Re: [ccp4bb] Protein-DNA covalent bond refinement

[Robert Nicholls]

Dear Cristina,

In CCP4 you've got two options that use the modern approach (Acedrg):

(1) CCP4i2 -> Ligands -> Make Covalent Link.

(2) Coot -> Calculate -> Modules -> CCP4. This will make a "CCP4" menu appear 
that contains the option: Make Link via Acedrg.

Hopefully it's pretty self-explanatory from there - you'll end up with a CIF 
dictionary containing the link description, which you can then pass to your 
refinement program. Both of these options require the latest version of CCP4 
(7.1).

Regards,
Rob


> On 2 Jul 2020, at 15:59, 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:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 
> 



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

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/

Re: [ccp4bb] Refmac Ideal Geometry Library

[Robert Nicholls]

Hi Dale,

Yes Refmac uses the dictionaries found in lib/data/monomers
Of course these targets may differ from those used by validation tools.
If you identify anything that's clearly wrong then do let us know!

Regards,
Rob


> On 25 Jul 2020, at 17:22, Dale Tronrud  wrote:
> 
> Hi,
> 
>   I'm seeking insight into some geometry outliers in my Refmac refined
> model.  It would be nice to have confidence in the target values used by
> Refmac.  Does Refmac use the library distributed by CCP4 in
> lib/data/monomers, or do it have its own library squirreled away somewhere?
> 
> Dale Tronrud
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> 
> 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/



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

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/

Re: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement

[Robert Nicholls]

To paraphrase from our 2018 Acta Cryst D74(6) paper:

“There has been debate as to whether refinement should be performed in real 
space or reciprocal space; both approaches have advantages and disadvantages. 
In real space, refinement can be performed locally, which has advantages for 
computational speed and parallelisation.
[Update: Refmac now has the option to perform “local refinement”, which means 
focusing on just a localised region in order to speed things up – this option 
is available from within the CCP-EM interface.]
Refinement in reciprocal space allows accounting for errors in Fourier 
coefficients more accurately. And, although errors in neighbouring Fourier 
coefficients are correlated, they are less correlated than between proximal 
points in real space. Furthermore, the degree to which errors are correlated 
will depend on the nature of the underlying data (i.e. on the reconstruction 
methods). There is a common misconception that the original data are in real 
space. Reconstructions are typically performed in Fourier space using the 
projection-slice theorem, before the reconstructed maps are subsequently 
calculated in real space. Alternatively, 3D reconstruction may be performed in 
real space using back-projection, noting that this results in a large 
correlation radius of errors in real space. The procedures in real and 
reciprocal space are mathematically equivalent; in both cases refinement should 
be equivalent (apart from details of implementation). The community would 
benefit from clarification about best practice on this controversial topic, 
thus proper analysis will be required in future."

Indeed, until works have been published on the objective comparative analysis 
of the performance of real vs reciprocal space refinement of models against 
cryo-EM maps, it seems premature to sit too strongly on one side of the fence 
or the other. Until then, I'd recommend people to try different tools and use 
what works best for them, without too much preconception.

Regards,
Rob


Dr Rob Nicholls
Senior Investigator Scientist
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH



