Re: [ccp4bb] Resolution discrepancy between MTZ file and Refinement

[Pavel Afonine]

Hi,
every time you run phenix.refine it may exclude some reflections from
refinement per Read 1999 (Acta Cryst. (1999). D55, 1759-1764). Usually the
number of reflections omitted ranges from none to just a few, however, this
may be just enough to make the resolution statistics look slightly
different.
Also, if you use Iobs as input data, internally they are converted into
Fobs using F method, and some reflections may not "survive" this
conversion. This may be another reason for discrepancies.
Pavel

On Fri, Apr 5, 2024 at 9:45 PM venkatareddy dadireddy 
wrote:

>
> Thank you very much Garib.
>
> Venkat
>
> On Sat, Apr 6, 2024 at 1:12 AM Garib Murshudov 
> wrote:
>
>> Unless you are confident that twin exists you should not use twin
>> refinement (Occam’s razor)
>>
>>
>>
>> On 5 Apr 2024, at 17:24, venkatareddy dadireddy 
>> wrote:
>>
>> CAUTION: This email originated from outside of the LMB:
>> *.-owner-ccp...@jiscmail.ac.uk -.*
>> Do not click links or open attachments unless you recognize the sender
>> and know the content is safe.
>> If you think this is a phishing email, please forward it to
>> phish...@mrc-lmb.cam.ac.uk
>>
>>
>> --
>> Hi Kay and Garib,
>>
>> Thank you for your input.
>> It is actually the twin refinement that gave rise to resolution
>> discrepancy.
>> For what reason I don't remember that I have turned the twin refinement ON
>> and the same job was cloned again and again.
>> With the twin refinement OFF, it gave rise to resolution present in the
>> MTZ (2.0 A).
>> From Xtriage: *the correlation between*
>> *the intensities related by the twin law 1/2*h-3/2*k, -1/2*h-1/2*k,-l
>> with an estimated twin*
>> *fraction of 0.10 is most likely due to an NCS axis parallel to the twin
>> axis*.
>> The statistics independent of twin laws show no twinning (more close to
>> untwinned than perfect twin).
>> Please suggest to me on how I proceed with refinement (twin OFF or ON).
>>
>> Thank you,
>> Venkat
>>
>>
>>
>>
>>
>>
>>
>> On Fri, Apr 5, 2024 at 1:00 AM Garib Murshudov 
>> wrote:
>>
>>> Did you use twin refinement (is it really twin if you used that).
>>> If twin refinement was used then twin related intensities might have
>>> different resolution, in case when your crystal are pseudomerohedral
>>> twinned.
>>>
>>> Regards
>>> Garib
>>>
>>> On 4 Apr 2024, at 18:40, venkatareddy dadireddy 
>>> wrote:
>>>
>>> CAUTION: This email originated from outside of the LMB:
>>> *.-owner-ccp...@jiscmail.ac.uk -.*
>>> Do not click links or open attachments unless you recognize the sender
>>> and know the content is safe.
>>> If you think this is a phishing email, please forward it to
>>> phish...@mrc-lmb.cam.ac.uk
>>>
>>>
>>> --
>>> Hi Kay,
>>>
>>> Thank you very much for your insights.
>>> Following are the cell parameters from mtz and pdb header.
>>> *MTZ: 117.8560   66.1700   70.9040   90.   91.4240   90.000*
>>>
>>> *CRYST1  117.856   66.170   70.904  90.00  91.42  90.00 C 1 2 1*
>>>
>>> The only difference is in the 3rd decimal point.
>>>
>>>
>>> Thank you,
>>> Venkat
>>>
>>>
>>>
>>> On Tue, Apr 2, 2024 at 10:10 PM Kay Diederichs <
>>> kay.diederi...@uni-konstanz.de> wrote:
>>>
 Hi Venkatareddy Dadireddy,

 do the unit cell parameters of your MTZ file and PDB file agree exactly
 ?

 Take for example a cell of (100,110,120,90,90,90) in the header of the
 MTZ file,
 and (97,110,120,90,90,90) in the CRYST1 line of the PDB file.

 In this example, the (50,0,0) reflection would be at 2.0A resolution if
 using the cell from the MTZ file,
 but it would be at 1.94A resolution if calculating the resolution based
 on the cell from the PDB file.

 So perhaps REFMAC5 takes the cell from the PDB file, and phenix.refine
 takes the cell from the MTZ?
 I didn't check but it may be worth finding out.

 HTH,
 Kay


 On Tue, 2 Apr 2024 21:16:57 +0530, venkatareddy dadireddy <
 venkatda...@gmail.com> wrote:

 >Hi,
 >
 >The resolution range in my MTZ file is 70.88 - 2.0 A. When I refined my
 >structure using REFMAC5, the resolution that it gives is 70.88 - 1.94
 A,
 >the difference of 0.04 A. I also used Phenix.refine which gives the
 >resolution output as it is in the MTZ file. Again, EDS (validation
 report)
 >gives the right resolution. What could be the possible reason for this
 >discrepancy? I have the structure deposited in the Protein Data Bank
 and it
 >is on hold. Thank you in advance for your help.
 >
 >Thank you,
 >
 >
 >
 >
 >
 >
 >*Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of
 >Physics,IISc, Banglore.Cell: 07259492227*
 >

 >
 >
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Re: [ccp4bb] About Staraniso

[Pavel Afonine]

Dear All,

What follows below is not very specific to the particular program
(STAIRSANISO) nor the original questions, but nonetheless, I believe it is
relevant.

In the past, performing any adjustments to the diffraction data intended
for solving and refining atomic models was more or less considered taboo.
When cryo-EM emerged as a competitor to x-ray crystallography, the paradigm
began to shift. In cryo-EM, manipulations applied to the data (the map) are
a standard practice. The map can be boxed, filtered (sharpened, blurred,
etc.), modified (e.g., setting something outside the molecular region), and
so forth; you name it. One might wonder why the same isn't done to x-ray
data. Historical analogies include truncating data beyond 6-8Å resolution
to avoid dealing with the bulk solvent or default sharpening (a feature
available in X-plor for some time, then removed for obvious reasons,
AFAIK), choosing resolution limits (PAIRREF), and anisotropic data
massaging by the UCLA server as a more recent example. STAIRSANISO is the
leader in doing things along these lines as of today.

Indeed, why not if this is helpful to solve the structure? However, it's
important that the deposition clearly contains and annotates at least the
following:

- the original unmanipulated data;
- modified data (by whatever method or program);
- accessible information about the data that was used to obtain the final
deposited atomic model.

Note *accessible* above as this is the key for what follows below.

Let's consider this example: https://files.rcsb.org/download/6R72-sf.cif ,
which is representative of the class of problems I'm trying to convey here.

The file has everything, kudos to the authors: The original data, the
manipulated data and a whole lot more.

Are these data accessible?

YES, if you download the file, open it in your favorite text editor, and
carefully scroll and read through its 76,566 lines and use your best guess
to infer what are the original data arrays, what are the modified data
arrays and so on.

NO, absolutely NO, if you parse data files in PDB automatically with a
script, and attempt to extract particular data (eg., original unmanipulated
data). And this is what I find problematic, especially given 215+k entries
in PDB as of today.

Hope someone does something about it!

All the best!
Pavel



On Tue, Feb 13, 2024 at 4:57 PM Arpita Goswami  wrote:

> Dear all,
>
> Good day. Thank you all for the very extensive discussions. Both on- and
> off-list discussions were very helpful.
>
> Thank you and a very happy Valentine's day to all..
>
> Best regards,
> Arpita
>
>
> On Wed, Feb 14, 2024, 01:25 Kay Diederichs 
> wrote:
>
>> Dear readers of CCP4BB,
>>
>> for various reasons I don't feel inclined to reply to this.
>>
>> I'm really sorry,
>> Kay
>>
>> On Tue, 13 Feb 2024 15:25:03 +, Gerard Bricogne <
>> g...@globalphasing.com> wrote:
>>
>> >Dear Kay,
>> >
>> ...
>>
>> 
>>
>> To unsubscribe from the CCP4BB list, click the following link:
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>> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
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>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] program to complete (or to change) side chains to specific rotamers

[Pavel Afonine]

Hi Jorge, it'd take about 1 hour of my time to put together a cctbx-based
script to do that, or about 2 hours to make an end-user program in Phenix
that'll be available in tomorrow's build. But I'm not sure how of general
interest this might be to warrant investing time into this. So here is an
alternative... If you are familiar with Python coding and cctbx, and are
motivated enough to have such a program, I can help navigate you where to
look in the cctbx codebase for relevant examples so you can use them to
make that program! We can continue discussing this off-list if interested!
Cheers, Pavel


On Tue, Feb 6, 2024 at 6:22 AM Jorge Iulek  wrote:

> Dear all,
>
>
> As I cannot find by myself, maybe someone can indicate here.
> I would like a program, command line - not gui, that I would
> simply
> indicate residues and the desired rotamer, and so it will change the
> coordinate file to complete/change side chains accordingly, say, somehow
> like I say for Glu251 make it rotamer tp10, and so on.
> Less desirable would be to indicate a reference structure for the
> specific rotamer. But I would really prefer that I indicate the specific
> residue and the rotamer I want.
> Is there such a program or any ideas for a combination of programs?
> Thanks,
>
> Jorge
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Identify mysterious density in CryoEM map

[Pavel Afonine]

Hello Jan,
you can convert your cryo-EM problem into crystallographic one by
FT'ing your map into a set of structure factors "Fobs" and then giving the
atomic model and MTZ with these "Fobs" to CheckMyBlob. If it's cool enough,
it should not make much difference if your "Fobs" come from X-ray or
cryo-EM!
Please share your experience if you choose to give it a try.
Good luck!
Pavel

P.S.: Phenix way to convert map to structure factors (example):
phenix.map_to_structure_factors map.mrc d_min=2.3


On Fri, Jan 26, 2024 at 10:27 AM Jan van Agthoven  wrote:

> Dear all,
>
> We’re working on an unknown ligand density in our CryoEM structure. Is
> there a program that uses deep-learning to fill uncharacterized electron
> density similar to Checkmyblob but for CryoEM maps?
>
> Thanks,
>
> Jan
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
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Re: [ccp4bb] Resolution Discrepancy in Data Set

[Pavel Afonine]

Hi Liliana,

a few things to consider:

0) There is a Phenix mailing list for Phenix specific questions (phenixbb);

1) Bin completeness depends on (obviously) how binning is done (number of
reflections per bin or number of bins or binning in d^2 or d^3 spacing or
log-binning etc etc etc) -- all of this will affect the number of
reflections in the "highest resolution bin" and corresponding statistics.
And..."how binning is done" wildly differs by the program used or even
within the same program!

2) Refinement (in Phenix) as well as many other Phenix tools apply
temporary reflection omission according to the Read (1999) paper (Acta
Cryst. (1999). D55, 1759-1764). This means while you have so many
reflections in your input reflection file, the actual reflections used in
calculations and reported statistics may be different. Most logs files keep
a good record of this, meaning you can track this down by inspecting logs
files carefully.

Let me know if you need more assistance with this issue.

Cheers,
Pavel

On Wed, Jan 17, 2024 at 1:43 PM David Briggs 
wrote:

> Hi Liliana,
>
> Two things leap out at me when I look at your data summary.
>
> (1) Your data probably do not go to 1.77Å. The CC1/2 in your outer shell
> is below any of the usual thresholds. There are discussions to be had about
> what the threshold is, but normally CC1/2 values of 0.5 or sometimes 0.3
> are used. You should also consider I/sigI.
>
> (2) I believe that by default Phenix.refine excludes weaker reflections
> from refinement, which leads to the discrepancy in completeness statistics.
> As your data do not extend as far as what is contained in your mtz file,
> Phenix excludes those essentially "empty" reflections. Judging by the
> Phenix refine output, I would estimate your data goes to somewhere around
> 1.9Å
>
> The program Pairef can help inform your choice of high-resolution cutoff.
>
> This can be run from CCP4cloud, but is also available for Phenix, I
> believe.
>
> See https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248825/
>
> Hope this helps,
>
> Dave
>
>
> *Dr David C. Briggs CSci MRSB*
>
> Principal Laboratory Research Scientist
>
> Signalling and Structural Biology Lab
>
> The Francis Crick Institute
>
> London, UK
>
> ==
>
> about.me/david_briggs
> --
> *From:* CCP4 bulletin board  on behalf of Liliana
> Margent 
> *Sent:* Wednesday, January 17, 2024 9:19:36 PM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] Resolution Discrepancy in Data Set
>
>
> External Sender: Use caution.
>
>
> Hello all,
>
> I hope this message finds you well.
>
> In my current data set, I’ve encountered a discrepancy between the
> completeness in the high-resolution shells in merged statistics vs the
> refinement statistics. For example, when I look at my merged statistics
> file, output by Xia2 dials, the completeness in the high-resolution shells
> are 97.6%. When I take this data and subsequently refine it in PHENIX I get
> extremely different completeness ranges in the high-resolution shells, but
> I cannot figure out why this large difference is occurring. I’m reaching
> out to you, our esteemed community, for any insights or advice you might
> have. Has anyone else faced a similar challenge? If so, how did you
> navigate through it?  Your experiences and suggestions could be invaluable
> in helping me understand and resolve this issue.
>
> Thank you in advance for your time and expertise.
>
> Best regards,
> Liliana
>
> see a side-by-side image of the files I mention in the message in
> https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fdrive.google.com%2Ffile%2Fd%2F1Y783MzlnqVwXRCtiLeV0Y2EpmkeDL2nd%2Fview%3Fusp%3Dsharing=05%7C02%7C%7Cb15f2dcfd7804db15e8808dc17a23ef1%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C638411232808490947%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C=5XcApcqvUkuvjz9oGUsaFpvnfY2p2X1LDE8G4XhFkuo%3D=0
> 
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
>
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> 
>
> This message was issued to members of
> 

Re: [ccp4bb] Poor correlation coefficient of model to cryo-EM map.

[Pavel Afonine]

Hi Ketul,

CCmask evaluates the cross-correlation between your experimental map and
the map that is calculated from the model. If you look at the formula, the
map calculated from the model involves, among other things, atomic
coordinates, B factors, and occupancies. This means that even if the model
visually appears to fit the experimental density, but its occupancies
or/and B factors are incorrect (e.g., not refined), then the CCmask can be
very low.

I can try to help more if needed, but since this is going to be more Phenix
specific (and this is the CCP4 mailing list!), I'd prefer to do it either
off-list or on phenixbb.

All the best,
Pavel

On Tue, Jan 9, 2024 at 9:57 PM Ketul Saharan 
wrote:

> Dear CCP4 community,
>
> I am building a structure model from ~3.0 Å resolution cryo-EM map. The
> structure consists of seven chains, with each chain containing an N-,
> middle, and C-terminal domain. Although I attempted to directly fit the
> Alfa-fold model, it became evident that the protein exhibited some
> movement, leading to poor fitting of N-terminal. To improve the fitting, I
> segmented the alfa-fold model into two parts: i) the N-terminal and ii) the
> middle and C-terminal domain. These fragments were then fitted into the
> map. After a few rounds of refinement using coot and phenix, the model
> effectively fitted all seven chains.
>
> The refinement resulted in a model to map correlation (CC mask) of over
> 60% for the N-terminus. *However, even though the model appeared to fit
> well inside the map, particularly in the middle and C-terminus regions, the
> refining consistently resulted in a map to model correlation of 0%.*
>
> For your perusal, I have included the snapshot of the phenix refinement
> results, the correlation graph, and the fitted model within the map
> (displaying one chain out of seven).
>
> I am not able to figure out why the correlation is so poor even after fine
> fitting of model to map.
>
> Any support in resolving this issue would be much appreciated.
>
>
> Thank,
>
> Ketul Saharan
>
>
>
> --
>
> Ketul Saharan
>
> Senior Research Fellow (Ph.D. Scholar)
>
> Laboratory of Macromolecular Crystallography (Lab-8)
>
> Institute of Life Sciences
>
> Nalco Square, Chandrasekharpur
>
> Bhubaneswar – 751023
>
> Odisha State, INDIA
>
>
>
> Phone: +91 8708290889
>  124.png
> 
>  correlation.tif
> 
>  map to model.tif
> 
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Can Refmac5 refine temperature factor residue by group?

[Pavel Afonine]

Hi All,

I believe it depends on the resolution. At sufficiently low resolution, it
may not be too unreasonable to assume that the main chain atoms of a
residue have the same B factors, and its side chain atoms also share the
same B factor (different from the main chain). Why? Simply because your
low-resolution data cannot resolve the B-factor difference between, for
example, CA and C atoms! This is much like expecting low to almost zero
deviations from ideal bonds and angles at low resolution, because your
low-res data simply cannot resolve a fraction of an Angstrom deviation in
bond lengths.

The bottom line, in my opinion, is that there is no black-and-white answer
to the "grouped vs. individual" B-factor refinement question. Depending on
a) the data resolution, and b) your experimentation with both individual
and group options, you may choose one over the other.

Back in the day (when working on the implementation of B-factor refinement
in phenix.refine), I conducted a test where I re-refined a sample,
consisting of about 50,000 models from the PDB, using 1) individual, 2)
group with one B per residue, and 3) group with two B per residue (one for
the main and one for the side chain). There were clear clusters of results
where each of the three parameterizations outperformed the others, and this
was heavily resolution-dependent. I regret not publishing that result,
mostly because I thought I could always re-do it any time later.. but as
Ben said "Don't put off until tomorrow what you can do today"!

Regarding TLS, since someone mentioned it, I prefer to see TLS as a more
physically realistic atomic vibration model, rather than a magic solution
to reduce R-factors or a way to decrease the number of refinable parameters
(which, in fact, is not the case because normally residual isotropic B
factors are always refined on top of TLS anyway, in Phenix at least).

All the best!
Pavel

On Sat, Jan 6, 2024 at 3:41 PM Robbie Joosten 
wrote:

> Hi Tom,
>
> I think restraints do change the data/parameter ratio, but how much is not
> straightforward. In, at least, the context of the Hamilton test restraints
> change the degrees of freedom which translates into a change of the
> effective data/parameter ratio. It is treated as adding extra observations
> albeit with some unknown weight. Ethan Merritt's way of handeling this
> unknown weight (which we implemented in bselect) is setting an upper limit
> for the weight and thus bracketing the value.
>
> Lets assume we have glycol. Adding individual B-factors instead of one
> overall B adds 3 extra parameters. We also add B-factor restraints: 3
> bonded atom 1-2 restraints plus 2 angled atoms 1-3 restraints (in Refmac's
> implementation). So 3 extra parameters, 5 extra restraints. These
> restraints can at best nullify the effect of the extra parameters so their
> effective weight is maximum 3/5, but probably less. The weight of the
> restraints must be somewhere between 0 (e.g when the B-factor restraint
> weight in Refmac is set to zero) and 0.6 (the restraint weight is huge).
> Assuming that the restraints do something, the degrees of freedom go up
> with less than 3 for our glycol refinement. How much less is related to the
> restraint weight we set in Refinement.
>
> Cheers,
> Robbie
>
>
>
>
> On 6 Jan 2024 23:26, Tom Peat  wrote:
>
> Hello Robbie,
>
> Thanks for the stats and description of what has been done.
> I think this puts us back into the realm of restraints versus constraints
> and what is possible when trying to reduce the number of parameters to be
> refined. Although grouped B-factors don't capture the reality of side
> chains being more mobile, it is a constraint that reduces the number of
> parameters being refined and helps highlight regions of the structure which
> are more mobile (which a flat or average B-factor would not do).
> Making tighter restraints on the system as a whole doesn't change the
> data/parameter ratio, which can lead to its own issues, but is certainly
> better than just letting things go wild.
> As is often the outcome, we state 'it depends on your individual
> situation' and generally suggest looking at various possibilities until one
> finds some compromise which works. Not as intellectually gratifying as
> having a cut and dry answer to these questions that come up rather
> frequently.
> Thanks again for the stats and description.
> cheers, tom
>
> --
> *From:* Robbie Joosten 
> *Sent:* Sunday, January 7, 2024 8:24 AM
> *To:* Tom Peat ; CCP4BB@JISCMAIL.AC.UK <
> CCP4BB@JISCMAIL.AC.UK>
> *Subject:* RE: [ccp4bb] Can Refmac5 refine temperature factor residue by
> group?
>
> [You don't often get email from robbie_joos...@hotmail.com. Learn why
> this is important at https://aka.ms/LearnAboutSenderIdentification ]
>
> Hi Tom,
>
> At 3A the median number of reflections per atom is 3.4 which is indeed
> lower than 4. So in unrestrained refinement the data/parameter ratio is
> indeed worse than 1. This 

Re: [ccp4bb] nearestcell

[Pavel Afonine]

Hi Kay,
to address "(...) not elsewhere (...)", in Phenix GUI go to Tools then down
to "Search PDB symmetry...". I've never used it myself nor do I know who
added it in Phenix but from the description it looks like what you're
looking for. Let us know if you have any problems with it.
All the best,
Pavel


On Wed, Dec 27, 2023 at 10:39 AM Kay Diederichs <
kay.diederi...@uni-konstanz.de> wrote:

> Dear all,
>
> I seem to remember a tool called "nearestcell", a command-line equivalent
> of the Oxford Nearest-Cell web server which appears to be offline.
> However I cannot locate that tool in CCP4 nor elsewhere. Can anyone point
> me to it or give alternatives, please? (I did try the PDB's advanced search
> but it is not made for this purpose)
>
> Thanks, and a happy and successful 2024 to everybody!
>
> Kay
>
> 
>
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Re: [ccp4bb] Occupany refinement protocol amd best practices

[Pavel Afonine]

Hi Matt,

Im wondering about occupancy refinements - both what's going on under the
> hood and what are best practices.
>

since you are quoting Phenix I suggest this bit of reading that is somewhat
relevant to your questions:
https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12
This documents 13 typical occupancy refinement scenarios and how they can
be handled in Phenix.


