates and B factors). Unrefined B
factors even for perfectly fitting model (coordinates wise) can result
in high R factors.
- Why we calculate and try to interpret R factors at all given it is
cryo-EM case (no Fobs)?
Pavel
On 6/9/22 05:56, Tom Terwilliger wrote:
> Hi Daniel,
>
> Oops...way too much jargon in my reply. "Cut the resolution" means
> "Use a lower resolution cutoff". Of course that could mean a bigger
> or smaller number. It means "Use a bigger number for the value of
> resolution" The idea here is to select a resolution range in which
> most of the data are reasonably accurate, where the estimated
> correlation of Fourier terms with the true ones is something like 0.5
> (the standard half-map correlation cutoff of 0.143), but it is not
> obvious exactly what quality cutoff and therefore what resolution
> cutoff should be used, so a few tries never hurt.
>
> You might try boxing your map yourself, using varying values for the
> buffer around the model (from 5 to 10 A). Then tell resolve_cryo_em
> not to box the incoming map. This way you can actually vary the
> solvent content.
>
> Using the model in density modification could help but keep in mind
> that you should always also do it without the model so that you have
> some map that you know does not have any model bias. (The model-based
> density modification does not seem to have model bias in my experience
> but it always could).
>
> All the best,
> Tom T
>
> On Wed, Jun 8, 2022 at 11:04 PM Daniel Larsson
> <daniel.larsson at icm.uu.se> wrote:
>
> Hi Tom,
>
> Thank you so much for the feedback.
>
> By “cutting the resolution” does that mean that I should put a
> smaller or higher number for the density modification resolution?
> I guess I should try both lower and higher...
>
> The solvent content using default settings is very high, more than
> 97%. My guess is that this is because there is a lot of nucleic
> acid and perhaps the density is being more localized (e.g. to the
> phosphate groups) than what is normal for proteins. I tried to
> force a lower solvent content, such as 90%, but that did not
> change the R-value. I will try with model-based density
> modification and see if that helps.
>
> Regards,
> Daniel
>
>
>> On 8 Jun 2022, at 21:39, Tom Terwilliger
>> <tterwilliger at newmexicoconsortium.org> wrote:
>>
>> Hi Daniel,
>>
>> We don't have a standard way to interpret the R value for density
>> modification...hence the rough guide of "0.25 is good and 0.5 is
>> poor" but no documentation. This applies to both X-ray and cryoEM
>> density modification.
>>
>> This R value describes the mismatch between measured and
>> map-based structure factor amplitudes. Usually these match
>> pretty well in cases where the map is improved a lot and not when
>> the map does not improve, but there is no obvious and simple
>> relationship.
>>
>> My recommendation would be to use the estimate of map resolution
>> and visual quality as your guide as to whether it has improved
>> your map, and the R value in density modification as a possible
>> indication that some change of parameters might help if things
>> are not going well (not that it tells you what to change, but a
>> good one is always resolution and another is solvent content.)
>>
>> In your case I might try cutting the resolution or randomly
>> changing solvent content and testing...but as the map looks ok I
>> would not try too hard.
>>
>> I hope that helps!
>> All the best,
>> Tom T
>>
>> On Wed, Jun 8, 2022 at 14:25 Daniel Larsson
>> <daniel.larsson at icm.uu.se> wrote:
>>
>> Hi all,
>>
>> I tried to do density modification using the resolve_cryo_em
>> tool. The resolution improves quite a lot from around 2.60 to
>> around 2.35 and the density looks significantly better than
>> the autorefined original map. However, the R-values is close
>> to 0.5, which seems very high. How should the R-value
>> generated by this tool be interpreted? It is very poorly
>> documented and not mentioned in the paper or on the manual
>> page, but Tom Terwilliger mentioned in a workshop that “low
>> number is good” and “point two-four is a very good number,
>> point five is not a good number”, which makes me worried.
>>
>> Regards,
>> Daniel
>>
>>
>>
>>
>>
>>
>>
>>
>>
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>> --
>> Thomas C Terwilliger
>> Laboratory Fellow, Los Alamos National Laboratory
>> Senior Scientist, New Mexico Consortium
>> 100 Entrada Dr, Los Alamos, NM 87544
>> Email: tterwilliger at newmexicoconsortium.org
>> Tel: 505-431-0010
>>
>
>
>
> --
> Thomas C Terwilliger
> Laboratory Fellow, Los Alamos National Laboratory
> Senior Scientist, New Mexico Consortium
> 100 Entrada Dr, Los Alamos, NM 87544
> Email: tterwilliger at newmexicoconsortium.org
> Tel: 505-431-0010
>
>
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