> On 30 Jul 2020, at 13:28, Schreuder, Herman /DE  
> wrote:
> 
> Hi everybody,
> 
> Thank you very much for the many tips! I discovered that in Coot by clicking 
> on the N- and C-terminus of a protein chain, one can indeed run a real-space 
> refinement on the complete chain and the same is true for a rigid body fit. 
> However, subsequent refinement with Refmac did not converge, so it looks like 
> I still have to go manually through the chains. ☹
> Still on my to do list are:
> - test Isolde
> - test Main
> - test the wiggle-fit option in Coot.
> 
> Although the Refmac procedure consisting of converting the cryo-EM map into 
> structure factors and running a "crystallographic" refinement on it seems to 
> work, in my opinion the future will be a robust real-space refinement on the 
> cryo-EM map itself, instead of a refinement on derived structure factors. 
> However, this is my personal opinion.
> 
> Best regards,
> Herman
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board  Im Auftrag von Tom Burnley - 
> UKRI STFC
> Gesendet: Mittwoch, 29. Juli 2020 18:51
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement
> 
> EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   
> 
> 
> 
> Hi Herman,
> 
> 
> There is lots of information on CCP-EM's modelling tools here:
> 
> 
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ccpem.ac.uk_training_icknield-5F2019_icknield-5F2019.php&d=DwIFAg&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=aaxzRy8JT0AM0_MqNvU2fr6IB4wIbkY8SsgvQkYcw_w&s=D5VOMK2Fz8AOV1PCvJpD9tm914KUxJlsRNRWXYPb7tI&e=
> 
> 
> This includes a tutorial on Refmac.  As you note this is done in reciprocal 
> space but the CCP-EM pipeline handles conversions from MRC to MTZ format (and 
> runs Refmac via CCP4).
> 
> 
> All the best,
> 
> 
> Tom
> 
> 
> From: CCP4 bulletin board  on behalf of Boaz Shaanan 
> 
> Sent: 29 July 2020 16:31:46
> To: ccp4bb
> Subject: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement
> 
> Hi,
> Try searching ccp-em.
> Boaz
> 
> Boaz Shaanan, Ph.D.
> Department of Life Sciences
> Ben Gurion University of the Negev
> Beer Sheva
> Israel
> 
> On Jul 29, 2020 18:29, "Schreuder, Herman /DE"  
> wrote:
> Coot does it, but if one wants to do it for a complete protein, it is a lot 
> of clicking. On the other hand, I have not tried to just select the N- and 
> C-terminus for real-space refinement to see what happens. I would prefer to 
> have some script to do it.
> Best, Herman
> 
> Von: Eleanor Dodson 
> Gesendet: Mittwoch, 29. Juli 2020 17:23
> An: Schreuder, Herman /DE 
> Cc: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] Re: [ccp4bb] real real-space-refinement
> 
> 
>

Re: [ccp4bb] AW: Going back to Coot 0.8

[Robert Nicholls]

Hello Dirk,

Upon reviewing this thread I saw your comment:

> On 11 Sep 2020, at 08:41, Dirk Kostrewa  
> wrote:
> 
> I'm still convinced since the beginning of Coot, that an implementation of 
> hydrogen-bond potentials in the RSR would give a huge improvement and 
> probably renders the use of secondary structure restraints unnecessary.

Yes you can do this! If using CCP4 then you can generate h-bond restraints with 
prosmart (externally) and then read them into Coot. 

If wanting to use the command line then the simplest command would be: 
"prosmart -p1 [model filename] -h". The -h flag tells prosmart to generate 
backbone hydrogen bond restraints. There are lots of other keywords, e.g. to 
control sigmas etc. see prosmart --help for the full list. There are also 
interfaces to prosmart in the various CCP4 GUIs.

To get the correct menu in Coot, go to: Calculate -> Modules -> Restraints. 
This will cause a new "Restraints" menu to appear at the top. Then select "Read 
Refmac Extra Restraints..." and select the restraints file output by prosmart. 
Coot will display these restraints (although you can undisplay them), and Coot 
will use them in RSR. 

Best regards,
Rob




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

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/

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Robert Nicholls]

Dear all,

Coming back to map blurring...

> On 24 Nov 2020, at 16:41, John R Helliwell  > wrote:
> 
> [...] Coot’s blurring and sharpening tool is tethered directly to one’s 
> measured diffraction data. 
> [...] But, I have just rechecked the Coot manual and see no reference.


The use of Coot's blurring tool is briefly recommended in the context of ligand 
binding analysis on page 12 of this publication: 
https://doi.org/10.1107/S2059798316020143 
. This is so useful in cryo-EM that 
the technique of simultaneously viewing multiple maps with different levels of 
blurring/sharpening as a visual confirmatory aid has become routine 
(https://doi.org/10.1107/S2059798318007313 
). More generally, map blurring can 
be useful for exploring evidence for the presence of structure in the crystal 
that is currently missing from the model in both MX and cryo-EM.