> In the example I have, there is a ligand found in two distinct, partially
> overlapping sites that can be modeled is some confidence, but likely there
> are very low occupancy additional poses that blurs the electron density.
> The modeled poses are known from prior work, so even though there is
> smearing we know the ligand is in the modeled conformation. After
> perturbing the crystal these I am trying to decide what the best approach
> is to get some sort of numerical occupancy value to describe the
> distribution.
>

I apologize in advance for this trivial statement, but refinement +
validation are the tools to answer your question.
If you can model these poses with an atomic model and prove they match the
experimental diffraction data ("The modeled poses are known from prior
work" doesn't count in this regard), then you are all good! Depending on
the size ration ligand:whole structure, R factors may or may not be useful
quantifiers of the modeling choices. So your best bets may be local
quantities, such as refined ligand group occupancy, flatness of Fo-Fc map
(assessed as map values at atom positions), local map-model CC, etc. Try to
challenge your modeling decisions by placing similar to expected answers
but knowingly wrong models and see how that changes the fit/quality
criteria -- that may give you a way to assess uncertainty.

1.  In Phenix, how is the occupancy number determined?


Please refer to the paper I mentioned above, and let me know if you have
additional questions.


> Is there a real-space correlation between the experimental density and the
> model(weighted to occ) that is optimized?


Yes, but very indirectly through optimization of overall standard ML target
function.


>   How can this go wrong?


It can go wrong in as many ways as the refinement target function has local
minima, that is in many thousands ways!


> I fear that the smearing and heterogenous nature will through the
> refinement off (over or underfitting to periphery density rather than
> hyper-localized position of the model)
>

That is a valid concern that is good to keep in mind but that is not a
show-stopper!


> 2.  Are there errors associated with the occupancy numbers?
>

Yes, like with any model refinable parameter. For some discussion please
see:

https://www.nature.com/articles/s41467-018-06957-w


>
> 3.  For my own testing, I did 5% increments and manually observed Fo-Fc
> and 2Fo-Fc maps and selected a value that resulted in the lowest amount of
> both positive and negative Fo-Fc peaks.  This is how we submitted the work
> to journal but reviewer wants it to be automatically calculated.


Above mentioned paper is now more relevant in light of this question! Yes,
you can do manual sampling to get starting values (because the closer they
are to the true values, the better chances refinement is successful), then
do some refinement starting with these values.

Good luck!
Pavel



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Re: [ccp4bb] skewness in a good EM map?

[Pavel Afonine]

Hi Rob,
the fundamental property of crystallographic or cryo-em maps -- the
presence of high density points -- is what skewness depicts. So I can't see
why the idea of using skewness to quantify maps would be any different for
x-ray vs cryo-em. I've just downloaded a cryo-EM map for this (PDB: 8d3y,
EMDB: 27168) entry. The histogram (and its log for easier plotting) is:

-0.0816 16 1.204119983
-0.0707 107 2.029383778
-0.0597 458 2.660865478
-0.0488 1643 3.215637563
-0.0378 4954 3.694956002
-0.0269 21438 4.331184267
-0.0159 819411 5.91350179
-0.005 9110776 6.959555369
0.006 569464 5.755466274
0.0169 73635 4.867084291
0.0279 22271 4.347739718
0.0388 10113 4.004880007
0.0498 5938 3.773640193
0.0607 3752 3.57426283
0.0717 2189 3.340245762
0.0826 1106 3.043755127
0.0936 471 2.673020907
0.1045 185 2.267171728
0.1155 61 1.785329835
0.1264 12 1.079181246

The skew is 4.2 which is well far from zero and positive!

More on map histograms in the context of cryo-EM :
https://www.nature.com/articles/s41592-020-0914-9
and references therein.

Good luck!
Pavel


On Tue, Nov 7, 2023 at 6:07 AM Robert Oeffner  wrote:

> Hi,
>
> In crystallography one of the features of a good map is that the histogram
> of map values has skewness. Does this also apply to EM maps? Whereas X-ray
> maps depicts the electron density EM maps depicts the electrostatic
> potential as I understand it. Any pointers about skewness or not in EM maps
> is greatly appreciated.
>
> thanks,
>
> Rob
>
> 
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Re: [ccp4bb] Occupancy Refinement limitation

[Pavel Afonine]

Hi Matt,
I believe figure 3 here:
https://www.nature.com/articles/s41467-018-06957-w
is relevant to your question.
Pavel


On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod  wrote:

> Hi all,
>
> I am trying to get some insight in the accuracy/precision of occupancy
> refinements.  I have done some 2-state occupancy refinements and have
> observed the refinement achieving ~0.25-0.3 occupancy for the minor
> population.  This population, when observing the electron density maps, had
> essentially no evidence for it being present.  I was wondering:
>
> What are the errors in the reported occupancies?
>
> Is there a lower and upper limit to occupancy refinements?  As in, if you
> occupancy refine two states and one is imaginary will it refine to
> approximately 1 and 0?  Or does the background noise always given a
> positive number to the imaginary set?  This would, to me at least, be the
> lower and upper limits to the occupancy refinements and could be used as a
> normalization factor for other atoms.  Maybe my logic is off...
>
> Any insight or literature would be appreciated!
> Matt
>
> 
>
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Re: [ccp4bb] quantifying electron density

[Pavel Afonine]

Hi Jon,
not really the answer to your question but.. This may be very tricky to do
because what you look at is not an electron density map but its Fourier
image of finite resolution phased by crystal model (that has errors), plus
experimental errors, and missing F000 (which is not measured as part of
your diffraction experiment). So.. if such a software exists I'd be very
cautious interpreting the results you get from it!
Pavel


On Wed, Jun 28, 2023 at 9:16 AM Hughes, Jonathan <
jon.hug...@bot3.bio.uni-giessen.de> wrote:

> hello everyone,
> is there software that can use an electron density map to quantify the
> number of electrons in a selected volume somewhere in a protein?
> cheers
> jon
>
> --
> Professor Jon Hughes, BSc, PhD
> Department of Physics
> Free University of Berlin
> Arnimallee 14
> 14195 Berlin
> Germany
> mobile:   (+49/0)1757929098
> email: lv...@posteo.de
> homepage:
> http://www.uni-giessen.de/fbz/fb08/Inst/pflphys
> Sent without the use of Apple products
>
> 
>
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Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Pavel Afonine]

Hi Stuart,
the answer, I think, is here:
https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12
Pavel


On Wed, Feb 15, 2023 at 8:37 AM Stuart McQuarrie <
974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk> wrote:

> I fit a large cyclic ligand cA6 (cylic hexa-adenylate) and after some
> refinement have noticed partial occupancy of it's hydrolysed form 2x A3,
> which has a cyclic 2'-3' phosphate on the terminal ribose.
>
> I tried fitting both ligands with 50% occupancy, but refinement doesn't
> allow them to occupy the same space.
>
> I subsequently tried text editing the pdb file, adding altloc identifiers
> to the ca6 and the 2 A3 molecules, making sure they were same chain and
> residue number as per this ccp4bb archive:
> http://www.phenix-online.org/pipermail/phenixbb/2011-March/016768.html.
> However, before I refined I checked in coot and all the ligand atoms are
> scattered and disconnected.
>
> I have thought about using coot to generate an alt conformation of ca6 and
> then text editing the B conformation to be split and have a 2'-3' cyclic
> phosphate. I am not sure if this is the correct way because it is a
> different chemical structure, so I could use some advice.
>
> Kind regards,
> Stuart
>
> 
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Re: [ccp4bb] Future Diffraction Methods

[Pavel Afonine]

Nukri,

IMO, the idea of cross-discipline meetings is great conceptually, at least
for reasons you pointed out, but utopical in practice. When we attend our
field-specific meetings we meet colleagues we know, we talk to
collaborators from the past or find new ones, we have things in common that
we can talk about to forge something new, we meet authors of papers we were
excited to read, and so on, and so on.
I once attended a meeting of some chemistry society, well, which is not too
far from what we are doing, really, as interpreting atomic models is
essentially putting your chemistry knowledge into production. And, at that
meeting I felt like I'm alone in a dark forest.
Now, I imagine, if you bring two (or more) groups of people to your meeting
from two different domains, well, I guess you will end up having two
bubbles of people clustered by their field of interest.

Same disclaimer goes here as yours -- no offence to any one, just thinking
out loud...

All the best!
Pavel

On Sun, Jan 29, 2023 at 6:09 AM Nukri Sanishvili  wrote:

> Hi James,
> This meeting has indeed been one of the best ones by its format, content,
> and atmosphere. Many thanks to all the organizers and attendees of the
> past. Nevertheless, it is not surprising that it was cancelled, given the
> trends in structural biology research. Straightforward evolutionary
> pressure to adapt or else...
>
> Throughout my career I was always amazed (dare I say, annoyed?) how
> scientists from different fields, or even the same field but different
> methods, speak different languages. How little they understand each other,
> become entrenched in their own methods and how much of the
> collaboration/cooperation opportunities are wasted.
>
> IMO, having a conference on "Complementary Methods in Structural Biology"
> with the emphasis on complementarity and not on individual methods, would
> be a great benefit in the long run. Hopefully it would give good examples
> to young researchers to help them develop a collaborative mindset.
>
> If I offended anyone, it was not intentional, I promise, and apologize in
> advance.
> Best wishes to all and best of luck to all who continue the effort for the
> benefit of the whole community.
> Nukri
>
>
>
>
>
> On Fri, Dec 16, 2022 at 4:11 PM James Holton  wrote:
>
>> I want to thank everyone who attended the 2022 Gordon Research
>> Conference and Gordon Research Seminar on Diffraction Methods in
>> Structural Biology, as well as all those who contributed to these great
>> gatherings in the past.  It was an outstanding meeting if I do say so
>> myself. Not just because it had been so long without in-person
>> interaction, not just because we had zero covid cases (which I see as no
>> small feat of Mind over Virus), but because of this amazing community.
>> It is rare in this world to have such a strong spirit of collaboration,
>> camaraderie and openness in undertakings as high-impact as this.
>> Surmounting the barriers to atomic-detail imaging of biological systems
>> has never been more exciting and more relevant.  I am proud to be a part
>> of it, and honored to have served as Chair.
>>
>> It is therefore with heavy heart that I report to this community that I
>> was the last Chair of the Diffraction Methods GRC.
>>
>> The GRC Conference Evaluation Committee
>> (https://www.grc.org/about/conference-evaluation-committee/) voted this
>> year to discontinue the Diffraction Methods GRC and GRS. This ends a
>> 46-year tradition that I feel played a vital, and vibrant role in the
>> work of the people who answer questions on this BB.  The reason given
>> was insufficient attendance.  All other metrics, such as evaluation
>> surveys and demographics were very strong. I have tried to appeal, but
>> I'm told the vote was unanimous and final. I understand that like so
>> many conference organizing bodies the GRC is having to make tough
>> financial decisions. I must say I disagree with this one, but it was not
>> my decision to make.
>>
>> Many of the past and elected Chairs have been gathering and discussing
>> how to replace the Diffraction Methods GRC/GRS going forward. Many great
>> ideas, advice and perspectives have been provided, but that is a select
>> group. I feel it is now time to open up this discussion to the broader
>> community of structural methods developers and practitioners. There are
>> some important questions to ask:
>>
>> * How do we define this community?
>>  Yes, many of us do cryoEM too, but is that one methods meeting?
>> or two?
>> * Does this community need a new diffraction methods meeting?
>>  As in one meeting or zero?
>> * Should we merge with an existing meeting?
>>  It would make logistics easier, but a typical GRC has 22 hours
>> of in-depth presentations over 5 days.  The GRS is 7 hours over 2 days.
>> As Chair, I found that was not nearly enough.
>> * Where do you think structural methods are going?
>>  I think I know, but I may be biased.
>> * Should 

Re: [ccp4bb] Map superposition!

[Pavel Afonine]

Hi Rams,

phenix.match_maps

will superpose maps and models. Totally general: target/moving boxes (unit
cells) and symmetry can be different, gridding can be different too.

I can help off list should you need any assistance!

Pavel


On Wed, Jan 25, 2023 at 6:30 AM Subramanian, Ramaswamy 
wrote:

> Hi All,
>
> I have two maps - both cryo-EM.  One with and one without ligand.  I am
> trying to superpose them and I cannot seem to get ChimeraX to work.  I then
> tried VESPER and that also does not work.
>
> The built models superpose very well.
>
> What is the best way to superpose maps.
>
> Thank you.
>
>
> Rams
> subra...@purdue.edu
>
>
>
>
> --
>
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Re: [ccp4bb] outliers

[Pavel Afonine]

>
> This is best illustrated by Ramachandran "outliers",
> which are perfectly supported by electron density.
>

Indeed, and 3NOQ is one of my favorite examples of that, an outlier isn't
necessarily equates to wrong! However, I think torsion angles (eg, phi/psi)
are much more flexible than covalent angles/bonds and so they can possibly
afford larger deviations compared to covalent bonds/angles.

The strain caused by any one of them will distribute itself
> over all neighbouring bond lengths and angles as well as
> over the torsion angles.
>

I wonder if there is a documented study that actually shows this happening?
Clearly this must take place one way or another, but I wonder if anyone
"measured" the effect and documented it..

Pavel



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[ccp4bb] ISO model with large groups of atoms in alternative conformations

[Pavel Afonine]

Dear community,



I’m looking for an example of a crystal structure where a large group of
atoms (as large as a whole chain or even a domain) have more than one
distinct conformation that would require modeling of such chain/domain as
more than one individual copy, with each copy having partial occupancy. I’m
not sure if that even exists but if someone can share an example that'd be
very much appreciated!



Thanks!
Pavel



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Re: [ccp4bb] Quantifying electron density inside of a given volume

[Pavel Afonine]

Hi James,

- Where exactly inside the blob of density do you place these dummy atoms?
>
> Where? At the peaks.
>

Peaks? This means you need to have atomic resolution data and also blobs
representing ordered atoms, so you actually have peaks!


> What I usually do is pick peaks, put atoms at the highest ones, then
> either refine for a bit or simply subtract a "divot" of density around each
> added atom and then look for new peaks. Once the height of after-refinement
> positive difference features drops to an insignificant level I stop.  NB
> that "significance" of any sigma level depends on the number of voxels in
> the map, but its usually between 3.5 and 4.5 sigmas.
>  One can also start with a 3D "grid" of dummy atoms.  Gaussians spaced
> within 0.8*fwhm of each other form a pretty flat density profile.  In such
> cases refining just one overall B factor and individual occupancies with no
> xyz motion is pretty effective all by itself.
>

Dummy atoms on a 3D grid approach in fact has a documented instance of a
successful application (although in a slightly different context):

 "Local improvement of electron density maps". (1997).  Acta Cryst., D53,
540-543.

- I guess O or C as an atom type should do it, but what about B factor
> (would you refine B as well?)?
>
> I find oxygen will do in peak-picking cases, but for grid atoms under very
> smooth density and closely-spaced atoms I have found the individual
> occupancies get rather small, the round-off error of occupancy from 0.01 to
> 0.00 creates a granularity of ~0.1 electron, and this can start creating
> artificial noise in the fit.  For cases like this I have gone to "liquid
> helium", or modelling dummy atoms as He. Yes, you might think that hydrogen
> would be better, but H atoms have so many special checks and whiz-bangs for
> how they are treated I eventually gave up and went to He.  (One could also
> argue that at low enough temperature He atoms are allowed to "overlap"
> anyway. ;) )
>

Yes, H (Acta Cryst. 1997. D53, 540-543) or O (Acta Cryst. 1993. D49,
129-147) depending on whether you do peak picking or refine DAs on the grid.
And I see why He may be a better choice, yes, hydrogens are too special
these days!

Pavel



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Re: [ccp4bb] Quantifying electron density inside of a given volume

[Pavel Afonine]

Hi James,

I like your approach with dummy atoms and occupancy refinement. Dealing
with actual maps sounds like hell to me indeed (especially given that we
deal with weighted Fourier maps!). Reading this as someone who immediately
translates this into a computer code (in my mind), a few things that aren't
clear to me:
- Where exactly inside the blob of density do you place these dummy atoms?
- How many do you place?
- Is there a risk of placing them too close to the boundary of the blob (in
which case the question remains: what is the boundary?)?
- I guess O or C as an atom type should do it, but what about B factor
(would you refine B as well?)?
- if you refine B, how do you deconvolute occupancy from refined B values
(and eventually from effects of positional errors of your DAs)?
-
- How all these choices are going to affect the result?

All the best!
Pavel


On Mon, Aug 15, 2022 at 4:38 PM James Holton  wrote:

> There are several programs for integrating electron density, but please
> let me assure you that it is almost always the wrong thing to do.
>
> A much better strategy is occupancy refinement.  Throw in dummy atoms,
> turn off non-bonded interactions to them, and refine their occupancy until
> it a) stops changing (may be more than one round), and b) there are no
> Fo-Fc differences left in the region of interest.  Then all you do is add
> up the occupancies, multiply by the relevant atomic number (usually 8), and
> voila! you get the best-fit number of electrons in your blob. You may want
> to try re-running with random starting points to get an idea of the error
> bars.
>
> What is wrong with integrating density?  Well, for one, it is hard to know
> where to set the boundaries. Integrated density can be VERY sensitive to
> the choice of radius, making your arbitrary decision of which radius to use
> a source of error. Too small and you miss stuff. Too big and you add
> unnecessary noise. Also, neighboring atoms have tails, and if you don't
> subtract them properly, that is another source of error. Also, because of
> the missing F000 term, there is an offset, which adds a term proportional
> to the integration volume.  For example, an integral resulting in zero
> "electrons" does NOT mean you have vacuum. It just means that the area you
> integrated has the same average density as the entire map. This may not be
> the number you want.
>
> The beauty of occupancy refinement is that it automatically handles all
> these problems. The "vacuum level" and F000 are known quantities in the
> calculated map. The B factors given to the dummy atoms als o allow the
> borders of your integration region to be "soft": down-weighting the
> contribution of map noise far from your region of interest.  And, finally,
> by including atoms in the green density, neighboring atoms won't be sucked
> into it.
>
> Think of it as fitting a smooth curve to noisy data and the number of
> electrons is just a parameter in that fit, rather than trying to integrate
> the noisy data itself.  This is not an analogy. Refinement programs are
> really just very sophisticated curve-fitting programs.  And if you have a
> forest of overlapping peaks and you are trying to de-convolute the
> area/volume of one peak, it is best to include that peak in the fit, rather
> than leave it out. Shoulder peaks especially tend to get "eaten" by large
> neighboring peaks.
>
> How do you turn off non-bonds? Well, there is documentation for refmac:
> http://www.ysbl.york.ac.uk/refmac/data/refmac_keywords.html
> and phenix:
> https://phenix-online.org/documentation/reference/refinement.html
>
> All that said, to answer the original question:
>  One very easy thing to do within the CCP4 suite is to use "mapmask" to
> make a mask corresponding to your "sphere", or other region of interest.
> Perhaps place a water at the center of your peak, and either use the
> "border" feature of mapmask, or use "sfall" to compute a calculated map and
> convert that to a mask using "threshold" in mapmask.  This mask should have
> values of 0 or 1 at every voxel. (or, if you feel like being clever,
> something between 0 and 1 to reflect how much you want to weight a given
> voxel). You can check it in coot. If you then multiply this mask by your
> mFo-DFc map the result will have a non-zero average value. This will be
> printed out in the log file. Multiply this average value by the volume of
> the unit cell and you have your integrated number of electrons. Yes, its
> that simple.
> One issue you may have is map parameter compatibility (grid spacing, axis
> order, xyz limits, etc.). You get around these by using the same grid in
> all your fft or sfall runs, and then use mapmask to make the axis and
> limits match before you multiply the map and mask.  The only other issue
> here might be the average value being a very small number and rounded off
> by the default print precision. You can fix this by multiplying the map by
> a large constant (again, using mapmask), 

Re: [ccp4bb] How to cut fragment of map

[Pavel Afonine]

Hi,
this can be a start:
http://cci.lbl.gov/docs/cctbx/doc_maps_boxing/
There are a lot of boxing options: with and without masking (with many
masking options: smooth masks, etc), around atom selection, per NCS etc..
Pavel

On Fri, Aug 5, 2022 at 2:30 AM Evgenii Osipov  wrote:

> Dear CCP4 community,
>
> I am looking for a way to cut a fragment of electron density around
> specified set of atoms using Python script. In CCP4 I found MAPMASK, which
> could cut fragment of electron density within specified region around input
> PDB:
> #!/bin/bash -f
>
> $CCP4/bin/mapmask \
> MAPIN tmp.map \
> MAPOUT masked.map \
> XYZIN ligand.pdb \
> <<
> eor
> BORDER 3
> eor
>
>  Of course I could call this shell script from Python script using
> subprocess, but it looks like unnecessary overcomplication.
>
> I wonder if the same task could be completed by Python script using either
> CCTBX or Gemmi. May be someone had this problem in the past and could share
> their example?
>
>
>
> Kind regards,
>
> --
>
> Evgenii Osipov
>
> Laboratory for Biocrystallography,
> Department of Pharmaceutical Sciences,
> KU Leuven O
>
> mobile: +32 484 38 26 01
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Help with One dimensional electron density calculation

[Pavel Afonine]

For some perhaps relevant discussion please see Appendix B here:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096492/pdf/d-74-00531.pdf

Pavel

On Sat, Jul 16, 2022 at 6:17 AM Marcin Wojdyr  wrote:

> On Thu, Jul 14, 2022 at 3:15 AM Pavel Afonine  wrote:
>
> > Map values are calculated using tricubic interpolation. Optionally you
> can choose a less accurate eight-point interpolation (use interpolation
> keyword for this).
> >
>
> I've been wondering which one is better. Tri-cubic is more accurate
> for smoothly changing values, but for noisy data it will amplify the
> noise. Did anyone test if the choice of interpolation method
> (tri-cubic vs tri-linear) makes a practical difference in any
> crystallographic application?
>



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Re: [ccp4bb] Help with One dimensional electron density calculation

[Pavel Afonine]

Hi Ravikumar,

next nightly build of Phenix (dev-4661 and up, likely becomes available
tomorrow) found in the usual place

https://phenix-online.org/download/nightly_builds.cgi?show_all=1

will contain the new command line program
called phenix.map_values_along_line.
The program inputs the map (real-space map in mrc format) and Cartesian
coordinates of two points in space, and it outputs map values along the
line connecting these two points.