Regards,
Rob


> On 27 Nov 2020, at 08:41, Clemens Vonrhein  wrote:
> 
> Dear Nika,
> 
> as a possible alternative or second opinion, you could have a look at
> the ligand-detection modes [1] in BUSTER too - see e.g.
> 
>  https://www.globalphasing.com/buster/wiki/index.cgi?LigandDetectionModes
> 
> It's very similar to the Phenix Polder maps - so might not tell you
> anything different, but looking at results from different
> implementations can be useful as a check.
> 
> Anyway, one of the main problems handling partially occupied ligands
> (and let's ignore the possibility of partial disorder for
> "simplicity") is that you might have a mixture of three models within
> your binding site: (1) compound, (2) waters (at ordered positions when
> the ligand is not bound) and (3) bulk solvent (at disordered positions
> when the ligand is not bound).
> 
> What can be useful is to include at least the alternative ordered
> water model (from a well refined APO model?) and then refine the
> (grouped) occupancies:
> 
>  OCC(compound) + OCC(waters) = 1.0
> 
> This way you give the refinement the option to chose between two
> alternative interpretations [2]. To avoid any bias in that refinement
> procedure, you could start from two extremes: once with
> OCC(compound)=0.9 and once with 0.1. If the OCC(compound) refines to a
> small value (say <0.2 or such) in both cases, it is possibly not really
> there.
> 
> Cheers
> 
> Clemens
> 
> [1] Vonrhein, C. and Bricogne, G., 2005. Automated structure
>refinement for high‐throughput ligand detection with
>BUSTER‐TNT. Acta Crystallogr. Sect. A, 61, p.c248.
> [2] 
> http://www.globalphasing.com/pipermail/buster-discuss/2015-August/000255.html
> 
> On Tue, Nov 24, 2020 at 11:28:42AM +, Nika Žibrat wrote:
>> Hello,
>> 
>> 
>> I have a question about protein-ligand, of which ligand displays an 
>> ambiguous electron density. I am solving a structure of protein with ligand  
>> which was obtained via soaking. Structural characteristics indicate the 
>> ligand is present however the electron density is quite vague and too small 
>> for the size of the whole ligand. I did a Polder map which showed much 
>> larger area of green density. After insertion of my ligand into the green 
>> density in Polder I ran phenix.refine and there is a lot of red on the spot 
>> where the ligand is which was to be expected. This leaves me wondering how, 
>> if even do I incorporate the polder map data into my refine input.
>> 
>> 
>> My question is, how do I continue refining and validating the structure in 
>> this case?
>> 
>> 
>> Thank you,
>> 
>> 
>> Nika Žibrat
>> 
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>> 
>> 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/
> 
> -- 
> 
> *--
> * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
> * Global Phasing Ltd., Sheraton House, Castle Park 
> * Cambridge CB3 0AX, UK   www.globalphasing.com
> *--
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> 
> 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/




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

This 

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Robert Nicholls]

Dear all,
 
I feel the need to respond following last week’s critique of the use of Coot’s 
map blurring tool for providing diagnostic insight and aiding ligand 
identification…

> On 24 Nov 2020, at 16:02, Dale Tronrud  > wrote:
> 
> To me, this sounds like a very dangerous way to use this tool decide if a 
> ligand has bound.  I would be very reluctant to modify my map with a range of 
> arbitrary parameters until it looked like what I wanted to see.  The 
> sharpening and blurring of this tool is not guided or limited by theory or 
> data.

I disagree with this, subject to the important qualification that care is 
needed with interpretation. Blurring isn't a crime - it merely involves 
adjusting the weighting given to lower versus higher resolution reflections, 
and thus allows relaxation of the choice of high-resolution limit, and 
facilitates local investigation of regions that exhibit a poor signal-to-noise 
ratio. This is particularly pertinent to liganded compounds, which are 
typically present with sub-unitary occupancies.
 
Coot's blurring merely involves convolution of the whole map with an isotropic 
3D Gaussian, with a parameter (B-factor) to control the standard deviation of 
the Gaussian. This corresponds to reweighting the structure factors in order to 
give higher weight to lower-resolution reflections. This approach is guided by 
a very simple theory: higher resolution structure factors (SFs) are typically 
noisier, with a worse signal-to-noise ratio than lower resolution SFs (due to 
increased errors in both observed higher-resolution reflections and calculated 
phases). Consequently, increasing the blurring B-factor reduces the effect of 
the noisier higher-resolution SFs. This results in a map that should be more 
reliable, but at the expense of reduced structural detail due to artificially 
reducing the effective resolution.
 