Example:

phenix.map_values_along_line map.mrc point_1="1.123 2.455 3.765"
point_2="4321 5.567 6.987"

Optionally, you can specify the step that map values are computed at going
from one point to another, by default it is set to 0.01 (use step parameter
for this).
Map values are calculated using tricubic interpolation. Optionally you can
choose a less accurate eight-point interpolation (use interpolation keyword
for this).

Those interested in CCTBX implementation of underlying functionality of
this program grep for 'map_values_along_line_connecting_two_points' that
will find you the function and the test that exemplifies the use of this
function.

Hope this solves your problem, Ravikumar.

Pavel

On Wed, Jul 13, 2022 at 11:19 AM Ravikumar  wrote:

>
> Dear all,
>
>
> I would like to calculate One-dimension electron density profiles of the
> different number of ions bound to an ion channel. Are there any programs in
> CCP4/ Phenix which can do the same job as the MAPMAN (UPPSALA electron
> density server) program to extract the electron density values to plot
> electron density values versus pore axis? I have attached an example
> figure.
>
>
> Thank you,
>
> Ravikumar.
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Help with One dimensional electron density calculation

[Pavel Afonine]

Hi Ravikumar,
this can be easily done in CCTBX, but requires knowing CCTBX and Python
scripting. I can write a script-example for you, if interested, or feel
free to give it a try yourself and let me know if you need any help.
Pavel

On Wed, Jul 13, 2022 at 11:19 AM Ravikumar  wrote:

>
> Dear all,
>
>
> I would like to calculate One-dimension electron density profiles of the
> different number of ions bound to an ion channel. Are there any programs in
> CCP4/ Phenix which can do the same job as the MAPMAN (UPPSALA electron
> density server) program to extract the electron density values to plot
> electron density values versus pore axis? I have attached an example
> figure.
>
>
> Thank you,
>
> Ravikumar.
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Regarding File conversion

[Pavel Afonine]

Hi,
Phenix has a collection of tools like this:

phenix.cif_as_mtz
phenix.cif_as_pdb
phenix.pdb_as_cif
phenix.mtz_as_cif

Pavel

On Tue, Apr 19, 2022 at 7:52 AM Marcin Wojdyr  wrote:

> Plenty of options. I'll add one maintained by me. Type:
> gemmi convert file.cif file.pdb
> or use it here:
> https://project-gemmi.github.io/wasm/convert/cif2pdb.html
>
>
> Marcin
>
> On Tue, Apr 19, 2022 at 2:01 PM Abhilasha Thakur 
> wrote:
> >
> > Hello!!
> > Greeting of the day,
> >
> > I want to know regarding file conversion from mcif file format to PDB
> format of proteins.
> > Is there any program or software that can be used to change one format
> to another format. KIndly guide me about programs used for file conversion
> or any other medium like python script or R script to convert the file
> format without changing the coordinates  of the file.
> > Thankyou
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> 
>
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Re: [ccp4bb] Coot 1

[Pavel Afonine]

It's April 1st today, isn't it? -;)


On Fri, Apr 1, 2022 at 3:15 AM Paul Emsley 
wrote:

> Coot 1
>
> 18 years after the release of Coot 0 it's time that I actually released
> Coot 1.
>
> Coot 1 is a major change beyond Coot 0. It has required a lot of writing
> and rewriting [1]
> and has been the preponderance of my work since 2017. I have had to
> learn how to program
> graphics from scratch using the new style. Much of the GUI internals has
> been
> rewritten [2].
>
> My experience with 0.9 was that I sat on it, bug-fixing for a long time,
> releasing it
> too late. I don't want to repeat that mistake. So here we are, there's a
> lot of good new
> stuff in Coot 1, but it's not as slick as it might be (or will become).
>
>o Update graphics to use OpenGL 3.3
>o Update Python to use version 3.9
>o Update GTK+ to use version 3
>
> While many of the features that were available in 0.9.x have been
> reworked or
> re-implemented, there are some gaps. Dropped features include
> Cross-hairs, Stereo, Pisa
> interface, built-in key-bindings, NCS Ghosts, Edit Phi/Psi,
> anti-aliasing, Kleywegt
> plots, the clipping dialog, Chemical Feature Clustering,
> Dynamically-Transformed/NCS
> Maps, LSQ plane distances, dynamic distances, CABLAM-markup, the test
> suite,
> user-defined colour schemes, anisotropic atoms, CURLEW, Scheme GUI
> scripting,
> Skeletonization and Baton-Building.
>
> "So, is there anything that _does_ work?" - you might ask...
>
> The GUI has been updated, I have tried to cut down on the number of
> dialogs, the Real
> Space Refinement in particular has seen quite a lot of work. The GUI now
> uses dark mode
> if the theme is installed [3]. Several menu items have been relocated.
> More menus are now
> alphabetically ordered [4].
>
> The graphics is, by default, now based on triangles instead of lines
> which affords a
> more pleasing representation (density maps can be represented using both
> modes) and one
> can have a more expeditious if not pleasant experience with a fast
> graphics card and a
> big screen. The various graphics effects and filters can be tailored to
> some extent by
> changing the configuration using the GUI or by editing/replacing the
> shaders.
> Full-screen mode is now an option [5].
>
> Overall, the GUI has only had light testing. At the moment, it's
> probably best to
> avoid closing dialogs using the window manager. The "OK" button now
> appears after the
> "Cancel" button in dialogs.
>
> Python scripting now has uses a history from previous sessions and the
> functions need
> to be used with namespaces/modules (e.g. "coot", or "coot_utils"). Coot
> (or coot) is
> now a module that can be imported into python.
>
> This build compiles with the RDKit and optionally MoleculestoTriangles
> from Martin Noble.
>
> The map and the model can now be exported to glTF files (for use in
> Blender and other
> 3D modelling software).
>
> The build script for Coot 1, called build-it-3-3 can be found in the
> "Build from Scratch"
> menu on the web page (you will need to have already installed cmake and
> Gtk+). The catch
> though is that (at least in my hands) Python and friends are
> frustratingly difficult to
> install, so it's possible/likely that the build script won't work.
> Likewise,
> the script doesn't work for Mac OS either. But it is currently the best
> method to get
> binaries so I will support it if you try it [6]. (Homebrew might be
> another method.)
>
> Bernhard Lohkamp has been working on the WinCoot version - I will defer
> WinCoot
> questions to him.
>
> Mac Coot is now native (no X11/XQuartz needed). I would be interested to
> see how a
> natively-compiled [7] version works on an M1 Max processor [8,9].
>
> Judging from previous experience, a few rapid iterations of bug-fix
> releases will be
> needed. After this, the version numbers will become sane - it's my plan
> to release a new
> major version every year or so.
>
>
> https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/source/releases/coot-1.tar.gz
>
> Normally I don't cross-post releases, but this one I thought I should.
>
>
> [1] new as in OpenGL 3.3 or later - using shaders - I don't mean Vulkan
> (that's for the
>  future)
> [2] the patch from the 0.9.x version is 285k lines
> [3] I recommend it
> [4] rather than chronologically
> [5] double-tap Esc key (the first time) to revert to standard display
> [6] I have be working with CCP4 collaborators to use their system to
> build Coot binaries.
>  Hopefully the binaries will be available stand-alone, as well as
> integrated into
>  the CCP4 Suite via that method
> [7] so that's a different meaning of "native"
> [8] the little testing I have done on a recent intel MacBook Pro shows
> unimpressive
>  performance (Iris graphics, retina display)
> [9] Thanks to my Mac-using colleagues for their feedback.
>
> 
>
> To unsubscribe from the CCP4BB list, click the 

Re: [ccp4bb] Ligand occupancy refinement

[Pavel Afonine]

For the record, occupancy refinement scenario you're looking for is
described here:

https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12

Cheers,
Pavel

On Thu, Mar 3, 2022 at 7:09 AM Jon Cooper <
488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello, the ligand needs to be treated as one occupancy group since
> refining individual occupancies would be a case of refining too many
> parameters, unless it was a very fragmentary compound!! It is one keyword
> in refmac, but I can't remember for phenix, sorry! Ta jc
>
>
> Sent from ProtonMail mobile
>
>
>
>  Original Message 
> On 3 Mar 2022, 14:15, Akanksha Tomar < akankshat...@gmail.com> wrote:
>
>
> Hi everyone,
>
> I am trying to refine the occupancy of a bound ligand. After fixing the
> protein model and water I fitted the ligand into it. Currently, I am using
> Phenix Refine with occupancy refinement for individual atoms switched on.
> After the refinement, the overall occupancy of the ligand is 0.7 and the
> RSCC value is 0.86. The resolution of the structure is 2.1 Å.
>
> Now the problem is that the program has assigned different occupancies to
> different atoms of the ligand. For some cases, it has assigned 0
> occupancies to atoms for which there is a clear positive peak.
>
> Why it has been done so and is it acceptable?
>
> Any help would be greatly appreciated.
>
> Thank you.
>
> --
> Best Regards,
> Akanksha Tomar
> Pre-Doctoral Fellow,
> C\o Dr. Arockiasamy Arulandu,
> Membrane Protein Biology Group,
> International Center for Genetic Engineering and Biotechnology,
> New Delhi, India
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> --
>
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>
>



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Re: [ccp4bb] Phenix/refmac incompatibility?

[Pavel Afonine]

Hi All,
who produced this file? If phenix.refine added water with chain S given
that protein chains named S were already in that model, then it is a bug
that I need to fix.
Cheers,
Pavel

On Thu, Dec 30, 2021 at 11:05 AM Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> I am not sure whether I have this straight but someone has sent a pdb file
> from phenix refinement with these atoms in the pdb file..
> 
> ATOM   5580  N   GLY S  18  36.182  44.368  56.021  1.00 79.25
>   N
> ATOM   5581  CA  GLY S  18  37.168  44.349  57.091  1.00 74.78
>   C
> ATOM   5582  C   GLY S  18  37.048  43.211  58.097  1.00 73.20
>   C
> ATOM   5583  O   GLY S  18  37.043  42.043  57.734  1.00 74.31
>   O
> ATOM   5584  N   SER S  19  36.992  43.568  59.375  1.00 75.40
>   N
> ...
>
> HETATM11068  O   HOH S  16  68.933  60.684 119.353  1.00 32.35
>   O
> HETATM11069  O   HOH S  17  17.772  20.649  91.306  1.00 40.79
>   O
> HETATM11070  O   HOH S  18  23.684  50.229  65.614  1.00 41.00
>   O
> HETATM11071  O   HOH S  19  45.488  71.114 105.890  1.00 51.50
>   O
>
> Then REFMAC gets upset because "residue" S 18 appears twice..
>
> Doesnt PHENIX worry about this? or has the user edits these HOH atoms into
> the file?
>
> And what should we do about it
> Eleanor
>
> 
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
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Re: [ccp4bb] Validation of structure prediction

[Pavel Afonine]

Hi Reza,

If you think about it this way... Validation is making sure that the model
makes sense, data make sense and model-to-data fit make sense, then the
answer to your question is obvious: in your case you do not have
experimental data (at least in a way we used to think of it) and so then of
these three validation items you only have one, which, for example, means
you don’t have to report things like R-factors or completeness in
high-resolution shell.

Really, the geometry of an alpha helix does not depend on how you
determined it: using X-rays or cryo-EM or something else! So, most (if not
all) model validation tools still apply.

Pavel

On Mon, Dec 20, 2021 at 8:10 AM Reza Khayat  wrote:

> Hi,
>
>
> Can anyone suggest how to validate a predicted structure? Something
> similar to wwPDB validation without the need for refinement statistics. I
> realize this is a strange question given that the geometry of the model is
> anticipated to be fine if the structure was predicted by a server that
> minimizes the geometry to improve its statistics. Nonetheless, the journal
> has asked me for such a report. Thanks.
>
>
> Best wishes,
>
> Reza
>
>
> Reza Khayat, PhD
> Associate Professor
> City College of New York
> Department of Chemistry and Biochemistry
> New York, NY 10031
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Different conformation of chains

[Pavel Afonine]

Hi,

if I understood your question correctly, then one of 13 scenarios described
here:

http://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12

fits your situation and you should be able to handle it in refinement all
right.

For more specific advice please feel free to contact me directly (off list)
with relevant files.

Good luck!
Pavel

On Wed, Oct 20, 2021 at 5:53 PM S  wrote:

> Dear All,
>
> I am working with a protein, the structure of which is already published
> in the PDB (2 chains in asu) The published structure has a break of around
> 20 residues.
>
> I have crystallized the same protein and saw some density near the gap (20
> residues break).
> I could build 3-4 residues in ChainA and 8 residues in ChainB (in the 20
> residue break region). After refinement these showed good density and b
> factor.
> But the issue is that in ChainA, it goes straight. While in ChainB, the 8
> residues converge into a loop and goes to the other side.
>
> The protein is a monomer in solution, is it possible to have different
> conformation in different chains that is completely opposite.
> Also the 8 residues (ChainB) that is build now occupies the site where
> ligand was placed in the published structure. I am not sure how to
> interpret this (since in ChainA that site is unoccupied as contrast to
> ChainB).
>
> Any thoughts/suggestions will be really helpful.
>
> Thanks you
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
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Re: [ccp4bb] AI papers in experimental macromolecular structure determination

[Pavel Afonine]

One more:

BraggNet: integrating Bragg peaks using neural networks
B. Sullivan, R. Archibald, J. Azadmanesh, V. G. Vandavasi, P. S. Langan, L.
Coates, V. Lynch and P. Langan
J. Appl. Cryst. (2019). 52, 854-863

Pavel

On Tue, Aug 3, 2021 at 4:53 AM Thorn, Dr. Andrea <
andrea.th...@uni-hamburg.de> wrote:

> Dear colleagues,
>
> I have compiled a list of papers that cover the application of AI/machine
> learning methods in single-crystal structure determination (mostly
> macromolecular crystallography) and single-particle Cryo-EM. The draft list
> is attached below.
>
>
>
> If I missed any papers, please let me know. I will send the final list
> back here, for the benefit of all who are interested in the topic.
>
>
>
> Best wishes,
>
>
>
>
>
> Andrea.
>
>
>
>
>
> __
>
> General:
>
> - Gopalakrishnan, V., Livingston, G., Hennessy, D., Buchanan, B. &
> Rosenberg, J. M. (2004). Acta Cryst D. 60, 1705–1716.
>
> - Morris, R. J. (2004). Acta Cryst D. 60, 2133–2143.
>
>
>
> Micrograph preparation:
>
> - (2020). Journal of Structural Biology. 210, 107498.
>
>
>
> Particle Picking:
>
> - Sanchez-Garcia, R., Segura, J., Maluenda, D., Carazo, J. M. & Sorzano,
> C. O. S. (2018). IUCrJ. 5, 854–865.
>
> - Al-Azzawi, A., Ouadou, A., Tanner, J. J. & Cheng, J. (2019). BMC
> Bioinformatics. 20, 1–26.
>
> - George, B., Assaiya, A., Roy, R. J., Kembhavi, A., Chauhan, R., Paul,
> G., Kumar, J. & Philip, N. S. (2021). Commun Biol. 4, 1–12.
>
> - Lata, K. R., Penczek, P. & Frank, J. (1995). Ultramicroscopy. 58,
> 381–391.
>
> - Nguyen, N. P., Ersoy, I., Gotberg, J., Bunyak, F. & White, T. A. (2021).
> BMC Bioinformatics. 22, 1–28.
>
> - Wang, F., Gong, H., Liu, G., Li, M., Yan, C., Xia, T., Li, X. & Zeng, J.
> (2016). Journal of Structural Biology. 195, 325–336.
>
> - Wong, H. C., Chen, J., Mouche, F., Rouiller, I. & Bern, M. (2004).
> Journal of Structural Biology. 145, 157–167.
>
>
>
> Motion description in Cryo-EM:
>
> - Matsumoto, S., Ishida, S., Araki, M., Kato, T., Terayama, K. & Okuno, Y.
> (2021). Nat Mach Intell. 3, 153–160.
>
> - Zhong, E. D., Bepler, T., Berger, B. & Davis, J. H. (2021). Nat Methods.
> 18, 176–185.
>
>
>
> Local resolution:
>
> - Avramov, T. K., Vyenielo, D., Gomez-Blanco, J., Adinarayanan, S.,
> Vargas, J. & Si, D. (2019). Molecules. 24, 1181.
>
> - Ramírez-Aportela, E., Mota, J., Conesa, P., Carazo, J. M. & Sorzano, C.
> O. S. (2019). IUCrJ. 6, 1054–1063.
>
> - (2021). QAEmap: A Novel Local Quality Assessment Method for Protein
> Crystal Structures Using Machine Learning.
>
>
>
> Map post-processing:
>
> - Sanchez-Garcia, R., Gomez-Blanco, J., Cuervo, A., Carazo, J. M.,
> Sorzano, C. O. S. & Vargas, J. (2020). BioRxiv. 2020.06.12.148296.
>
>
>
> Secondary structure assignment in map:
>
> - Subramaniya, S. R. M. V., Terashi, G. & Kihara, D. (2019). Nat Methods.
> 16, 911–917.
>
> - Li, R., Si, D., Zeng, T., Ji, S. & He, J. (2016). 2016 IEEE
> International Conference on Bioinformatics and Biomedicine (BIBM), Vol. pp.
> 41–46.
>
> - Si, D., Ji, S., Nasr, K. A. & He, J. (2012). Biopolymers. 97, 698–708.
>
> - He, J. & Huang, S.-Y. Brief Bioinform.
>
> - Lyu, Z., Wang, Z., Luo, F., Shuai, J. & Huang, Y. (2021). Frontiers in
> Bioengineering and Biotechnology. 9,.
>
> - Mostosi, P., Schindelin, H., Kollmannsberger, P. & Thorn, A. (2020).
> Angewandte Chemie International Edition.
>
>
>
> Automatic structure building:
>
> - Alnabati, E. & Kihara, D. (2020). Molecules. 25, 82.
>
> - Si, D., Moritz, S. A., Pfab, J., Hou, J., Cao, R., Wang, L., Wu, T. &
> Cheng, J. (2020). Sci Rep. 10, 1–22.
>
> - Moritz, S. A., Pfab, J., Wu, T., Hou, J., Cheng, J., Cao, R., Wang, L. &
> Si, D. (2019).
>
> - Chojnowski, G., Pereira, J. & Lamzin, V. S. (2019). Acta Cryst D. 75,
> 753–763.
>
>
>
> Crystallization:
>
> - Liu, R., Freund, Y. & Spraggon, G. (2008). Acta Cryst D. 64, 1187–1195.
>
> - (2004). Methods. 34, 390–407.
>
> - Bruno, A. E., Charbonneau, P., Newman, J., Snell, E. H., So, D. R.,
> Vanhoucke, V., Watkins, C. J., Williams, S. & Wilson, J. (2018). PLOS ONE.
> 13, e0198883.
>
>
>
> Crystal centering:
>
> - Ito, S., Ueno, G. & Yamamoto, M. (2019). J Synchrotron Rad. 26,
> 1361–1366.
>
> - Crystal centering using deep learning in X-ray crystallography.
>
> - Elbasir, A., Moovarkumudalvan, B., Kunji, K., Kolatkar, P. R., Mall, R.
> & Bensmail, H. (2019). Bioinformatics. 35, 2216–2225.
>
>
>
> Diffraction image analysis:
>
> - Czyzewski, A., Krawiec, F., Brzezinski, D., Porebski, P. J. & Minor, W.
> (2021). Expert Systems with Applications. 174, 114740.
>
>
>
> Peak search in serial crystallography:
>
> Ke, T.-W., Brewster, A. S., Yu, S. X., Ushizima, D., Yang, C. & Sauter, N.
> K. (2018). J Synchrotron Rad. 25, 655–670.
>
>
>
> Space group assignment from diffraction image (small molecules):
>
> Aguiar, J. A., Gong, M. L., Unocic, R. R., Tasdizen, T. & Miller, B. D.
> (2019). Science Advances. 5, eaaw1949.
>
>
>
> Data quality assessment in MX:
>
> - Vollmar, M., Parkhurst, J. M., Jaques, D., Baslé, A., Murshudov, 

Re: [ccp4bb] AI papers in experimental macromolecular structure determination

[Pavel Afonine]

Andrea,

you may want to include this one:

Using support vector machines to improve elemental ion identification in
macromolecular crystal structures. Morshed N, Echols N, Adams PD Acta
Cryst. D71, 1147-58 (2015).