It should be noted that this does assume that lower resolution reflections are 
more reliable than higher resolution ones. So, good low-resolution data quality 
and completeness is important.
 
Unfortunately, determination of an optimal B-factor parameter is not presently 
automated. Consequently, users are currently expected to trial different values 
in the Coot slider tool in order to maximise information and gain, for want of 
a better word, intuition. Furthermore, due to the spatially heterogeneous 
nature of atomic positional uncertainty in macromolecular complexes, it can be 
that different B-factor parameters are of optimal usefulness in different local 
regions of the map that exhibit different signal-to-noise ratios. Such issues 
are on-going areas of research.
 
The main problem is that interpretation is subjective. In difficult cases, it 
is necessary to obtain as much information and insight as possible in order to 
gain a good intuition. If you can't see a ligand in the "standard" maps, but 
you can see evidence for a ligand in blurred density (or difference density) 
maps of the various types, then it means that careful exploration of those 
avenues is required. Any "evidence" from viewing such maps and map types should 
serve to guide intuition, and should be digested along with all other available 
information. Such complementary maps should be seen as diagnostics to gain 
intuition, rather than something that can be used as an unequivocal argument 
for ligand binding.
 
Ultimately, the presence of significant density in a blurred map means that 
there is something substantial present. Or in a blurred difference density that 
there is something missing from the current model. This could be a missing 
ligand, or it could be a mismodelled region of the macromolecule, or it could 
be mismodelled solvent (in which case re-evaluating any solvent mask may be 
worthwhile). Ultimately it is down to the practitioner to explore all potential 
explanations for any such behaviour, in order to maximise intuition and 
convince themselves of the crystal's structural composition.
 
In some cases the presence of density in a blurred map might be sufficient to 
convince the practitioner that it is worth pursing investigation of binding. 
This may take various forms: hypothesising an approximate pose for the ligand; 
the nature of interactions in the structural environment of the macromolecule; 
re-evaluation after modelling and refinement; or simply stating that there may 
be evidence of binding. In many cases, the latter is the appropriate action, 
and, as Robbie quite rightly pointed out: "in a scientific setting this digging 
is not to come to a strong conclusion, but only to see if you should pursue the 
project and do additional experiments".

> On 24 Nov 2020, at 16:02, Dale Tronrud  > wrote:
> [...] to avoid bias in the interpretation of the results, all of the 
> statistical procedures are decided upon BEFORE the study is even began. This 
> protocol is written down and peer reviewed at the start

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Robert Nicholls]