Pavel

On Tue, Aug 3, 2021 at 4:53 AM Thorn, Dr. Andrea <
andrea.th...@uni-hamburg.de> wrote:

> Dear colleagues,
>
> I have compiled a list of papers that cover the application of AI/machine
> learning methods in single-crystal structure determination (mostly
> macromolecular crystallography) and single-particle Cryo-EM. The draft list
> is attached below.
>
>
>
> If I missed any papers, please let me know. I will send the final list
> back here, for the benefit of all who are interested in the topic.
>
>
>
> Best wishes,
>
>
>
>
>
> Andrea.
>
>
>
>
>
> __
>
> General:
>
> - Gopalakrishnan, V., Livingston, G., Hennessy, D., Buchanan, B. &
> Rosenberg, J. M. (2004). Acta Cryst D. 60, 1705–1716.
>
> - Morris, R. J. (2004). Acta Cryst D. 60, 2133–2143.
>
>
>
> Micrograph preparation:
>
> - (2020). Journal of Structural Biology. 210, 107498.
>
>
>
> Particle Picking:
>
> - Sanchez-Garcia, R., Segura, J., Maluenda, D., Carazo, J. M. & Sorzano,
> C. O. S. (2018). IUCrJ. 5, 854–865.
>
> - Al-Azzawi, A., Ouadou, A., Tanner, J. J. & Cheng, J. (2019). BMC
> Bioinformatics. 20, 1–26.
>
> - George, B., Assaiya, A., Roy, R. J., Kembhavi, A., Chauhan, R., Paul,
> G., Kumar, J. & Philip, N. S. (2021). Commun Biol. 4, 1–12.
>
> - Lata, K. R., Penczek, P. & Frank, J. (1995). Ultramicroscopy. 58,
> 381–391.
>
> - Nguyen, N. P., Ersoy, I., Gotberg, J., Bunyak, F. & White, T. A. (2021).
> BMC Bioinformatics. 22, 1–28.
>
> - Wang, F., Gong, H., Liu, G., Li, M., Yan, C., Xia, T., Li, X. & Zeng, J.
> (2016). Journal of Structural Biology. 195, 325–336.
>
> - Wong, H. C., Chen, J., Mouche, F., Rouiller, I. & Bern, M. (2004).
> Journal of Structural Biology. 145, 157–167.
>
>
>
> Motion description in Cryo-EM:
>
> - Matsumoto, S., Ishida, S., Araki, M., Kato, T., Terayama, K. & Okuno, Y.
> (2021). Nat Mach Intell. 3, 153–160.
>
> - Zhong, E. D., Bepler, T., Berger, B. & Davis, J. H. (2021). Nat Methods.
> 18, 176–185.
>
>
>
> Local resolution:
>
> - Avramov, T. K., Vyenielo, D., Gomez-Blanco, J., Adinarayanan, S.,
> Vargas, J. & Si, D. (2019). Molecules. 24, 1181.
>
> - Ramírez-Aportela, E., Mota, J., Conesa, P., Carazo, J. M. & Sorzano, C.
> O. S. (2019). IUCrJ. 6, 1054–1063.
>
> - (2021). QAEmap: A Novel Local Quality Assessment Method for Protein
> Crystal Structures Using Machine Learning.
>
>
>
> Map post-processing:
>
> - Sanchez-Garcia, R., Gomez-Blanco, J., Cuervo, A., Carazo, J. M.,
> Sorzano, C. O. S. & Vargas, J. (2020). BioRxiv. 2020.06.12.148296.
>
>
>
> Secondary structure assignment in map:
>
> - Subramaniya, S. R. M. V., Terashi, G. & Kihara, D. (2019). Nat Methods.
> 16, 911–917.
>
> - Li, R., Si, D., Zeng, T., Ji, S. & He, J. (2016). 2016 IEEE
> International Conference on Bioinformatics and Biomedicine (BIBM), Vol. pp.
> 41–46.
>
> - Si, D., Ji, S., Nasr, K. A. & He, J. (2012). Biopolymers. 97, 698–708.
>
> - He, J. & Huang, S.-Y. Brief Bioinform.
>
> - Lyu, Z., Wang, Z., Luo, F., Shuai, J. & Huang, Y. (2021). Frontiers in
> Bioengineering and Biotechnology. 9,.
>
> - Mostosi, P., Schindelin, H., Kollmannsberger, P. & Thorn, A. (2020).
> Angewandte Chemie International Edition.
>
>
>
> Automatic structure building:
>
> - Alnabati, E. & Kihara, D. (2020). Molecules. 25, 82.
>
> - Si, D., Moritz, S. A., Pfab, J., Hou, J., Cao, R., Wang, L., Wu, T. &
> Cheng, J. (2020). Sci Rep. 10, 1–22.
>
> - Moritz, S. A., Pfab, J., Wu, T., Hou, J., Cheng, J., Cao, R., Wang, L. &
> Si, D. (2019).
>
> - Chojnowski, G., Pereira, J. & Lamzin, V. S. (2019). Acta Cryst D. 75,
> 753–763.
>
>
>
> Crystallization:
>
> - Liu, R., Freund, Y. & Spraggon, G. (2008). Acta Cryst D. 64, 1187–1195.
>
> - (2004). Methods. 34, 390–407.
>
> - Bruno, A. E., Charbonneau, P., Newman, J., Snell, E. H., So, D. R.,
> Vanhoucke, V., Watkins, C. J., Williams, S. & Wilson, J. (2018). PLOS ONE.
> 13, e0198883.
>
>
>
> Crystal centering:
>
> - Ito, S., Ueno, G. & Yamamoto, M. (2019). J Synchrotron Rad. 26,
> 1361–1366.
>
> - Crystal centering using deep learning in X-ray crystallography.
>
> - Elbasir, A., Moovarkumudalvan, B., Kunji, K., Kolatkar, P. R., Mall, R.
> & Bensmail, H. (2019). Bioinformatics. 35, 2216–2225.
>
>
>
> Diffraction image analysis:
>
> - Czyzewski, A., Krawiec, F., Brzezinski, D., Porebski, P. J. & Minor, W.
> (2021). Expert Systems with Applications. 174, 114740.
>
>
>
> Peak search in serial crystallography:
>
> Ke, T.-W., Brewster, A. S., Yu, S. X., Ushizima, D., Yang, C. & Sauter, N.
> K. (2018). J Synchrotron Rad. 25, 655–670.
>
>
>
> Space group assignment from diffraction image (small molecules):
>
> Aguiar, J. A., Gong, M. L., Unocic, R. R., Tasdizen, T. & Miller, B. D.
> (2019). Science Advances. 5, eaaw1949.
>
>
>
> Data quality assessment in MX:
>
> - Vollmar, M., Parkhurst, J. M., Jaques, D., Baslé, A., 

Re: [ccp4bb] writing coordinates of full biomol into one (PDB) file

[Pavel Afonine]

Hi Frank,

phenix.pdb.biomt_reconstruction command should do it.

Pavel

On Tue, May 25, 2021 at 12:44 PM Frank von Delft <
frank.vonde...@cmd.ox.ac.uk> wrote:

> Hello all - this presumably has a really simple solution:
>
> For a PDB file with a (correct) biomolecular assembly record (REMARK
> 350), what program do I use to generate and write out the coordinates of
> the biomolecular assembly (or one of them).
>
> Thanks
> Frank
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
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Re: [ccp4bb] phenix refinement for bent DNA

[Pavel Afonine]

Hi Dhiraj,

I have structure with bent DNA. I am trying to refine the structure
> using phenix. do I need to turn off the DNA secondary structure restraints
> during refinement?
>

probably not, unless you have reasons to do so otherwise.

P.S.: There is a Phenix mailing list for Phenix specific questions. This is
CCP4 mailing list!



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Re: [ccp4bb] Student Question--Negative Difference Density in some Histidine side chains in Iron Coordination complex in 2XGF T4 Phage Model Structure

[Pavel Afonine]

Hi,

as others pointed out electron rich elements tend to amplify imperfections
visible in Fo-Fc maps. Consider:
- refining occupancy of Fe, the site may be partially occupied;
- refining f' and f'' if data are anomalous;
- surrounding histidines may 'see' this Fe as a nonbonded interaction and
thus repulsion terms may kick in and push Fe from its position. Try
defining weak coordination bonds between Fe and respective nitrogens;
- refining anisotropic ADP of Fe only.

Good luck!
Pavel

On Mon, Feb 22, 2021 at 7:27 AM Patrick Needham 
wrote:

>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] increased Rfree after ligand fitting refinement

[Pavel Afonine]

Hi Wajahat,

first off, there is a dedicated mailing list for Phenix specific questions (
http://phenix-online.org/).

You need to run more than one refinement cycle before you try making sense
of R factors.

Pavel

On Wed, Jan 20, 2021 at 10:05 AM Wajahat ali khan 
wrote:

> Dear all,
>
>
> I have just started with X-ray diffraction.
> I would appreciate any ideas as to why after one cycle of phenix.refine ,
> the Rfree value increased for my ligand fitted model molecular replacement
> solution model.
>
> Thanks
>
> Wajahat
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
> available at https://www.jiscmail.ac.uk/policyandsecurity/
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Re: [ccp4bb] Rama-Z, Ramachandran plot validation in PDB-REDO

[Pavel Afonine]

Rama-Z is implemented in CCTBX meaning it is available to CCP4 and Phenix.
Also it is reported in validation and refinement in Phenix (you get the
number every time you run real- or reciprocal-space refinement!).
Pavel

On Mon, Jan 18, 2021 at 1:58 PM Boaz Shaanan  wrote:

> Hi,
> Will it be possible to include the rama-z analysis in Coot (perhaps as a
> plugin)?
> Boaz
>
> Boaz Shaanan, Ph.D.
> Department of Life Sciences
> Ben Gurion University of the Negev
> Beer Sheva
> Israel
>
> On Jan 18, 2021 22:47, Robbie Joosten  wrote:
> Dear all,
>
> During the last CCP4 meeting, Oleg presented our collaboration with the
> Phenix team, about the Ramachandran plot Z-score (or Rama-Z). Since then,
> some asked for a convenient way to get this score. You are now welcome to
> use: https://pdb-redo.eu/tortoize
>
> This is a quick and easy way to check you model. Just upload your
> structure model (and restraints if you have non-standard compounds) and
> press "calculate". You get the Ramachandran Z-score with an error margin
> and you get the side-chain equivalent (also known as the Chi-1/Chi-2
> Z-score in WHAT_CHECK) for free.
>
> There are two more ways to get the score, that might be relevant for
> specialized use:
> 1) use the webservice to get the same values and the per-residue values in
> an easy-to-parse JSON file. For example through curl with the command: curl
> -F data=@1cbs_final.pdb -F
> dict=@/zata/ccp4-7.1/lib/data/monomers/a/ALA.cif
> https://pdb-redo.eu/tortoize. Note that the "dict" value is optional. Any
> other POST on https://pdb-redo.eu/tortoize would also work.
>
> 2) to analyse a very large group of models you are encouraged to install
> tortoize locally. It's available on https://github.com/PDB-REDO/tortoize
> with a BSD license.
>
> The scores are also of course available after each PDB-REDO run: the
> Rama-Z is one of the model quality metrics. All  methods take PDB and mmCIF
> formatted files as longs as they are valid, i.e. fit the current format
> specification (mmCIF) or contain at least a CRYST1 record (PDB). As always,
> constructive feedback is appreciated.
>
> If you use Rama-Z, please do cite:
> Sobolev et al. A Global Ramachandran Score Identifies Protein Structures
> with Unlikely Stereochemistry; Structure;
> https://doi.org/10.1016/j.str.2020.08.005
>
> All the best on behalf of Team REDO,
> Robbie
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
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>
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Re: [ccp4bb] how to swap chain IDs

[Pavel Afonine]

Hi Christian,

you can do it in Phenix PDBTools: GUI->Model Tools-> load files then
Options->Other modifications look for Rename chain ID.

Pavel

On Mon, Dec 7, 2020 at 9:49 AM Christian GALICIA <
christian.galicia.diaz.sant...@vub.be> wrote:

> Hello,
> I'm trying to swap the chain IDs of a structure. I tried changing the IDs
> in coot and with PDBset, both change labels of the chains but not the atom
> positions nor numbering.Also, after the pdb file is converted to cif for
> deposition it displays in pymol in the original position making chain A not
> to be at the beginning of the sequence. I would appreciate if you would
> suggest a good way to achieve this. The structure is otherwise finished and
> will no go any other rounds of refinement. Thanks
>
> Christian
> --
> *Christian Galicia*
> E-mail: cgali...@vub.be
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Phenix refine distorting a sidechain despite correct density

[Pavel Afonine]

Hi Folmer,

I would choose to not do the real space refinement in phenix.refine during
> the last rounds of refinement of a model, when sidechain positions are
> essentially correct.
>

by design it is supposed to place and fit side chains as good as possible,
satisfying both map fit and geometry criteria (such as rotameric states and
NCIs). If it does not perform up to your expectations it is best to report
this to us and I will make sure the issue is taken care of immediately. I
wonder if you wouldn't mind explaining your choice and if possible argue it
with specific examples (preferable all done off mailing list as this gets
way too narrow and specific!). -Thanks!

Pavel



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Re: [ccp4bb] over-fitting? over-refinement?

[Pavel Afonine]

Hi Sam,


> Hi, the question may be a bit weird, but how do you define 'over-fitting'
> in the context of structure refinement? From users' perspective the
> practical aspect is to 'fit' the model into the density. So there comes
> this question from our juniors: fit is fit, how is a model over-fit?
>

this is a good question for which there is an answer. I suggest reading
classics on the matter:

https://www.nature.com/articles/355472a0
https://atbweb.stanford.edu/atb_publications/brunger_kleywegt_struct_1996.pdf
https://www.sciencedirect.com/science/article/pii/S0076687997770216
https://pubmed.ncbi.nlm.nih.gov/15299543/
https://journals.iucr.org/d/issues/1998/04/00/ad0030/ad0030.pdf

and numerous references therein. That should set the scene for the next
questions to ask.

Good luck!
Pavel



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Re: [ccp4bb] Tyr pushed out during refinement

[Pavel Afonine]

Hi All,

there was a bug at some point that could potentially lead to this.

This all should be fixed in current Phenix nightly builds:
http://phenix-online.org/download/nightly_builds.cgi

If not, get back to us (phenixbb or Phenix help lists or reply directly to
me).

Pavel

On Mon, Oct 19, 2020 at 11:52 AM Boniecki, Michal 
wrote:

> Hello,
> I have a problem during refining with one of the Tyr residues. It is
> constantly pushed out of the position during refinement in all 4 chains in
> ASU.
> I have tried to exclude it from refinement in phenix but it is refined
> anyway out of the position with wrong geometry. Is there a possibility to
> fix it during refinement?
> The closest contact it has, is 2.4A between hydroxyl group and C=O of Pro,
> but Tyr itself is inside hydrophobic cleft (Ile,Pro,Leu)
> Thank You for all help. Stay safe,
> Michal
>
>
>



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Re: [ccp4bb] Protein-DNA covalent bond refinement

[Pavel Afonine]

Hi Cristina,

in Phenix it is:

Refinement settings -> Select Atoms -> Custom Geometry Restraints : you can
define bonds, angles, torsions, planes, etc...

Pavel

On Thu, Jul 2, 2020 at 8:11 AM Cristina Machon  wrote:

> Dear all,
>
> I am writing regarding a problem we are facing with the refinement of a
> structure. We would really appreciate it if anybody could suggest how to
> set up geometrical restraints for a protein-DNA covalent bond in Refmac or
> Phenix?
>
> Thanks in advance,
>
> Best wishes,
>
> Cristina
>
>
> --
> Cristina Machón Sobrado, PhD
> Instituto de Biología Molecular Barcelona-CSIC
> Parc Científic de Barcelona
> c/ Baldiri Reixac 10
> 08028 Barcelona
> Spain
>
> Phone: +34934034957
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Heavy atom vs light atoms density

[Pavel Afonine]

Hi Vito,

   many of us have probably experienced that, in the
> diffraction of protein ligands containing heavy atoms (cisPt, I3C, etc),
> the overwhelming electron density of the metal can totally flatten that of
> the light atoms around (or rather make it look insignificant).
>

I wonder what FEM map shows in your case (
https://doi.org/10.1107/S1399004714028132)? By design it is supposed to
address exactly this issue.
Pavel



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Re: [ccp4bb] visual mask editor - why

[Pavel Afonine]

Hi Bernhard,

"Like comparing these map regions, excluding

intrusion of a solvent mask, etc.":

You didn't say much about the context.. So I'd say Polder map approach
comes to mind first based on these keywords. Next is "map comparison" (
https://doi.org/10.1107/S1399004714016289).

If none of the above: what we (or you) are missing?

Pavel

On Thu, May 28, 2020 at 11:17 AM Bernhard Rupp 
wrote:

> Maybe I should explain an example: Say coot detects an unmodelled blob
> (maybe a ligand). Now, I would like to do
>
> a number of things without biasing towards a model. Like comparing these
> map regions, excluding
>
> intrusion of a solvent mask, etc.
>
>
>
> Now could coot for example just generate a mask around what it already
> knows are blobs?
>
> Possible useful items could be a solvent mask not including that regions,
> or a density map
>
> that includes only features with a certain boundary around that blob.
>
>
>
> I pilfered some kludges together from different sources, but let’s just
> say inelegant would be a compliment.
>
>
>
> Best, BR
>
> 
>
> Brief question: Does something like a visual density mask editor exist?
>
> Thx, BR
>
> --
>
> Bernhard Rupp
>
> http://www.hofkristallamt.org/
>
> b...@hofkristallamt.org
>
> +1 925 209 7429
>
> +43 676 571 0536
>
> --
>
> Many plausible ideas vanish
>
> at the presence of thought
>
> --
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] How to compare between electron density maps?

[Pavel Afonine]

phenix.match_maps can overlay model B and map B onto model A and map A. A
and B can be any symmetry and box (unit cell) dimensions. Model A and map A
stay in its original frame of reference. Let me know should you have
questions.

Pavel

On Mon, May 11, 2020 at 3:19 PM Murpholino Peligro 
wrote:

> I want to compare electron density features of map A from protein A and
> map B from protein B...
>
> Because each map has a different rmsd level...
>
> ...what is the best way to compare electron density between maps?
>
> Is there a way to normalize maps or something like that?
>
> Thanks
>
> --
>
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Re: [ccp4bb] [3dem] Which resolution?