iffy 
> new map calculation and you want to try it.  While you certainly can 
> calculate the map, you have no intuition on how to interpret it.  You have 
> not practiced with that type of map.
> 
>   It may look similar to the maps you've looked at before, but that 
> similarity can be a trap.  By now a large number of us here on the BB have 
> had the experience of looking at a high resolution electrostatic potential 
> (ESP) map and "feeling" that something is wrong with it.  The carbonyl oxygen 
> bumps are too small and the acid groups are oddly weak.  Wow, those magnesium 
> ions really stand out -- Maybe they're potassium instead?  No, there is 
> nothing wrong with the ESP map.  The fault is with our intuition which was 
> based on many, many hours of looking at ED maps.  To interpret ESP maps you 
> have to practice with a bunch of ESP maps first.
> 
>   You cannot develop intuition for the spiffy map calculated from your 
> project's data since you don't know its correct interpretation -- It cannot 
> give you feedback.  Before you calculate this map for your data you should 
> calculate versions for many other *completed* projects and get a "feel" for 
> what that kind of map shows under different circumstances.  Practice, 
> practice, practice, then you will be ready to return to your little mystery 
> and be able to apply your, newly acquired, intuition.
> 
>   Yes, I try new refinement programs - But first I run refinement with them 
> on familiar proteins. Yes, I try new styles of map calculations - But first I 
> calculate those maps for cases where I know the answer. I've refined a fair 
> number of structures, probably not as many as most of you, but at the end of 
> a refinement I take the answer and go back to the original maps.  Looking at 
> those maps in light of the answer is what improves my map interpretation 
> skills, such as they are, the most.
> 
>   All of my practice has been with ED (and some ESP) maps of better than 3 A 
> resolution.  Despite all the intuition I can bring to bear on them, when it 
> comes to a 4 A resolution map I'm no better than an undergrad.
> 
>   Your first experience with a new technique should never be with your 
> current project's data.  You should work to add that technique to your tool 
> box, and then move back to your data.  Practice, and more practice will build 
> that squishy neural network in your head.
> 
> Descending from soapbox,
> Dale Tronrud
> 
> 
> On 12/1/2020 8:31 AM, Robert Nicholls wrote:
>> Dear all,
>> I feel the need to respond following last week’s critique of the use of 
>> Coot’s map blurring tool for providing diagnostic insight and aiding ligand 
>> identification…
>>> On 24 Nov 2020, at 16:02, Dale Tronrud >> <mailto:de...@daletronrud.com>> wrote:
>>> 
>>> To me, this sounds like a very dangerous way to use this tool decide if a 
>>> ligand has bound.  I would be very reluctant to modify my map with a range 
>>> of arbitrary parameters until it looked like what I wanted to see.  The 
>>> sharpening and blurring of this tool is not guided or limited by theory or 
>>> data.
>> I disagree with this, subject to the important qualification that care is 
>> needed with interpretation. Blurring isn't a crime - it merely involves 
>> adjusting the weighting given to lower versus higher resolution reflections, 
>> and thus allows relaxation of the choice of high-resolution limit, and 
>> facilitates local investigation of regions that exhibit a poor 
>> signal-to-noise ratio. This is particularly pertinent to liganded compounds, 
>> which are typically present with sub-unitary occupancies.
>> Coot's blurring merely involves convolution of the whole map with an 
>> isotropic 3D Gaussian, with a parameter (B-factor) to control the standard 
>> deviation of the Gaussian. This corresponds to reweighting the structure 
>> factors in order to give higher weight to lower-resolution reflections. This 
>> approach is guided by a very simple theory: higher resolution structure 
>> factors (SFs) are typically noisier, with a worse signal-to-noise ratio than 
>> lower resolution SFs (due to increased errors in both observed 
>> higher-resolution reflections and calculated phases). Consequently, 
>> increasing the blurring B-factor reduces the effect of the noisier 
>> higher-resolution SFs. This results in a map that should be more reliable, 
>> but at the expense of reduced structural detail due to artificially reducing 
>> the effective resolution.
>> It should be noted that this does assume that lower resolution reflections 

Re: [ccp4bb] PROSMART spectrum bar

[Robert Nicholls]

Hi Chen,

> could anybody suggest me an easy way to generate the spectrum bar for the 
> color-coded residue-wise scores? Generating a spectrum bar in Pymol seems 
> nontrivial to me…

There are some HTML/javascript files that will allow you to reproduce the 
horizontal and vertical legends seen in the ProSMART publications. I've made 
them available from our website, but here's a direct link:

http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/prosmart/docs/prosmart_legends.tar.gz

You can open the "legend.html" file in a web browser, then zoom in and/or 
export to whatever format you want. You can edit the "legend.css" file if you 
want to adjust the text font. You can edit the values in the top of the 
"legend.js" file in order to change the legend scale and colours so that they 
correspond to your ProSMART run (if you have adjusted these parameters). I hope 
this is an acceptable solution for you. Let me know if you need further help 
with this.

> BTW, here is a rather unrelated question. what does the white color represent 
> (in default color setting)? Does it mean the residues that are not included 
> in the analysis?

You are correct. The white colour represents residues that were not included in 
the analysis - these were not matched to any residues in the other model.

> I might be careless, but I failed to find a documentation for this.

I'd say that the best resource for providing practical usage info is the 
tutorial that's available from our website (see part 2 of the tutorial):
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
I'll try to update and expand on the documentation soon to help clarify such 
issues.