[Pavel Afonine]

Randy Read's paper in latest Acta D:

Measuring and using information gained by observing diffraction data
http://journals.iucr.org/d/issues/2020/03/00/ba5308/index.html

seems very relevant to this discussion!

Pavel


On Fri, Mar 6, 2020 at 8:44 AM James Holton  wrote:

> Thank you Kay,
>
> Very good points, as always.  I was thinking there must be a better
> apodization filter than cutoffs and B factors.  I'll have to try a
> CC1/2-based roll-off.  But, I wonder if this could be done better on a
> per-reflection basis?  Taking advantage of the sigmas?  I have tried
> using 1/sigma^2 as a weight in map calculation, and that makes the map
> look really weird.  Your idea makes more sense.
>
> On the other hand, the French-Wilson (F) truncation procedure is
> supposed to come up with the maximum-likelihood Fourier coefficient
> given the observed intensity and sigma(intensity).  So, the "F" values
> we get from truncate or xdsconv should already be "good"?  Maybe the
> problem is that we are sharpening after the F step, rather than
> before?  Or maybe the problem is that F bottoms out around F=sig(F).
> Your proposed weight might finish the job...
>
> As for sigmaA, one thing I do know is that refmac5 now uses experimental
> sigmas by default.  Phenix.refine does not.
>
> One thing is for sure, sharpening the data before refinement is not
> going to become a popular strategy.  This is because pre-sharpened data
> will make Rwork and Rfree higher than they are with the "natural" B
> factor.  You can also make your Rwork Rfree much lower by applying a
> positive B factor to your data before starting refinement.  This comes
> with absolutely no improvement in model quality, so please don't try
> this at home.
>
> -James Holton
> MAD Scientist
>
> On 3/5/2020 9:38 PM, Kay Diederichs wrote:
> > Dear James,
> >
> > important and educational points! This triggers some thoughts ...
> >
> > The one point where I don't quite agree is with "What about filtering
> out the noise?  An ideal noise suppression filter has the same shape as the
> signal (I found that in Numerical Recipes), and the shape of the signal
> from a macromolecule is a Gaussian in reciprocal space (aka straight line
> on a Wilson plot). This is true, by the way, for both a molecule packed
> into a crystal or free in solution.  So, the ideal noise-suppression filter
> is simply applying a B factor.  "
> >
> > I think we can do better than that. We should use the knowledge about
> the actual signal and its noise (which we measure) as a weighting factor,
> rather than that of the theoretical signal (the straight line in the Wilson
> plot), for the purpose of noise suppression. Formula 13.3.6 in Numerical
> Recipes (3rd ed., 2007), which gives the optimal (Wiener) filter to be used
> for weighting, is phi(f) = S(f)^2/(S(f)^2 + N(f)^2)  . But at high
> resolution, this is just CC1/2: see Box 1 of reference (1) - the formula
> CC1/2 = 1/(1 + 2/(I/sigma)^2 can be written as CC1/2 = ^2/(^2 + 2
> ^2) where sigma is the estimate of the noise in I ; don't know right
> now why there is a factor of 2).  This goes to zero at the resolution where
> the signal goes to zero, and is near one in the resolution range in which
> we have good knowledge of the signal.
> > (I only thought about this today, and I also considered CC* as a
> weighting factor, as I understand is suggested by Rosenthal and Henderson,
> J.Mol.Biol. 2003, but I cannot convince myself currently that this is
> right. Anyway, the shape of the CC* curve as a function of resolution
> matches that of CC1/2)
> > In other words, we should be able to suppress the noise by multiplying
> the Fourier coefficients used for map calculation with (a smooth
> resolution-dependent approximation of) CC1/2. This should allow to sharpen,
> with the best noise suppression we can get.
> >
> > Thinking about this, we are already typically using weighted Fourier
> coefficients of the form 2mFobs-DFcalc for map calculation. Aren't these
> already weighted in the correct way? I think not - those m and D weights
> are calculated from estimates of model (in-)accuracy and (in-)completeness,
> but don't properly take the measurement errors into account. Of course,
> since noisy data make the sigmaA values worse, the noise in the data
> influences sigmaA, but not in the functionally correct form. To my
> understanding, the correct way to take account of both model and data
> errors is given by reference (2), which - to my knowledge - is not yet
> implemented except in PHASER.
> >
> > Hope this makes sense!
> >
> > Kay
> >
> > References:
> > (1) Karplus & Diederichs (2015) Assessing and maximizing data quality in
> macromolecular crystallography.
> > Curr. Opin. Struct. Biol. 34, 60-68 . PDF at
> https://www.biologie.uni-konstanz.de/typo3temp/secure_downloads/82815/0/2b10c9e6f9a28129e1b119d21aeeab217c918bb1/Karplus2015_CurrOpinStructBiol.pdf
> > (2) RJ Read, AJ McCoy (2016) A log-likelihood-gain intensity target for
> 

Re: [ccp4bb] Hydrogens in PDB File

[Pavel Afonine]

Clearly, it is a good idea to keep hydrogens:

http://phenix-online.org/presentations/hydrogens.pdf

Not sure why this keeps coming up as a topic given how much it was said
about it in the past, all the MolProbity arguments, etc..

Issue of missing side chains and loops is tricker indeed.

Pavel

On Mon, Mar 2, 2020 at 11:33 AM Dale Tronrud  wrote:

> On 3/2/2020 10:12 AM, Alexander Aleshin wrote:
> > Dear Dale,
> > You raised a very important issue that has been overly ignored by the
> crystallographic community. The riding hydrogens are just a tip of an
> iceberg. It is absolutely unclear even to an experienced crystallographer
> how to treat poorly ordered side chains or even whole residues. As a matter
> of fact,  their models are "riding atoms", and consumers have no clue how
> much they can trust our modeling.
>
>Oh no!  Now I've opened up this can of worms.
>
>The matter of describing completely disordered side chains has been
> discussed heavily on this BB, along with the advantages and shortcomings
> of overloading the meaning of "B factor" or "Occupancy" to describe this
> situation.
>
>Using one data item to describe multiple things is never a good idea,
> in my opinion.  The move to mmCIF for model storage does open the
> possibility of adding new tags to uniquely describe model properties.
> Creating such a tag for "place-holder" side chain atoms was one of the
> recommendations in "Outcome of the First wwPDB/CCDC/D3R Ligand
> Validation Workshop" (https://www.ncbi.nlm.nih.gov/pubmed/27050687).  I
> don't know the status of the implementation of any of these
> recommendations.  The wheels of the wwPDB grind exceedingly slowly.
>
>This is just another part of the huge problem of describing the
> nature of the deposited model and the origin of the information
> supporting all of its parts.
>
> 1) Riding Hydrogen atoms vrs free-floating and refined
> 2) Placeholder side chains vrs visible in density
> 3) Placeholder loops vrs visible in density
> 4) TLS anisotropic B's vrs restrained individual atom aniso vrs
> unrestrained individual - The options here are many and multiple types
> may be present in one model
> 5) NCS restraint/constraint - The options here are many and multiple
> types may be present in one model
> 6) Concerted alternative conformation spread over multiple residues
> 7) Sequence heterogeneity
>
> These are just the topics that bother me with almost every model I
> download.  I'm sure there are plenty of other topics that don't come to
> mind at the moment.
>
>With the move to mmCIF we now have the opportunity to create
> descriptions of these model properties w/o just adding more and more
> REMARK cards.
>
>Until that wondrous day arrives we are stuck with trying to create
> models that are useful to the general community and provide minimal
> opportunity for confusion.  We can argue as to where that line is, but
> shouldn't loose sight of the ultimate goal.
>
>Ethan and I disagree over the relative damage caused by the inclusion
> of riding hydrogen atom positions vrs the confusion that results from
> their absence.  I think we agree strongly that all of the list items
> above need to be tackled by the wwPDB and are of extreme importance.  I
> think we need a comprehensive solution, not a piecemeal, special case,
> for each.
>
> Dale Tronrud
>
> > Moreover, some programs (including the version of Pymol that I use), get
> confused when they detect residues with multiple conformations. Like my
> Pymol version fails to build cartoon elements in those areas, and it is not
> obvious for a beginner how to remove the alternative conformations. I
> presume many consumers just ignore such structures and pick up analogues
> that are displayed without problems.
> >
> > Pymol developers, is it so difficult to report a user, when a structure
> is loaded that it has residues with alternative conformations, and one of
> conformers should be hidden for a correct presentation of the secondary
> structure elements?
> >
> > Alex
> >
> >
> >
> > On 3/2/20, 12:57 AM, "CCP4 bulletin board on behalf of Dale Tronrud" <
> CCP4BB@JISCMAIL.AC.UK on behalf of de...@daletronrud.com> wrote:
> >
> > [EXTERNAL EMAIL]
> >
> > Dear Tim,
> >
> >I am in agreement with Ethan and you that a complete description
> of
> > the restraints and constraints applied to the model should be
> included
> > in the deposition.  This is currently a major failing of the wwPDB.
> For
> > hydrogen atoms we, at least, have the "Riding hydrogen atoms were
> added"
> > remark but that simple statement is inadequate to allow anyone (or
> > program) to reproduce what the depositor had on disk before the
> hydrogen
> > atoms were redacted.  We know that shelxl and MolProbity produce
> > hydrogen models that differ, and that shelxl requires additional
> > information about the temperature of the molecule at least.
> >
> >How could someone hope to 

Re: [ccp4bb] [3dem] [ccpem] Which resolution?

[Pavel Afonine]

Interesting conversation! I see the 2017 paper is on bioRxiv. I wonder if
it ever made into a peer reviewed journal (couldn't find quickly)?
@Tim Gruene  : have a look at d_model in
https://www.ncbi.nlm.nih.gov/pubmed/30198894 which is sort of along similar
lines of what you are hinting here.
Pavel

On Wed, Feb 12, 2020 at 3:07 PM Marin van Heel <
057a89ab08a1-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi Tim,
> Good to hear from you!  No longer at PSI??
> See... You are already touching upon one of the logical breaking points in
> the resolutiton story...!  X-ray crystallography resolution criteria like
> R-factors make absolutely no sense outside the field of crystallography and
> of structural biology.  It is the result of a hybrid iterative optimisation
> process between the phases of a model structure and the measured amplitudes
> of a diffraction experiment!  The FRC/FSC resolution criteria, in contrast,
> are universal quality metrics not at all coupled to Cryo-EM or structural
> biology.  Using structural biology arguments like how well I see an alpha
> helix or how well I see the hole in an aromatic ring as an assessment
> criterion of whether a metric is good or not is a waste of time!  (Moreover
> filtering a map can completley change its appearance without changing its
> information contents). Even some my own (ex-)students and (ex-)postdocs
> sometimes completely miss this fundamental point. The FRC and FSC criteria
> are now used as quality metrics in all walks of image science like X-ray
> tomography and super-resolution light microscopy, fields of science where
> atomic coordinates of proteins are not an issue. The FRC / FSC functions
> are universal and very direct metrics that compare both the amplitudes and
> the phases of two independent measurements of images or 3D-densities of the
> same object. For more details, see the 2017 bioRxiv paper and references
> therein (https://www.biorxiv.org/content/10.1101/224402v1) and check my
> #WhyOWhy tweets (@marin_van_heel). See also: van Heel - Unveiling
> ribosomal structures: the final phases - Current opinion in structural
> biology 10 (2000) 259-264.
>
> Cheers,
> Marin
>
>
> On Wed, Feb 12, 2020 at 11:22 AM Tim Gruene 
> wrote:
>
>> Dear Marin,
>>
>> I did not read the enire thread, nor the manuscript you point at -
>> apologize
>> in case this has been discussed before.
>>
>> What about a practical approach to determine the resolution of a cryoEM
>> map:
>> one could take a feature with scales of interest, e.g. an alpha-helix,
>> and
>> shift and/or rotate it in steps of, say, 0.3A in several directions to
>> see, at
>> which magnitude (degree / distance) refinement does not take the helix
>> back to
>> its original position (within error margins).
>>
>> One could also take a Monte-Carlo approach and do an arbitrary number of
>> random re-orientations of such a helix, refine, and calculate the
>> variation in
>> position and rotation.
>>
>> This would reflect my understanding of resolution, much more than any
>> statistical descriptor.
>>
>> Best regards,
>> Tim
>>
>> On Wednesday, February 12, 2020 1:46:48 PM CET Marin van Heel wrote:
>> > Hi Laurence,
>> >
>> > One thing is certain: the 0.143 threshold is RUBBISH and all CC50 etc
>> are
>> > also based on the same SLOPPY STATISTICS  as are all  fixed-valued  FSC
>> > thresholds. This controversy has been ragings for a long long time and
>> the
>> > errors made were extensively described (again) in our most recent paper
>> > (Van Heel & Schatz 2017 BioRxiv:
>> > https://www.biorxiv.org/content/10.1101/224402v1) which has been
>> downloaded
>> > more than 3000 times. Further papers on the issue are in the pipeline.
>> The
>> > math BLUNDER behind this controversy is simple:  the inner product
>> between
>> > a signal vector and a noise vector is NOT zero (but rather proportional
>> to
>> > SQRT(N) where N is the length of the vectors) and cannot be left out of
>> the
>> > equations. This error goes back to a paper published in Nature in 1975
>> and
>> > has since been repeated frequently, including in the first paper
>> promoting
>> > the erroneous 0.143 FSC threshold. The consequences of this blunder in
>> > current processing are serious especially when these erroneous metrics
>> are
>> > used as an optimisation criterion in iterative refinements at
>> resolutions
>> > close to Nyquist.  I get tired of facing this systematic misuse of the
>> FSC
>> > function, which I myself have introduced into the literature in
>> 1982/1986,
>> > and people nevertheless feel they know better (with no scientific
>> arguments
>> > to support!) and they feel justified to use it beyond its definition
>> range,
>> > and to continue to ignore the correct math. To counter this systematic
>> > abuse of my brain child - over decades - I feel the need to use CLEAR
>> > LANGUAGE!
>> > Have fun!
>> > Marin
>>
>> --
>> --
>> Tim Gruene
>> Head of the Centre for X-ray Structure Analysis
>> Faculty of Chemistry
>> 

[ccp4bb] Phenix workshop at ACA 2020 (San Diego)

[Pavel Afonine]

Dear Colleagues,

please make a note of upcoming Phenix workshop focusing on crystallography
and Cryo-EM tools for structure solution, August 2nd 2020 in San Diego,
California. This is a day-long satellite workshop prior to ACA meeting. For
schedule and registration see ACA 2020 web site: www.acameeting.com
  (Program -> Workshops).

Looking forward to see you there!
Pavel



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Re: [ccp4bb] refinement of 0.73A data in shelxl

[Pavel Afonine]

Hi Matthias,

did you use correct model parameterization and optimal refinement strategy
for the resolution? Such as:
- Add H atoms;
- Refine all but H atoms with anisotropic ADPs;
- Model alternative conformations (that one'd expect many at this
resolution);
- Add solvent (water, crystallization cocktail components if you see any);
- Relax restraints on geometry and ADPs;
 long list!

If not, then what you have in terms of R factors is more or less what I'd
expect.

In the absence of obvious data pathologies, I'd expect Rwork/Rfree in
10-15% range, and the Rfree-Rwork gap around 1-2% or less.

Since you mentioned Phenix refinement, I am happy to help you with details
etc off-list.

Pavel

On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias 
wrote:

> Dear ccp4 community
>
> Im having some problems solving a 0.73A structure. Spacegroup seems to be
> correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer
> shell CC1/2 24% and 90.4% complete.
>
> The model is nearly fully built, there is no remaining unmodelled areas.
> However, Rfact is stuck 27% in phenix, with a very distinct artifact in
> the electron map (see phenix.jpg). You can see difference density on
> various well defined sidechain atoms. Notably, they seem to follow a
> pattern: Nearly all Val CG have difference signal, as well as many backbone
> NH. Hence, I suspected that it might be a problem with the SF, since we
> recorded the DS at 0.86A.
>
>
> Hence I gave shelxl a shot:
>
> I used the refined model from phenix, converted it via pdb2ins and pasted
> the restraints created by prodrg.
>
> The shelxl hkl was produced by xdsconv, using the freeR flagging of the
> mtz used by phenix (no merge, friedel false).
>
> Interestingly, shelxl can bring Rfree down to 16% and almost all of
> the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg).
> Except one: the inhibitor contains a chlorinated phenylring (pdb ligand
> 2L5) which now shows massive difference density for Cl.
>
> I therefore suggested that I might deal with a wrong SF for Cl. Funny
> enough, pdb2ins does not produce a DISP line for Cl if converting the pdb
> that contains the inhibitor. Hence, I used pdb2ins and the pdb from
> PRODRG to produce SFAC for the inhibitor Cloride. I then pasted this line
>
> DISP $CL0.188450.21747   1035.16450
>
> into the .res file and updated the UNIT line. Shelxl runs through, and
> the density looks ok on the Chloride now. However Rfree is back up at 24%
> and the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg):
> now, very distincitvly, backbone carbonyls and NHs show difference density.
>
> Am I right in my assumption, that the SFAC of Cloride is not properly
> calculated at the given wavelenght? And if so, how do I guess it correctly?
>
>
> Thank you very much for your help!
>
> Best, matthias
>
>
>
>
> Dr. Matthias Barone
>
> AG Kuehne, Rational Drug Design
>
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
>
> Germany
> Phone: +49 (0)30 94793-284
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
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Re: [ccp4bb] Examples of EM models at 4.0Å or better with serious modelling errors?

[Pavel Afonine]

Dear Gerard,

a possible programmatic approach may be a loop over all (model, map,
resolution) or (model, map, half_map1, half_map2, resolution) from PDB/EMDB
and calling

phenix.validation_cryoem model. emdb_.map resolution=value
> .log
or
phenix.validation_cryoem model. emdb_.map
half_map1.map half_map2.map resolution=value > .log

The log file will have things like overall model counts:

DEVIATIONS FROM IDEAL VALUES.
  BOND  :  0.031   1.220   4166
  ANGLE :  3.271 102.317   5611
  CHIRALITY :  0.085   0.434629
  PLANARITY :  0.018   0.270730
  DIHEDRAL  : 28.082 179.891   1579
  MIN NONBONDED DISTANCE : 1.223

MOLPROBITY STATISTICS.
  ALL-ATOM CLASHSCORE : 20.08
  RAMACHANDRAN PLOT:
OUTLIERS :  3.29 %
ALLOWED  :  9.69 %
FAVORED  : 87.02 %
  ROTAMER OUTLIERS : 24.26 %
  CBETA DEVIATIONS :  0.00 %
  PEPTIDE PLANE:
CIS-PROLINE : 0.00 %
CIS-GENERAL : 1.59 %
TWISTED PROLINE : 0.00 %
TWISTED GENERAL : 1.39 %

and model-to-map fit:

  CC_mask  : 0.8818
  CC_box   : 0.7657

Also it checks the sanity of HELIX/SHEET records.

Then parsing these logs and taking top N entries having the worst metric
you focus on (like clashscore or map-model CC or anything else you choose)
will likely give you some good candidates for your tests. Of course this
won't help you with local modeling issues such as wrong register or similar.

Instead of dealing with text log files you can use pickle=true keyword in
the above commands. Then a Python pickle file will be created that contains
all the information. Once you iterated over all entries, then you can load
these pickle files and harvest information you need.

Also, you may find some examples here (but probably not a lot individual
ones):
http://journals.iucr.org/d/issues/2018/09/00/kw5139/index.html

I can help more with details, if needed.

Good luck!
Pavel

On Tue, Dec 10, 2019 at 5:26 AM Gerard DVD Kleywegt 
wrote:

> Dear colleagues,
>
> We are developing a new validation method that takes EM maps and models
> into
> account. In order to understand the potential, the applicability and the
> limitations of the method we are looking for good test cases, which turn
> out
> to be surprisingly hard to find.
>
> What we are looking for are EM structures with serious modelling errors,
> e.g.
> register errors (model sequence out-of-register with the map),
> connectivity
> errors (between (non-)adjacent secondary structure elements),
> directionality
> errors (e.g., a helix built backwards), substantial stretches of mistraced
> residues, etc.
>
> The structures should be publicly available in PDB (model) and EMDB (map),
> they should be at 4.0Å resolution or better, and ideally there should be a
> higher resolution improved structure (EM or X-ray).
>
> If the modelling errors have been confirmed and discussed in the
> literature
> that is ideal, but we are also interested if this is not the case.
>
> Feel free to reply either in confidence to me (mailto:ger...@ebi.ac.uk),
> or to
> the entire list.
>
> Many thanks in advance for any examples you can think of!
>
> PS: apologies for cross-posting to two other mailing lists.
>
> --Gerard
>
> ---
> Gerard J. Kleywegt, EMBL-EBI, Hinxton, UK
> Head of Molecular and  Cellular Structure
> ger...@ebi.ac.uk pdbe.org emdb-empiar.org
> PA: Roisin Dunloppdbe_ad...@ebi.ac.uk
>
> 
>
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Re: [ccp4bb] TLS parameters

[Pavel Afonine]

Dear Eleanor,

Phenix reads and writes TLS records, both PDB and mmCIF format. It can also
read (but not write) TLS records in REFMAC and BUSTER format.

In ATOM records Phenix outputs complete B factors, which includes both
individual and TLS components (this is why they have ANISOU).

Phenix won't re-define (or define from scratch) TLS groups automatically
unless it is asked to do so.

Pavel

On Tue, Nov 19, 2019 at 7:36 AM Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> And what about PHENIX?
> E
>
> On Tue, 19 Nov 2019 at 15:33, Pierre Rizkallah 
> wrote:
>
>> I can vouch for TLS blocks being used by REFMAC and the PDB validation
>> servers, after some frustration I had with a deposition recently:
>> Towards the end of a refinement, I renamed some chains, to make oligomers
>> in the a.u. contiguous in real space. Validation told me the centre of
>> gravity as declared in the header is not the same as that produced from the
>> coordinates. I edited the input TLS file, but it still produced the same
>> outcome. I eventually realised that REFMAC reads the TLS blocks from the
>> input PDB after reading all the other input instructions for the refinement
>> run. This happening last, it overrides the declarations in the input TLS
>> definitions file. In order to get the coordinates and the definitions to
>> match, I had to remove the TLS blocks, produced by an earlier run of
>> REFMAC, from the pdb input file, so that the new definitions can be
>> followed, and appropriate TLS blocks produced. REFMAC would use
>> pre-existing TLS blocks if they are in the PDB file.
>>
>> The mismatch notwithstanding, REFMAC still worked correctly, although the
>> shifts from the group origins would have looked strange if one tries to
>> analyse the TLS motions with TLSANL. I admit, I don't view them. Moral of
>> the story is, be careful when you rename chains at the end of the
>> refinement!
>>
>> Pierre Rizkallah
>> ***
>> Dr Pierre Rizkallah, Senior Lecturer Structural Biology
>> Institute of Infection & Immunology, Sir Geraint Evans Building,
>> School of Medicine, Heath Campus, Cardiff, CF14 4XN
>> email: rizkall...@cardiff.ac.ukphone: +44 29 2074 2248
>> http://www.cardiff.ac.uk/people/view/126690-rizkallah-pierre
>>
>> -Original Message-
>> From: CCP4 bulletin board  On Behalf Of Robbie
>> Joosten
>> Sent: 19 November 2019 15:14
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] TLS parameters
>>
>> Hi Eleanor,
>>
>> The blocks are reliably recorded in PDB entries but in some cases the
>> renumbering of residues was not pushed through to TLS groups. Certain
>> selections cannot be captured in the PDB format, for instance the split in
>> main chain and side chain that Refmac allows. Fortunately that feature is
>> hardly used.
>> Parsing TLS records is not straightforward, particularly the sets from
>> Buster suffered a lot from inconsistent manual editing in the early days of
>> TLS refinement. PDB-REDO's extractor does a decent job in getting
>> selections and changing those into Refmac format, but there are definitely
>> cases that it cannot do. We also have a tool that does this for mmCIF files
>> which is not written by me and (therefore) much more sophisticated in
>> handling more complicated cases.
>>
>> Cheers,
>> Robbie
>>
>> > -Original Message-
>> > From: CCP4 bulletin board  On Behalf Of Eleanor
>> > Dodson
>> > Sent: Tuesday, November 19, 2019 3:59 PM
>> > To: CCP4BB@JISCMAIL.AC.UK
>> > Subject: [ccp4bb] TLS parameters
>> >
>> > Does anyone know how reliably the different programs record and use
>> > these blocks from the PDB file?
>> >
>> > Eleanor
>> >
>> > 
>> >
>> >
>> > To unsubscribe from the CCP4BB list, click the following link:
>> > https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
>> > jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1data
>> > =01%7C01%7Crizkallahp%40CARDIFF.AC.UK%7Cf2a9834dd919481f28d508d76d030e
>> > 63%7Cbdb74b3095684856bdbf06759778fcbc%7C1sdata=BMwtQsLcQzHTeZcKqC
>> > Yg5W7MLDM7Phk0MUx8ohylApI%3Dreserved=0
>>
>>
>> 
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>>
>> https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1data=01%7C01%7Crizkallahp%40CARDIFF.AC.UK%7Cf2a9834dd919481f28d508d76d030e63%7Cbdb74b3095684856bdbf06759778fcbc%7C1sdata=BMwtQsLcQzHTeZcKqCYg5W7MLDM7Phk0MUx8ohylApI%3Dreserved=0
>>
>> 
>>
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>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> 

Re: [ccp4bb] Calculating RMSD of a loop

[Pavel Afonine]

Yet another way is:

phenix.superpose_pdbs fixed.pdb moving.pdb selection_fixed="chain A and and
resseq 1:10 and name CA" selection_moving="chain B and resseq 1:10 and name
CA"

or using the GUI.
Pavel

On Tue, Sep 17, 2019 at 8:06 AM Folmer Fredslund  wrote:

> Dear Kyle,
>
> As other non-CCP4 solutions have also been suggested, perhaps I can
> suggest using PyMOL?
> https://pymolwiki.org/index.php/Align
> Here's a nice wiki article about what you can do with the align command.
>
>
> If you're already familiar with scripting languages it's quite easy, and
> you can load your already superimposed structures and calculate on the
> selection you want.
>
>
> Hope this helps,
> Folmer Fredslund
>
>
> tir. 17. sep. 2019 15.42 skrev Kyle Gregory <
> 3632e92fcc15-dmarc-requ...@jiscmail.ac.uk>:
>
>> Hi all,
>>
>> What is the best/easiest way to calculate RMSD of a loop for 2 c-alpha
>> aligned structures?
>> Thought I could do this in Coot but I only see this if I align the
>> specific loops, which I don't want to do.
>>
>> Thanks,
>>
>> Kyle
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
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Re: [ccp4bb] I am doing phenix refinement now. Is it a problem if the r-work and r-free values are equal?

[Pavel Afonine]

Thanks for sharing log file. The answer is -- there are no valid free r
flags in your data file:

Number of work/free reflections by resolution:
 work  free  %free
  bin  1: 52.1844 -  3.0380 [4249/4297]  4249 0   0.0%
  bin  2:  3.0380 -  2.4114 [3632/4074]  3632 0   0.0%
  bin  3:  2.4114 -  2.1065 [3304/4043]  3304 0   0.0%
  bin  4:  2.1065 -  1.9139 [2664/3989]  2664 0   0.0%
  bin  5:  1.9139 -  1.7767 [1763/3961]  1763 0   0.0%
  bin  6:  1.7767 -  1.6720 [1304/3975]  1304 0   0.0%
  bin  7:  1.6720 -  1.5882 [ 891/3947]   891 0   0.0%
  bin  8:  1.5882 -  1.5191 [ 571/3912]   571 0   0.0%
  bin  9:  1.5191 -  1.4606 [ 293/3936]   293 0   0.0%
  bin 10:  1.4606 -  1.4102 [ 130/3913]   130 0   0.0%
overall 18801 0   0.0%

That's what log file is for: to check for obvious issues like this one!
Pavel

On Fri, Aug 30, 2019 at 7:59 AM Pavel Afonine  wrote:

> Please send me log file off-list and that may be a start. -Thanks!
> Pavel
>
> On Fri, Aug 30, 2019 at 6:48 AM Tung Thanh Dinh  wrote:
>
>> After phasing with phenix, i clicked on the phenix.refine ribbon. Since
>> then for every macrocycle of refinement, r-work and r-free values are
>> always the same. Is this a problem that I need to fix?
>>
>>
>> Kindest regards,
>> Tung Dinh
>>
>> PhD student
>> The University of Georgia
>> Department of Chemistry
>>
>> --
>>
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Re: [ccp4bb] I am doing phenix refinement now. Is it a problem if the r-work and r-free values are equal?

[Pavel Afonine]

Please send me log file off-list and that may be a start. -Thanks!
Pavel

On Fri, Aug 30, 2019 at 6:48 AM Tung Thanh Dinh  wrote:

> After phasing with phenix, i clicked on the phenix.refine ribbon. Since
> then for every macrocycle of refinement, r-work and r-free values are
> always the same. Is this a problem that I need to fix?
>
>
> Kindest regards,
> Tung Dinh
>
> PhD student
> The University of Georgia
> Department of Chemistry
>
> --
>
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> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] How to include in refinement high resolution shells with VERY low completeness ?

[Pavel Afonine]

Hi Ivan,


> My conclusion is that  "no fill-in" option might be tried at some
> stages, but with caution, especially for datasets with poor low res
> completeness.
>

I'm guessing you really meant *high*, not low. More or less repeating what
others said already.. Correcting for low res completeness doesn't add
high-res details and so has lesser risk of introducing model bias at atomic
level. Contrary, filling in high-res terms is likely to noticeably bias the
map at atomic level of detail.

Here is one of my favorite examples of what missing low-res data can do to
your map:
http://cci.lbl.gov/~afonine/tmp/1nh2.pdf

Pavel



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Re: [ccp4bb] How to include in refinement high resolution shells with VERY low completeness ?

[Pavel Afonine]

For the record, phenix.refine always produces two versions of 2mFo-DFc
maps, with and without filling in missing Fobs. The one that opens in Coot
by default is the "filled" map, but you can always load the other one for
comparisons.

Pavel

On Tue, Aug 6, 2019 at 8:34 AM Ivan Shabalin 
wrote:

> Dear Clemens,
>
> Thanks!
>
> It sounds like a good test to do on the output .mtz and see if there are
> any significant changes in maps after these "filled in" coefficients are
> removed.
>
> Ivan
>
> With best regards,
> Ivan Shabalin, Ph.D.
> Research Scientist,
> Department of Molecular Physiology and Biological Physics,
> University of Virginia,
> 1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
> Charlottesville, VA 22908
> https://www.linkedin.com/in/shabalinig/
> https://minorlab.org/person/ivan_s/
>
> On 8/6/19 08:59, Clemens Vonrhein wrote:
> > Dear Ivan,
> >
> > On Mon, Aug 05, 2019 at 05:44:56PM -0400, Ivan Shabalin wrote:
> >> Dear Kay,
> >>
> >> Thanks a lot for your answers!
> >>
> >> To my best understanding, REFMAC does not have an option of for
> restoring
> >> reflections only in certain resolution shells.
> >
> > Remember that you can always do some simple (but powerfull) operations
> > within SFTOOLS (*). So using something like
> >
> >read refmac.mtz
> >CALC col resol = 0.5 stol /
> >select col FP = absent
> >absent col FWT  if col resol < 3.0
> >absent col PHWT if col resol < 3.0
> >delete col resol
> >select all
> >write refmac_new.mtz
> >
> > might/should do the trick: setting all filled-in FWT/PHWT values to a
> > MNF (missing-number-flag) for reflections with resolution higher than
> > 3A. So you should only have filled-in 2mFo-DFc valeus for the low-res
> > data (3A and lower).
> >
> > But check those commands above (typed from memory)
> >
> > Cheers
> >
> > Clemens
> >
> > (*) there is very little one can't do with SFTOOLS
> >
>
> 
>
> 
>
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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Pavel Afonine]

Hard to see from a static image, but could it be an alternative
conformation?
Pavel

On Tue, Jul 9, 2019 at 2:32 AM Lumbini Yadav  wrote:

> Dear all,
>
>
>
> We have found a huge Fo-Fc density close to cysteine residue (see attached
> image) in the structure with resolution of 1.2A. In the crystallization
> condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and
> protein was in Tris and NaCl. Before freezing the crystals were soaked in
> mother liquor containing sodium dithionite.
>
>
>
> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in
> all the screenings we do see some part of Fo-Fc density unaddressed at 3
> sigma.
>
>
>
> Does anyone have an idea about what this density could be? Covalent
> modification?
>
>
>
> Thanks.
>
>
>
> Kind regards,
>
> Lumbini
>
> --
>
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Re: [ccp4bb] resolution

[Pavel Afonine]

Hi Sam Tang,

Sorry for a naive question. Is there any circumstances where one may wish
> to refine to a lower resolution? For example if one has a dataset processed
> to 2 A, is there any good reasons for he/she to refine to only, say 2.5 A?
>

yes, certainly. For example, when information content in the data can
justify it.. Randy Read can comment on this more! Also instead of a hard
cutoff using a smooth weight based attenuation may be even better. AFAIK,
no refinement program can do this smartly currently.
Pavel



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Re: [ccp4bb] Phenix / Coot neutron queries.

[Pavel Afonine]

Hi Jonathan,

send me files off list and I will have a look. From your description it
isn't clear to me what the problem is. You need to add H or D or H and D
only once (and phenix.ready_set is the right tool to do it!), then just do
refinement and all should work. Coot indeed may not play well with some of
exotic scenarios but so far that hasn't been a huge issue as phenix.refine
can handle this mostly automatically.

Pavel

On Thu, Jun 20, 2019 at 5:44 PM Jonathan Cooper <
0c2488af9525-dmarc-requ...@jiscmail.ac.uk> wrote:

> I am trying to refine a neutron structure for someone and I have come
> across a couple of things which I need help with.
>
> I am struggling to get the occupancy refinement of the
> hydrogens/deuteriums on the N-terminal nitrogens to behave right. Some of
> them get deleted by readyset but the trick seems to be to call them
> hydrogen in the atom name field yet say they are D in the element symbol
> field. Is that the best way?  Also, readyset seems to delete the D's in D2O?
>
> I would appreciate any tips on what is the best 'strategy' for refining
> with neutron data i.e. reciprocal- versus real-space or both, etc, because
> my R-free just seems to go up.
>
> Also, if I do a real-space refine on a D2O molecule (DOD) in Coot it
> stretches the O-D bond length from around 1 to 1.3 Angstrom and the bond
> angle from ~110 to about 120 degrees so it seems to be picking-up wrong
> geometry info from somewhere.
>
> --
>
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Re: [ccp4bb] Disulphide occupancies.

[Pavel Afonine]

Hi Jonathan,

this may also be a result of too strong SS bond restraints or/and
inaccurate SS bond restraints parameters or/and disorder (in some fraction
of unit cells there is no SS bond). More on the topic:

"Disulfide bond restraints" here:
http://phenix-online.org/newsletter/CCN_2015_01.pdf#page=13

Pavel

On Mon, May 27, 2019 at 6:20 AM Jonathan Cooper <
0c2488af9525-dmarc-requ...@jiscmail.ac.uk> wrote:

> When you refine structures with disulphide bridges you often get negative
> difference density for the sulphurs, presumably due to the well-known
> radiation damage effects. The negative difference density often won't
> disappear with usual B-factor refinement. However, it seems to go away if
> you refine the occupancy of the affected sulphur atoms e.g. to 0.9 or
> thereabouts. Would it be acceptable to publish/deposit structures where the
> sulphur occupancy is less than one, given a suitable REMARK in the pdb
> file? Thank you.
>
> --
>
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Re: [ccp4bb] On estimating Unit Structure Factor distribution

[Pavel Afonine]

Hi Andre,

here is the link to cctbx-based code that computes Uhkl according to your
formula below, using model mean B and F000 that accounts for atomic model
and bulk-solvent:

https://www.dropbox.com/sh/g7sp7pqxst4ldj0/AAD1whlVD2mvAGoRa5jOF-fla?dl=0

I leave it up to you to read and understand the script, as well as ensuring
it works as you expect (bug-free!). I hope it exemplifies enough for you to
pick up from there and modify it the way you wish. The only test I did is I
made sure it runs and outputs and MTZ file!

Good luck,
Pavel

On Wed, May 15, 2019 at 3:20 PM Andre LB Ambrosio  wrote:

> Dear all,
>
> We seek to calculate the distribution of Unitary Structure Factors, Uhkl,
> from a few datasets (at different maximum resolutions) for which the
> corresponding atomic models are already available at the PDB; this
> according to the formula (6.4), in the 2nd edition of Jan Drenth´s book:
>
> Uhkl = exp[B*(sin^2(theta)/lambda^2)] x FOBShkl / F(000)
>
> Hence, the following questions:
>
> - Is there any macromolecular crystallography software that can compute
> Uhkl as above, or equivalent?
>
> - If not, would it be more correct to use the Wilson-B or the mean B from
> the final model?
>
> - How can F(000) be best estimated from the final model, which is not
> necessarily always the most complete or best refined? Should we simply add
> together the number of electrons for all the atoms refined in the
> asymmetric unit (protein + ligands + solvent)?
>
> Many thanks in advance and best wishes,
>
> --
> Andre LB Ambrosio
>
> --
>
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Re: [ccp4bb] On estimating Unit Structure Factor distribution

[Pavel Afonine]

Hi Andre,


> - Is there any macromolecular crystallography software that can compute
> Uhkl as above, or equivalent?
>

I estimate this can take about 10 minutes to script in CCTBX. I can write a
script for you, if interested, and send off list.


> - If not, would it be more correct to use the Wilson-B or the mean B from
> the final model?
>

I'd guess mean B from the model is a better estimate.

- How can F(000) be best estimated from the final model, which is not
> necessarily always the most complete or best refined? Should we simply add
> together the number of electrons for all the atoms refined in the
> asymmetric unit (protein + ligands + solvent)?
>

See my previous email.

Pavel



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Re: [ccp4bb] On estimating Unit Structure Factor distribution

[Pavel Afonine]

Hi Andre,

- How can F(000) be best estimated from the final model, which is not
> necessarily always the most complete or best refined? Should we simply add
> together the number of electrons for all the atoms refined in the
> asymmetric unit (protein + ligands + solvent)?
>

the text here explains how to do this:

"On the analysis of residual density distributions on an absolute scale"
http://phenix-online.org/newsletter/CCN_2012_07.pdf

Also, there is a Phenix tool to do this:

phenix.f000

Good luck,
Pavel



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Re: [ccp4bb] “Bound ligand” versus “modified residue”

[Pavel Afonine]

Hi Ian,
perhaps there are as many answers to this as many subscribers to this list,
but personally "Cysteine with attachment" seems more logic and clear to me
than calling the whole thing a different name. Although I would also
understand arguments like if it is a CYS with an attachment it is not
really CYS any more and perhaps should be called a unique name. From
refinement viewpoint both a fine.
Pavel

On Wed, Apr 24, 2019 at 8:59 AM Ian Clifton 
wrote:

> Hello everyone,
>
> PDB structure 4qdu contains a “modified residue”, 30V. This is joined
> into the rest of the main chain by means of LINK records. In 5kwj, a
> similar type of modification is described as a cysteine with a
> side‐chain LINK to its “bound ligand”, 6Y3 . (These structures are just
> two clear examples we found to illustrate the question.)
>
> Is there any reason to prefer one of these approaches over the other?
> Does it just depend on what ligands are already in the PDB?
>
> Thanks,
> --
> Ian Clifton ⚗ ℡: +44 1865 275677
> Chemistry Research Laboratory ℻: +44 1865 285002
> Oxford University : ian.clif...@chem.ox.ac.uk
> Mansfield Road   Oxford OX1 3TA   UK
>
>
> 
>
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Re: [ccp4bb] High Rfree in last Shell

[Pavel Afonine]

Also, values in parentheses (high-res shell) depend on how you (the program
you use) do binning. Different programs do it differently and so these
values can vary quite substantially. With a little of trial-and-error
effort choosing the binning one can make these values farther or closer to
overall R. To me this is a hint that these numbers are not very useful. A
plot R (completeness, ..) vs resolution is much more useful than the values
for an arbitrarily defined resolution bin!
Pavel

On Wed, Apr 17, 2019 at 12:57 AM Jan van Agthoven  wrote:

> Hi everyone,
> I’m trying to publish two structures at 3.1Å resolution with the following
> refinement statistics:
>
> Resolution range (Å)   49.2-3.1
> 49.3-3.1
> *R*factor (%)24.0 (32.4)
> 23.4 (32.0)
> *R*free (%)  26.6 (29.2)
> 26.3 (31.6)
>
> *Data collection*
>
> Completeness  100 (100)
> 100 (100)
>
> Redundancy6.9 (7.0)
> 6.2 (6.3)
>
> Molecules in asymmetric unit  1
> 1
>
> Average* I*/σ 14.1 (1.7)
> 15.3 (2.0)
>
> *Rmerge *(%)  14.9 (100)
>  12.7 (100)
>
> *Rmeas* (%)16.2 (100)
>13.9 (100)
>
> *Rsym* (%)   6.2 (68.6)
>5.5 (57.1)
> Wilson *B*-factor 65.6
> 62.7
>
> I’ve been told that the Rfree factor in the* last shell* are too high.
> Does anyone know how I can improve these Rfree factors other then cutting
> the resolution, which already is rather low?
>
> --
>
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Re: [ccp4bb] Question about the electron density maps (.ccp4) in PDBe

[Pavel Afonine]

P.S.: while on the topic, it might be helpful to you to have a look at this
article ("On the analysis of residual density distributions on an absolute
scale", page 43) available here:

http://phenix-online.org/newsletter/CCN_2012_07.pdf

Pavel

On Tue, Mar 5, 2019 at 10:02 AM Pavel Afonine  wrote:

> Hi Sen,
>
> As Pavel mentioned, phenix.f000 will give you F(0,0,0) value, but I don't
>> see that information stored and easily calculated from .ccp4 data.
>>
>
>  phenix.f000 requires atomic model (PDB or mmCIF file) as input, not a
> map. If you want bulk-solvent to be added, then you need to give it mean
> solvent density.
>
> Pavel
>
>>



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Re: [ccp4bb] Question about the electron density maps (.ccp4) in PDBe

[Pavel Afonine]

Hi Sen,

As Pavel mentioned, phenix.f000 will give you F(0,0,0) value, but I don't
> see that information stored and easily calculated from .ccp4 data.
>

 phenix.f000 requires atomic model (PDB or mmCIF file) as input, not a map.
If you want bulk-solvent to be added, then you need to give it mean solvent
density.