Best regards,
Rob


--
Dr Robert A Nicholls
Career Development Fellow
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH
07729 202999


On 13 Dec 2014, at 23:55, Chen Zhao wrote:

> Hi all,
> 
> Sorry for bothering you with this small technical question. I just got to 
> know PROSMART as a handy local structural alignment tool. But could anybody 
> suggest me an easy way to generate the spectrum bar for the color-coded 
> residue-wise scores? Generating a spectrum bar in Pymol seems nontrivial to 
> me...
> 
> BTW, here is a rather unrelated question. what does the white color represent 
> (in default color setting)? Does it mean the residues that are not included 
> in the analysis? I might be careless, but I failed to find a documentation 
> for this.
> 
> Thank you so much in advance!
> 
> Best,
> Chen

Re: [ccp4bb] problen in installing ProSMART

[Robert Nicholls]

I'd recommend downloading the CCP4 suite, which contains pre-compiled ProSMART 
binaries:
http://www.ccp4.ac.uk/download/index.php#os=mac
Just download the Package Manager, which will automatically download and 
install CCP4 for you.

The problem is that the -static-libgcc flag is specific to the gcc compiler, 
but unfortunately your system is overriding gcc and using the clang compiler 
instead.

If you really want to try to compile ProSMART from source, you can try removing 
the -static-libgcc flag from the makefile. This may or may not work, depending 
on your system architecture. Alternatively, you can download and use the gnu 
gcc/g++ compiler instead of clang, which should be a more robust solution.

Regards,
Rob


On 28 May 2015, at 14:36, Kaushal, Prem (HEALTH) wrote:

> Hi
> I am trying to install ProSMART 
> (http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/), in MAC OXS Yosemite 10.10.3
>  
> It is giving following error.
> -
> ---
> Compiling prosmart_restrain
> clang: error: unsupported option '-static-libgcc'
> make: *** [restrain] Error 1
> make: *** Waiting for unfinished jobs
> 1 warning generated.
> 1 warning generated.
> 1 warning generated.
> 1 warning generated.
>  
> Your suggestion would be appreciated.
>  
> Thanks in advance
> Prem
> ---
> 
> 

Re: [ccp4bb] RMSD of side chains

[Robert Nicholls]

> Let me put it this way.  Suppose you were reading a paper about someone
> else's structures.  Which of these two statements would be more useful:
> 1) The RMSD for sidechain atoms between apo and holo was 0.678 Å.
> or
> 2) Only two residues exhibited a significant change of conformation:
>the Asn XXX carboxamide flipped 180 degrees allowing ND to act as 
>H-bond donor to ligand atom FOO;  the Lys YYY sidechain occluded
>the ligand binding site in the apo structure but extends into the
>solvent when the ligand is bound.

Certainly, in a paper, specific and detailed information regarding differences 
in particular side chain conformations would be more useful and biologically 
relevant than merely quoting an overall global measure of side chain 
dissimilarities. I am sure that no-one would think of making standalone 
statements such as "the average RMSD of side chain atoms is 0.678Å". However, 
it can be useful to calculate side chain RMSD for various purposes, whether in 
a publication, or simply for personal use during the course of a comparative 
structural analysis. It is more meaningful if side chain RMSD is calculated 
using a local coordinate frame (i.e. after local superposition), resulting in a 
measure which is independent of global conformation. This is only meaningful in 
cases where the backbone of the compared structures is locally very similar. 
For example, if this is performed for sequence-identical structures in 
different global conformations, then it is possible to use local side chain 
RMSD to identify and visualise the side chain conformational (dis)similarity 
between corresponding residues. Such information can be hard for humans to 
otherwise discern visually, since our interpretation is not independent of the 
superposition used to visualise the structures. Such information allows us to 
easily tell whether observed differences are due to differences in the backbone 
conformation, or differences in side chains, and can be easily visualised by 
colouring residues according to the side chain dissimilarity score. Certainly, 
if structures are practically near-identical in net local backbone structure, 
then interesting information can be achieved by moving to the higher level of 
structural resolution of side chain atoms. Furthermore, if wanting to compare a 
class of structures, all of which have highly conserved backbones, information 
regarding the overall similarity of side chain conformations may help in 
identifying or quantifying intraclass (dis)similarities.