Pavel

>



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Re: [ccp4bb] Question about the electron density maps (.ccp4) in PDBe

[Pavel Afonine]

Hi,


> Unfortunately not all structure
> factor programs will give you that F000.
>

phenix.f000 will give you F(0,0,0) value based on atomic model alone or
atomic model plus bulk-solvent.

Pavel



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Re: [ccp4bb] Electron scattering factors for SHELXL

[Pavel Afonine]

Hi,


> Or, alternatively, if anyone has used a different program to refine small
> molecule structures determined by ED, we would be happy to hear about that
> program too.
>

phenix.refine has an option to use electron scattering factors. I can
provide further assistance off-list or on phenix mailing list, if needed.

Pavel



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Re: [ccp4bb] Turning off the bulk solvent modelling in Refmac5 to generate Polder maps?

[Pavel Afonine]

Hi,
this is why Polder map tool also includes analysis of the map in question
to determine whether it looks like bulk-solvent or something else, as
described in paragraph 5 here:
http://journals.iucr.org/d/issues/2017/02/00/ba5254/ba5254.pdf
This analysis tells you in plain English what you are likely to look at.
Otherwise I agree that making sense of maps in solvent-excluded regions are
very tricky.
Pavel

On Fri, Feb 8, 2019 at 2:32 AM Bernhard Rupp 
wrote:

> Hi Fellows,
>
> I'd really like to emphasize the point in the Buster instructions "be
> careful when
> examining fo-fc at low levels" when solvent is excluded. If the solvent
> contribution
> is omitted where you suspect the ligand (e.g. occupancy 0.02 in Refmac),
> there
> will be a fo contribution there from the solvent that is there.
> Particularly if that solvent is a
> dense solution, that fo component will show up nicely and at not so low
> difference
> map levels, and in the shape of that 'excluded' ligand.
>
> Figure 2 in link illustrates that.
> https://febs.onlinelibrary.wiley.com/doi/epdf/10./febs.14320
>
> If you start to fill that void with multiple ligands of various low
> occupancies, you
> are effectively modelling disordered solvent. This is particularly tempting
> because
> I found multiple cases where the classical RSR and RSCC measures give
> acceptable stats for such models. The hunt for low occupancy ligands then
> quickly
> becomes murky density fishing business...
>
> Best, BR
>
>
> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Clemens
> Vonrhein
> Sent: Friday, February 8, 2019 09:53
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Turning off the bulk solvent modelling in Refmac5 to
> generate Polder maps?
>
> Dear Samuel,
>
> On Mon, Feb 04, 2019 at 11:39:58AM +, Samuel Davis (PG Research) wrote:
> > I'm wondering if anyone knows if it is possible to turn off the bulk
> > solvent modelling in Refmac5, for the purpose of generating Polder
> > maps? I know that an option for Polder maps is directly implemented in
> > Phenix, but we ideally want to use Refmac5, as we have used it for the
> > rest of our refinement and want to keep it consistent if possible.
>
> And if you want to try the original implementation of the underlying idea
> as
> an alternative, have a look at the ligand detection mode and maps [1]
> produced by BUSTER [2]. See also [3] and some early examples of their
> usefulness [4-5].
>
> Cheers
>
> Clemens
>
> [1]
> https://www.globalphasing.com/buster/wiki/index.cgi?LigandDetectionModes
> [2] https://www.globalphasing.com/buster/
> [3] Vonrhein, C., & Bricogne, G. (2005). "Automated Structure
> Refinement for High-throughput Ligand Detection with
> BUSTER-TNT". Acta Crysta A61, C248.
> [4] Thoma, Ralf, et al. "Insight into steroid scaffold formation from
> the structure of human oxidosqualene cyclase." Nature 432.7013
> (2004): 118.
> [5] Ekroos, Marika, and Tove Sjogren. "Structural basis for ligand
> promiscuity in cytochrome P450 3A4." Proceedings of the National
> Academy of Sciences 103.37 (2006): 13682-13687.
>
> --
>
> *--
> * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
> * Global Phasing Ltd., Sheraton House, Castle Park
> * Cambridge CB3 0AX, UK   www.globalphasing.com
> *--
>
> 
>
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>
> 
>
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Re: [ccp4bb] [offtopic for ccp4bb] Real space vs reciprocal refinement

[Pavel Afonine]

Hi Mahesh,

In the current version of phenix.refine contains the separate option for
> refinement: xyz (reciprocal space) and xyz (real space ).  What does it
> mean and how it differs from the previous versions which had xyz and real
> space instead.
>

All the same, different name. One refines model against Fobs, the other one
against 2mFo-DFc map (whole model and side chain rotamer fitting using grid
searches, if it is a protein).


> Can we run both of them simultaneously?
>

Both are enabled by default, so apparently the answer is yes.


> Is it advisable to do both refinement for DNA ?
>

Sure, why not?

Good luck!
Pavel



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Re: [ccp4bb] Experimental phasing vs molecular replacement

[Pavel Afonine]

Hi,


> Any time you do a thought experiment you make a fake-data data set, the
> "true" phases and "true" amplitudes become the ones you put into the
> simulation process.  This is by definition.  Is there potential for
> circular reasoning?  Of course!  But you can do controls:
>

this is so much true! This is what I've been doing for development and
testing all the time since started working for Phenix! Fully controlled
thought/numerical experiments done this way are super-helpful (but
obviously have limitations!).


>If you start with an ordinary single-conformer coordinate model and
> flat bulk solvent from refmac to make your Ftrue, then what you will
> find is that even after adding all plausible experimental errors to the
> data the final Rwork/Rfree invariably drop to small-molecule levels of
> 3-4%.  This is true even if you prune the structure back, shake it, and
> rebuild it in various ways.  The difference features always guide you
> back to Rwork/Rfree = 3/4%. However, if you refine with phenix.refine,
> you will find Rwork/Rfree stall at around 10-11%.  This is because Ftrue
> came from refmac and refmac and phenix.refine have somewhat different
> bulk solvent models.  If Ftrue comes from phenix and you refine with
> refmac you get similar "high" R values.  High for a small molecule
> anyway. And, of course, if you get Ftrue from phenix and refine with
> phenix you also get final Rwork/Rfree = 3/4%. If you do more things that
> automated building doesn't do, like multi-headed side chains, or get the
> bulk solvent from an MD simulation, then you can get "realistic"
> Rwork/Rfree in the 20%s.  All of this is the main conclusion from this
> paper: https://dx.doi.org/10./febs.12922


Even within Phenix alone this is true if you switch between different
scaling/bulk-solvent models or play with automation levels (such as
ignoring reflection otliers, etc).

Pavel



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Re: [ccp4bb] Polder or FEM

[Pavel Afonine]

Hi Markus,

I can only guess without seeing files (and link to files seems to be
broken).. So my guess is that the ligand density is weak enough so FEM
treats it as "near noise" and it wipes it. Polder decides it is likely
ligand because it uses correlations, and so even of two maps are weak but
similar the CC is going to be high. I can have a closer look if you send me
files (off mailing list!).

Pavel

On Fri, Nov 23, 2018 at 11:39 PM Markus Heckmann 
wrote:

> Dear Pavel,
>
> By coincidence today I was looking at a soak-dataset and got totally
> confused with FEM (map) and Polder now.
>
> I am working on a dataset that processed it at 2.7A (in summer with
> XDS) and there was weak density for (OCA-Octanoic acid) when looked
> with FEM. This week, I retried it with DIALS and got about 2.6A (for
> CC_half 0.5).  I ran FEM, and there was*no* density at all at OCA.
> After seeing this CCP4 answer, I ran Polder maps and it shows clear
> density for OCA. Now I am really confused why and what should I infer?
> Polder run also states "The polder map is likely to show the ligand."
> CC(1,2): 0.5639
> CC(1,3): 0.8722
> CC(2,3): 0.6193
>
>
> https://imagebin.ca/v/4NbuaSRUbc5t
> FEM in violet and Polder in Green
>
> Any guidance appreciated.
> Markus
>
> 
>
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Re: [ccp4bb] high SOLVENT content low RESOLUTION and a LIGAND to be found

[Pavel Afonine]

Hi Almudena,

I wonder to which extent I can trust the positive blobs or polder maps that
> I am generating...
>

the answer to this question is given in corresponding paper that describes
Polder map:
http://journals.iucr.org/d/issues/2017/02/00/ba5254/ba5254.pdf

Based on the analysis described in this paper the program states the
verdict in plain English (as opposed to a bunch of numbers).

For a lazy option you can watch the video tutorial

https://www.youtube.com/watch?v=TcTuMJayh5c=youtu.be

For even lazier option you can fast-forward to minute 4 of the video where
Dorothee shows what to watch for.


> Anybody that can make other suggestions concerning how to validate the
> ligand binding site and/or generate other maps that could be more helpful?
>

If you have another data set collected from the crystal without the ligand
and crystals are reasonably isomorphous, you can calculate Fobs-Fobs map.

Good luck,
Pavel



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Re: [ccp4bb] VERY old mtz file..

[Pavel Afonine]

Now I see the value of storing data in plain text files even more (mind
Shelx or X-plor formats, for example) -;)



On Fri, Nov 9, 2018 at 9:47 PM Clemens Vonrhein 
wrote:

> Hi Eleanor,
>
> You could try running the oldest MTZ2VARIOUS binary you can find -
> e.g.
>
>   wget ftp://ftp.ccp4.ac.uk/ccp4/4.2/binaries/ccp4-4.2_Linux.tar.gz
>   tar -xvf ccp4-4.2_Linux.tar.gz bin/mtz2various
>
>   bin/mtz2various hklin ...
>
> Any older binaries (ftp://ftp.ccp4.ac.uk/ccp4/4.0.1/) will require an
> SGI or Dec/Alpha machine ;-)
>
> If that doesn't help I would first check that it is actually a correct
> MTZ file: does the ASCII header (trailer) show up with
>
>   strings your.mtz
>
> towards the end?
>
> Cheers
>
> Clemens
>
> On Fri, Nov 09, 2018 at 12:47:09PM +, Eleanor Dodson wrote:
> > Anyone any idea what to do about this?? Created in 1992!!
> > Seems unreadable..
> >
> > No CTYP lines input for file:  1
> > Indices output even if all data items flagged "missing"
> >  Warning, NOT all LABOUT data lines given
> > Warning: Machine stamp corrupted? Assuming native format.
> > >> CCP4 library signal library_file:End of File (Error)
> >
> > 
> >
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>
> --
>
> *--
> * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
> * Global Phasing Ltd., Sheraton House, Castle Park
> * Cambridge CB3 0AX, UK   www.globalphasing.com
> *--
>
> 
>
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Re: [ccp4bb] transform NMR ensemble with pdbset

[Pavel Afonine]

Perhaps

phenix.pdbtools model.pdb rotate=... translate=...

which should work with any PDB or mmCIF file.

Pavel

On Thu, Nov 8, 2018 at 11:36 PM Tim Gruene  wrote:

> Dear Kaushik,
>
> you could try moleman2 from the Uppsala Software Factory,
> http://xray.bmc.uu.se/usf/moleman2_man.html - maybe it keeps those cards
> in the PDB file.
>
> Best,
> Tim
>
> On 11/8/18 4:23 PM, KAUSHIK H.S. wrote:
> > Hello,
> >
> > I want to transform (rotate+translate) structures determined by NMR.  I
> > tried using pdbset from the commandline.  The program seems to remove
> > "MODEL" and "TER" lines from the coordinate file.  Is there a way to
> > make pdbset retain these lines? or am I missing something obvious?
> >
> > I could split the ensemble into individual coordinate files, apply
> > transformation and merge them back.  However, is there an easier way out?
> >
> > Best wishes,
> > Kaushik
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
>
> --
> --
> Paul Scherrer Institut
> Tim Gruene
> - persoenlich -
> OSUA/204
> Forschungsstrasse 111
> CH-5232 Villigen PSI
> phone: +41 (0)56 310 5297
>
> GPG Key ID = A46BEE1A
>
>
> 
>
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Re: [ccp4bb] CC work / free

[Pavel Afonine]

Clément,
I'm guessing this is because it isn't clear what CCwork/CCfree can tell you
that Rwork/Rfree can not. Needless to say we all more or less have a good
idea about what the ok values for Rwork, Rfree and Rfree-Rwork (as function
of resolution) while it is much less clear (to me at least) when it comes
to CCwork/CCfree.
I think phenix.refine used to report CCwork/CCfree in some releases but I
removed it as useless.
All the best,
Pavel

On Thu, Nov 8, 2018 at 8:16 PM Clement Degut <
26de0a160250-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi all,
>
> This probably have already be discussed, but can't find trace of it :
> Is there any reason I am not aware of that refinement software (thinking
> of both phenix and refmac) do not output CC work / free ?
> We all accepted CC 1/2 for resolution cut (whether it's 30 or 10 %) which
> tend to lead to high Rfree ( last shells having very poor phases), at least
> high overall R free in regard to the majority same resolution structures as
> they been refined with I/sig > 3.
> Which in return push people to cut resolution AFTER refinement which
> obviously drastically decrease the R free and catapult your structure from
> worst of the PDB to best of the PDB. It should never be done but it is
> nonetheless.
> How to blame a "non expert"  to do that when changing the resolution from
> 1.49 to 1.5 change your Rfree by 2% (yes it exaggerated but you get the
> idea).
> Now it seems to me -but I may be very wrong as I am far from expert in the
> arcane math of these- that CC work and free would be more agnostic to this
> resolution cut off problem, as we compare it to the CC* value anyway ?
> thought it will never start to be used if refinement software don't output
> it as a standard (not talking about completely replacing Rfree here as it
> is obviously useful).
> Would CC work/free be a bad appreciation of the refinement process ?
>
> Thanks
>
> Clément
>
> --
>
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Re: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang)

[Pavel Afonine]

Hi Tony,

I always feel some people are too "greedy" with the resolution they want to
> achieve. I mostly find that extremely high density is a pain to work with
> as it's usually accompanied by many dual, triple conformers, a lot of noise
> in the solvent phase that is often difficult to interpret, etc... Often you
> spend more time on a high resolution structure that clearly shows your
> protein, as opposed to a low resolution structure where it's difficult to
> interpret parts of the map. Also, in most cases you REALLY don't need a 1
> Ang map to clearly show the overall structure of your protein, ligands,
> first shell of solvent molecules on the surface of your protein, etc...
>

higher resolution typically means you have a chance to obtain a more
accurate atomic model of a crystal structure, which in turn may lead to a
more accurate interpretation of this model. I fully agree that high
resolution requires more modeling effort. There is still a great room for
software developers to provide more automation.


> Your completeness is 90% in your high resolution shell, which is fine, but
> have you checked you can clearly see most reflections for h, k and l? Maybe
> you're missing many reflections for one of them. I would at least
> try cutting your data back to 1.1 or 1.2 Ang, as it might dramatically
> improve your R factors and still show everything you want to show.
>
>
> Also, did you try TLS refinement?
>

At resolutions like 1.2A or better one normally models ADPs as anisotropic
for all atoms (except H), so no need to use TLS.

Pavel



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Re: [ccp4bb] structure with missing density

[Pavel Afonine]

Hi Deepanshu,

how complete the data set? What's completeness across resolution zones?
Systematically missing reflections can have systematic impact in real space
(maps). I've seen entire ligands or domains disappear due to missing
low-resolution data.

Just another check-point to consider..

Good luck!
Pavel

On Wed, Oct 10, 2018 at 10:59 AM Deepanshu Choudhary <
deepanshui...@gmail.com> wrote:

> Dear all,
> I am working on a protein complex. After many efforts, I obtained crystals
> from one of the refinement screen (which took few weeks to grow) and I got
> a 3 Å dataset at synchrotron. I scaled the dataset in P21 spacegroup (which
> is also confirmed by Zanuda and Pointless). There is no twinning detected.
> I solved the phase using molecular replacement with a model of over 90%
> sequence identity. After several rounds of refinement with Refmac, the
> Rfree is 0.307 and Rfactor of 0.23.
> The density looks good and I can see everything that's important. But one
> of the proteins has missing density in 2 of its beta strands (corresponding
> to ~15%) and its not appearing upon several rounds of refinement. Also, the
> B factors are higher for this protein. The missing beta strands are not at
> the interface of the complex.
> I ran some crystals on the gel and did silver staining to find both the
> proteins and no degradation products. I doubt that there is any proteolytic
> cleavage because the protein is unlikely to remain folded if those beta
> strands are chopped out.
> I want to ask if such a structure with missing density and high B-factors
> (>100) can be deposited. Is it possible that some parts of the lattice
> don't have this protein with missing density which is resulting in high B
> factors?
> I would appreciate your efforts if someone can send me few references
> describing such type of structures. I would also welcome any other
> suggestions and recommendations.
>
> Thanks and regards,
> Deepanshu
>
> --
>
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Re: [ccp4bb] collective term for hydrogen bonds and salt bridges

[Pavel Afonine]

Nonbonded interactions? (if you approach this from classic geometry
restraints used in refinement programs)

Pavel

On Mon, Sep 17, 2018 at 2:09 PM Joel Tyndall 
wrote:

> Hi,
>
>
>
> Polar interactions seems to make the most sense. This is what Pymol uses
> as I don’t  think it differentiates
>
>
>
> J
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Sheila
> Boreiko
> *Sent:* Tuesday, 18 September 2018 8:06 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] collective term for hydrogen bonds and salt bridges
>
>
>
> Dear all,
>
>  I had some literature search, but could not find clearly. Would there
> be an appropriate term to call the sum of hydrogen bonds (HB) and salt
> bridges (SB)? What about "hydrophilic interactions" or "polar
> interactions"? I am analyzing the different number of theses interactions
> in different monomers of my protein, as a totality I wanted to cite
> (compare) the number of HB + SB, yet I think to specify them separately
> could take out some focus of the discussion.
>
>  Thank you,
>
>
> Sheila
>
>
> --
>
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>
> --
>
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Re: [ccp4bb] Electron density maps for Cryo-EM structures.

[Pavel Afonine]

P.S.: all questions are welcome of course, no labeling. It's just some of
them are so orthogonal to common sense that answers my be such as well.
Best,
Pavel

On Sun, Sep 9, 2018 at 9:38 PM Pavel Afonine  wrote:

> Hi,
>
> Is there any sever available to create electron density maps for cryo-em
>> structures?
>>
>
> The questions are nonsensical. Here is why:
>
> 1) In cryo-EM maps are not electron density maps but surfaces representing
> electric potential.
>
> 2) Creating such a map is essentially carrying on from cryo-EM experiment
> and obtaining the 3D reconstruction.
>
> Are you really sure about what you are asking for?
>
>
>> Or, we should create the maps from mmCIF.
>>
>
> mmCIF is a file format. It may contain representations of rabbits,
> boysenberries or some diffraction data. So.. how you think it may be
> related to cryo-EM, in your particular case?
>
>
>> I am particularly interested in those cryo-em structures with high
>> resolution, like 2.6~2.8A.
>>
>
> Sure, all are excited about high-res cryo-EM!!!
>
>
>> Please give me an education.
>>
>
> Sure. One of available universities can do this.
>
> Cheers,
> Pavel
>
>



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Re: [ccp4bb] Electron density maps for Cryo-EM structures.

[Pavel Afonine]

Hi,

Is there any sever available to create electron density maps for cryo-em
> structures?
>

The questions are nonsensical. Here is why:

1) In cryo-EM maps are not electron density maps but surfaces representing
electric potential.

2) Creating such a map is essentially carrying on from cryo-EM experiment
and obtaining the 3D reconstruction.

Are you really sure about what you are asking for?


> Or, we should create the maps from mmCIF.
>

mmCIF is a file format. It may contain representations of rabbits,
boysenberries or some diffraction data. So.. how you think it may be
related to cryo-EM, in your particular case?


> I am particularly interested in those cryo-em structures with high
> resolution, like 2.6~2.8A.
>

Sure, all are excited about high-res cryo-EM!!!


> Please give me an education.
>

Sure. One of available universities can do this.

Cheers,
Pavel



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Re: [ccp4bb] cryo-EM

[Pavel Afonine]

Hi Xavier,

> is there some kind of general trend if not consensus as how to refine
> cryo-EM structures?
>
Not really consensus, but my clearly biased contribution to the topic:

I'd say basic common sense for refinement applies:

- optimally sharpen the map (
https://journals.iucr.org/d/issues/2018/06/00/ic5102/);

- refine against the map using refinement parameterization appropriate for
the data (map) quality (resolution) (
https://journals.iucr.org/d/issues/2018/06/00/ic5103/);

- make sure to validate data (map), model, and model-to-map fit (
https://doi.org/10.1101/279844).

> What do people do for maps in the 3-4Å range,
>
3-4A may require secondary structure and rotamer restraints. Use "NCS" if
available. If map is symmetrized, then use "NCS" constraints, otherwise
restraints should be just fine. Typically, data at this resolution range
does not allow to confirm geometry validation outliers, so your aim is no
outleirs. Etc, etc. And again, validate the model and model-to-map fit,
locally (at atom/residue level) and globally (standard "Table 1" metrics,
discussed here https://doi.org/10.1101/279844 , for example).

> pure RSR against the untouched map and that's it?
>
Good idea, in my opinion.

> Reciprocal space refinement against the backtransformed structure factors
> from the map but without touching the map?
>
Personally don't like this idea.

> I've even heard that some people "dare" to calculate 2mFo-DFc-maps
>
Not a good idea, I'm pretty sure. Cryo-EM map is not biased by atomic model
(normally, unless a model was used in reconstruction). 2mFo-DFc map is
model biased by definition. Also needless to say that absence of "Fobs" in
cryo-EM is a good hint that this map isn't what you want -;)

All the best,
Pavel



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Re: [ccp4bb] Can H-clashes be ignored ?

[Pavel Afonine]

Hi,

also, it helps to keep in mind that some clashes may actually be valid
interactions that are labeled as 'clashes' by validation software that is
simply not sophisticated enough to distinguish between bad steric clashes
and chemically/physically favorable valid interactions. For an example,
please refer to results we show in our full quantum refinement of a small
structure done as part of quantum refinement project (www.qrefine.com):

http://scripts.iucr.org/cgi-bin/paper?S2059798317016746

All the best,
Pavel

On Fri, Aug 17, 2018 at 6:27 PM, Firdous Tarique  wrote:

> Hello everyone.
>
> I have a basic question. When a validation report of a coordinate is
> generated we often come across a term known as "Too-Close Contacts".
> First of all can somebody please explain me what is the shortest
> interatomic distance between the two atoms which is permissible ?
>  Next, in this list there are Non-H and H columns list, their Interatomic
> distance and Clash overlap. I could see  two types of clashes in my
> validation report. One in which interatomic distance between the two atom
> (one is always a modeled  H) ranges from 1.7-2.4A, and clash overlap from
> 04-1.0. The other in which the interatomic distance between two atom is
> greater than 2.2A and the clash overlap is between 0.4-0.6 (always between
> two non H-atoms).
>
> So my question is that out of so many clashes shown in the list which are
> one which actually need to be fixed and can't be ignored specially because
> one of my ligand is an amino acid which is showing lots of these H clashes
> (interatomic distance less than 1.5A).
>
> Regards
>
> Kahkashan
>
>
>
> --
>
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Re: [ccp4bb] identifying bound ions

[Pavel Afonine]

There is an option in phenix.refine to do this, described here:

Automated identification of elemental ions in macromolecular crystal
structures. Echols N, Morshed N, Afonine PV, McCoy AJ, Miller MD, Read RJ,
Richardson JS, Terwilliger TC, Adams PD Acta Cryst. D70, 1104-1114 (2014).

Pavel

On Tue, Jul 31, 2018 at 5:39 AM,  wrote:

> Dear BB,
>
>
>
> I know it has been discussed some time ago, but a google search did not
> come up with anything useful.
>
>
>
> I need a program which analyzes the bound waters and suggests whether a
> particular water might be a chloride, calcium, sulfate, sodium or something
> else. Preferably a program that can be run off-line (not a web server), but
> if there is no choice, we will use a webserver as well.
>
>
>
> Thank you for your suggestions!
>
> Herman
>
>
>
>
>
>
>
> --
>
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Re: [ccp4bb] wwpdb validation

[Pavel Afonine]

Phenix has its own way to do it which is "Comprehensive validation"
available from the GUI. This includes validation of model, data and
model-to-data fit. It is data-specific: there is one for crystallography
(X-ray or neutron) and one for Cryo-EM. For best results, you need the
latest version from nightly builds. Hope "wwpdb validation pipeline" is
something along the same lines, or close at least.
All the best,
Pavel

On Fri, Jul 27, 2018 at 9:24 AM, wtempel  wrote:

> Hi,
> did I hear correctly that the wwpdb validation pipeline can be accessed
> from within ccp4 and/or phenix? If so, how does one access that
> functionality in either software suite?
> Many thanks.
> Wolfram Tempel
>
> --
>
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Re: [ccp4bb] Help with omit map

[Pavel Afonine]

As others suggested, you can use Polder map:

- how-to video tutorial can be found here:

http://www.phenix-online.org/documentation/reference/tutorial_channel.html

- background is described here:

http://journals.iucr.org/d/issues/2017/02/00/ba5254/ba5254.pdf

Pavel


On Tue, Jul 24, 2018 at 3:26 PM, Vikram Dalal 
wrote:

> Hi everyone,
>
>
> I have to generate the omit map of metal and coordinating residues of
> protein structure for the figure.
>
> Which program can be used to generate the omit map for my requirement.
>
>
>
> Thanks & Regards,
>
>
>
>
>
>
>
>
> --
>
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>



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Re: [ccp4bb] R-flag choose

[Pavel Afonine]

It's important to remember that free-R reflections are not only used to
calculate Rfree, but also are used in calculation of m and D scales in
2mFo-DFc and mFo-DFc maps, as well as in likelihood-based refinement
targets. The fact is that you need to have a sufficient amount of free-R
reflections in each relatively thin resolution bin. Some authors estimate
this number as at lest 50 reflections per bin. In Phenix binning is defined
such that each sufficiently thin bin gets no less than 150 reflections.
Bernhard pointed out about "precision or significance of Rfee". Also, I
would add that too few free-R reflections can affect refinement progress
(not only robustness of Rfree value) (I've done relevant tests to convince
myself more than a decade ago)! Thus I favor having 10% just to be on a
safer side.

Pavel


On Tue, Jul 10, 2018 at 1:02 AM, 张士军 <21620150150...@stu.xmu.edu.cn> wrote:

> Dear all
>
> I am confusing the choose of free R-flags recently. Rfactor
> means calculatted from reflection not used in refinement,so what's big the
> difference between different percentage of R-flags,like it's about 5% in ccp4
> -refmac, while it is 10% in phenix-refinement,what'
> s the difference between them and how they affect the
> Rwork and Rfree values when do refinement. Thanks a lot !
>
> best regards
>
> shijun
>
> --
>
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Re: [ccp4bb] Clear segid

[Pavel Afonine]

This should work in latest nightly builds:

phenix.pdbtools model.pdb clear_seg_id=true

If it doesn't please report a bug (on appropriate Phenix lists).

Good luck!
Pavel


On Thu, Jul 5, 2018 at 4:33 AM, Eugene Osipov  wrote:

> Hello everyone,
> is there any simple way in CCP4 to clear segid field?
> I used phenix.pdbtools but version 1.13 does not work anymore.
>
> --
> Eugene Osipov
> Junior Research Scientist
> Laboratory of Enzyme Engineering
> Research Center of Biotechnology
> Russian Academy of Sciences
> Leninsky pr. 33, 119071 Moscow, Russia
> e-mail: e.m.osi...@gmail.com
>
> --
>
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>



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Re: [ccp4bb] disulfate bond ?

[Pavel Afonine]

More re disulfide bonds:
http://www.phenix-online.org/newsletter/CCN_2015_01.pdf#page=13

Pavel

On Tue, Jul 3, 2018 at 11:26 PM, 张士军 <21620150150...@stu.xmu.edu.cn> wrote:

> Hi all
>
> I got a structure which has COA in it, and the SH in the tail of COA
> is very close to the SH side chain of Cys in the structure. I don't know
> whether it is disulfate bond or not? I remember they
> should link together if they are disulfate bond,am I right?
>  so what could this be? Thanks a lot!!!
>
> best regards
>
> shijun
>
>
>
> --
>
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Re: [ccp4bb] B-factor standardization

[Pavel Afonine]

> If I am not wrong, I remember that someone proposed to standardize
> B-factors of protein atoms as “BS = B - Bave”, where Bave is the average
> B-factor of the protein.


This will make some of BS negative (if B

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[Pavel Afonine]

Just in case you find it helpful, you can get 100% complete set of
reflections (Fcalc) in specified resolution range using

phenix.fmodel model.pdb high_res=2.5 low_res=15

or if you leave out low_res it will go all the way up to theoretical limit
of low resolution.

If you have/use cctbx then I can show to do this at lower (Python) level.

Pavel

On Thu, Apr 5, 2018 at 3:52 AM, Frank von Delft  wrote:

> Hello - can anybody shed light on this mystery:
>
> We need (for PanDDA analysis) a lot of datasets each to have the complete
> set of low resolution indices, whether measured or not.  (Refmac adds the
> estimates as DFc, which is crucial when comparing maps.)
>
> In ccp4, there are two obvious ways to get these indices complete:
>
>- uniqueify
>- CAD using the keyword "RESOLUTION FILE 1 999 "  (999 is the
>low resolution limit).
>
> Mystifyingly, in ~1% of datasets, one or the other route misses one or two
> indices.  Our work-around is to go belt-and-braces and run both for each
> dataset.
>
>
> It does however remain a bug.  Does anybody have any idea what's
> happening?  We can send example datasets to any volunteers who want to
> fiddle with it.
>
> phx
>
>
>

Re: [ccp4bb] Looking at an EM map..

[Pavel Afonine]

This is discussed, for example, here:
http://www.pnas.org/content/114/12/3103

Also, here I calculated the distribution of map values (scaled in r.m.s.)
for four groups of atoms: main-chain atoms, side-chain oxygen atoms of ASP
and GLU (negatively charged OD1, OD2, OE1, OE2), side chain atoms of ARG
and LYS (positively charged NH1, NH2, NZ), and all other side-chain atoms.
Clearly side-chain oxygen atoms of ASP and GLU have indeed systematically
weaker density:

http://cci.lbl.gov/~afonine/tmp/fig.png

First picture: all maps from EMDB of resolution 3A or better. Second
picture: all maps from EMDB of resolution 3-4A.

All the best,
Pavel

On Thu, Mar 15, 2018 at 7:17 AM, Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> I am pig-ignorant about these ,, but this example has negative values as
> well as positive..
>
> What does this mean? I thought a well phased map would be pretty well all
> positive..
>
>  Eleanor
>

Re: [ccp4bb] building into EM map

[Pavel Afonine]

> I am new to building into an EM map, and I wonder if I could get a
> recommendation for some standard routine for programs to use. I tried "find
> helixes and strands" tool in phenix, it found some secondary structure
> elements, however, it seems that there is more that could be done
> automatically. Is there any standard set of tools to use for automatic
> building in EM map of about 4A resolution?
>


phenix.map_to_model may be able to do more:
https://www.phenix-online.org/documentation/reference/map_to_model.html

Pavel

Re: [ccp4bb] new ContaMiner features

[Pavel Afonine]

It's amusing how a seemingly innocent ad for a new tool can ignite a rather
prickly thread.. I see two keys to this.

Firstly, for those who are not familiar with the issue the add could be
better structured by providing a clearer statement of what the problem is
or why it is important (with appropriate citations as examples) and then
addressing how this is solved using the tool being advertised (ContaMiner).

Second, I can't agree more with with Gerard: "a word of warning" (concerns
about Staraniso use) is best to discuss with respective developers first
before going public. (A nuance though is: not all developers are keen to
open-access source code or even allow their software for benchmarking by
others in some instances. So this may be tricky sometimes.)

Radu's assessment is harsh to my taste.. I guess others commented on this
enough to explain the validity of effort. Plus, I find wording "on the
bizarre ContaMiner" isn't helpful.

All in all, intentionally or not, this made enough of advertisement for
ContaMiner and Staraniso! Win-win, I think!

All the best,
Pavel


On Thu, Nov 23, 2017 at 10:33 PM, Graeme Winter  wrote:

> Dear All,
>
> As someone who is both a user of external software and supports internally
> developed software to external users, I am quite familiar with both sides
> of this argument. From time to time someone will notice a "weird feature"
> in software - sometimes this is a bug, sometimes misuse (which is still by
> and large a bug, but in documentation) and sometimes a change in
> circumstances i.e. reasonable assumptions made in developing a package
> (e.g. background always over 1 count / all data sets have some reflections
> with I/sigI > 3 etc) become problematic.
>
> As an individual developing software, and a member of a team doing so, it
> is always more comfortable if a user comes back with a collegiate "quiet
> word" that the software did something odd. However, I suspect it would be
> for the greater good that a more public approach was taken in general,
> since the less attentive user may miss this odd feature and take the
> results as gospel - if the knowledge of the bug / feature whatever was not
> in the public domain. Despite appearances people do not like to contact
> authors of software packages to complain.
>
> To make a note that "this version of package xyz has been found to be
> sometimes unpredictable with version 123 of the pipeline" does not blame
> the package, or the pipeline, but says that you should be warned with this
> combination. Ideally the authors of package xyz and the pipeline would be
> alerted, by the other developers or users, but it is better (IMHO) to be
> open. Public bug trackers are a good example of this.
>
> I suspect this matter of anisotropy correction is a similar one. Here we
> have a change in circumstances - the actual intensity measurements are
> modified - and passed in as if they are the originals. It is reasonable
> that this may affect the outcome of subsequent analysis and it is hoped
> that this does change the outcome but in a positive manner. This does not
> appear to be the case here.
>
> I have been asked on several occasions to incorporate the anisotropy
> correction into xia2 as it 'always makes things better', and have resisted
> on the grounds that the purpose of the package is to faithfully analyse the
> data as provided and provide uncorrected intensities as output. The
> corrections should ideally be performed within the downstream software,
> since they then know exactly what has happened to the measurements and will
> make fewer (ideally no) incorrect assumptions.
>
> It's already routine to write out multiple versions of e.g. phases,
> weights, sigma values etc based on different assumptions and flag then
> accordingly - perhaps we should be doing the same with modified
> intensities, so that packages which require the unmodified values could
> ignore the corrected ones. That would avoid the need for any health
> warnings and ensure changes in the wider environment do not invalidate
> assumptions...
>
> Obviously, all of the above is my humble opinion and other opinions are
> equally valid.
>
> Best wishes Graeme
>
>
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Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

[Pavel Afonine]

Perhaps this can be automated:

https://www.phenix-online.org/papers/wd5073_reprint.pdf

Software doesn't get tired or bored, and thus potentially can try more and
produce more plausible interretations. Then one can hire a number of people
of various expertise to choose "best" result according to their subjective
interpretations.

Pavel

On Mon, Nov 20, 2017 at 12:25 PM, Shintaro Aibara  wrote:

> Dear All,
>
> Apologies for the slightly off-topic, but I was wondering if anybody knew
> of a paper/textbook where a protein model was built by multiple people
> (ranging from novice to experienced builders) and compared. I believe the
> conclusion was that while the overall trace was broadly correct,
> experienced builders maintained better geometry compared to beginners.
>
> I distinctly remember reading it a couple of years ago but cannot seem to
> find the figure. If anybody knows of this figure/paper/textbook it would be
> much appreciated if you could point me in the direction.
>
> Yours faithfully,
> Shintaro
>

Re: [ccp4bb] High R/Rfree

[Pavel Afonine]

Hi Radhika,

R-factor value is almost useless unless you know the resolution (which you
did not tell us): 26% is ok for 3A resolution and is nonsense for 1A, for
example. The Rfree-Rwork gap is obviously large, suggesting sub-optimal
refinement strategy. Try optimizing weights, let program update
(add/remove/refine) water. You can send me model and data files for further
diagnostics.

Pavel

On Mon, Nov 13, 2017 at 3:45 PM, Radhika Singh  wrote:

> Hello All,
>
> I am currently working on the structure of a DNA protein complex.  The
> data has been processed in space group P1 (53.042   59.527   78.526 105.24
> 98.03 106.99 P 1, Rpim 11.7%).  At this stage I have almost 85% model is
> complete but my R/Rfree is stuck as 26%/34%.
>
> I have some concerns and questions:
> * Xtriage says there are a large number of outliers; however no
> pseudotranslational symmetry is detected by the program.  What are the
> other reasons for outliers?
>
> * I am trying phenix.refine for refinement with the default settings. Is
> there any special setting that can help me?
>
> I would like to have some suggestions about my problem.
>
> Thanks in advance
>
> Radhika
>
>

Re: [ccp4bb] question about resolution bins in deposition

[Pavel Afonine]

See Table 1 and corresponding discussion here:

http://journals.iucr.org/d/issues/2013/04/00/dz5273/dz5273.pdf

Hope that hints you the answer. If not get back to me with questions.

All the best,
Pavel


On Mon, Nov 6, 2017 at 7:18 PM, Eze Chivi  wrote:

> Hi, my PDB file lists only 4 resolution bins (full range: 1.6-19.1A). This
> is the first time I noted this behavior (It's used to be 20 bins). It is OK
> for deposition? It is need to rearrange statistics? How I can do that?
> Thank you very much. (I did my refinements in phenix, oops!, but the ccp4bb
> people is so kind...)
>

Re: [ccp4bb] another unknown density problem

[Pavel Afonine]

If by "it was truncated at 5A for clarity" you really mean you truncated
all low-resolution data from 5A and lower then I am not surprised you see
funny densities all over or don't see density where it is expected. Why?
Consult a textbook for the answer.

All the best,
Pavel

On Fri, Nov 3, 2017 at 3:10 AM, Abhishek Anan 
wrote:

> Dear Prof Schreuder
>
> Here are another couple of perspectives from coot. The density is too far
> and isolated from the peptide chain to be an alternate conformation or
> conformational change. The density of the peptide chain does not look good
> because it was truncated at 5A for clarity.
>
> Best regards
> Abhishek
>
> On Fri, Nov 3, 2017 at 9:33 AM,  wrote:
>
>> Dear Abhishek,
>>
>>
>>
>> To me, it looks like an alternative conformation of the peptide chain or
>> maybe even a conformational change with respect to the starting model. The
>> peptide chain does not look too well defined, despite high resolution
>> electron density.
>>
>>
>>
>> Best,
>>
>> Herman
>>
>>
>>
>> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag
>> von *Abhishek Anan
>> *Gesendet:* Freitag, 3. November 2017 09:26
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [EXTERNAL] [ccp4bb] another unknown density problem
>>
>>
>>
>> Hi all,
>>
>> I have an "unknown" density in the map. I have tried to fit it to PEG but
>> it doesn't fit very well. I was wondering if there are other PEG-related or
>> other molecules I could try.
>>
>> The crystal grew in TRIS-HCl and PEG MME 2K.
>>
>> Thank you
>>
>> Abhishek
>>
>
>

Re: [ccp4bb] efresol download?

[Pavel Afonine]

Johan,

core functionality described in that paper is implemented in cctbx. Re the
stand-alone program -- I'm cc'ing to the author.

Pavel

On Thu, Oct 26, 2017 at 8:39 AM, Hattne, Johan 
wrote:

> Dear all;
>
> Would anybody know where I can find efresol (as detailed in
> http://dx.doi.org/10.1107/S0907444913016673) these days?  Googling lead
> me to a 2014 post (https://www.jiscmail.ac.uk/
> cgi-bin/webadmin?A2=ind1406=ccp4bb===155751), but those links
> result in 403 Forbidden as of right now.
>
> // Best wishes; Johan
>
>
>   Research Specialist @ Gonen Lab
> 
> Janelia Research Campus * 19700 Helix Drive
> Ashburn, VA 20147 * +1 (571) 209-4000 extension 3376
>