> For residues where the two sequences are
> not identical, how do you even calculate an RMSD for sidechain atoms?


For sure, comparing side chains becomes less meaningful if the sequences are 
not identical - this would not be useful in most cases. Nevertheless, if the 
backbone is practically identical then some researchers performing structural 
comparisons may want to achieve some way of visualising differences in general 
side chain orientations. This can be achieved by considering the difference 
between average positions of side chain atoms between the compared residues, 
after local backbone superposition. Whilst interpretation would have to be 
thought about very carefully, such analyses can provide useful information that 
would be very hard for us to otherwise obtain (e.g. regarding side chain 
signalling patterns).

Considering side chain RMSD requires careful consideration, but can provide 
useful information.

Regards
Rob


On 13 Jan 2012, at 18:40, Ethan Merritt wrote:

> On Friday, January 13, 2012 09:07:07 am Appu kumar wrote:
>> Firstly thanks to Robert Nicholls for making me aware of the software
>> necessary for side chain RMSD calculation. I have installed and now going
>> through manual to use it for exploiting the structural differences. Thanks
>> a lot.
>> 
>> Secondly, for Ethan Merritt, I am seeking the information for comparing the
>> side chains RMSD for better comparison of structure. Please correct me if i
>> am wrong, i want to elaborate more on what i am thinking. If we have refine
>> the structure well so that issue of rotamers are  fixed
> 
> Sorry, I don't know what you mean when you say "the issue of rotamers are 
> fixed".
> 
>> , then it is
>> possible to take the advantage  of side chain orientation for correctly
>> understanding the trivial differences between homologous proteins  and such
>> differences harbouring good piece information for understanding protein
>> structure-function relationship. Any kind of suggestion would be highly
>> appreciated.
> 
> Let me put it this way.  Suppose you were reading a paper about someone
> else's structures.  Which of these two statements would be more useful:
>  1) The RMSD for sidechain atoms between apo and holo was 0.678 Å.
> or
>

Re: [ccp4bb] Refmac and sigma value

[Robert Nicholls]

Hi Uma,

Altering sigma affects the strength of geometry restraints throughout the model 
- bonds, angles, etc. Choosing a very low sigma will cause geometry to be more 
tightly restrained towards "ideal" values, which is why you observe 
improvements in Coot validation.  Note that strengthening the geometry weight 
causes the observations (data) to be less influential in refinement. The "risk" 
of this is that your model may no longer appropriately/optimally describe your 
data. You can assess this locally by manual inspection of the electron density, 
and globally by considering overall refinement statistics (as reported at the 
bottom of the Refmac5 log file). Ideally, you want your model to both describe 
the data and have reasonable geometry.

Regards
Rob


On 26 Apr 2012, at 21:26, Uma Ratu wrote:

> Hi, Alex:
>  
> > Which sigma do you mean?
>  
> The one for automatic weight, not for Jelly-body refinement.
>  
> I did not turn the "Jelly-body refinement" on.
>  
> Thanks
>  
> Ros
> 
> On Thu, Apr 26, 2012 at 4:08 PM, aaleshin  wrote:
> Hi Uma,
> Which sigma do you mean? The one for Jelly-body refinement?
> J-B sigma=0.01 means very small fraction of the gradient will be used in each 
> step. It is used usually with very low resolution (less then 3A)
> 
> Alex
> 
> On Apr 26, 2012, at 11:38 AM, Uma Ratu wrote:
> 
> >
> > Dear All:
> >
> > I use Refmac5 to refine my structure model.
> >
> > When I set the sigma value to 0.3 (as recommended from tutorial), the 
> > resulted model has many red-bars by coot validation (geometry, rotamer, 
> > especially, Temp Facotr).
> >
> > I then lower the sigma value to 0.1, the resulted model is much improved by 
> > coot validation.
> >
> > I then lower the sigma value to 0.01, the resulted model is almost perfect, 
> > by coot validation and Molprobity.
> >
> > My question is: what is the risk for very low value sigma value?
> >
> > Thank you for your advice
> >
> > Ros
> 
> 

Re: [ccp4bb] NCS rotamers

[Robert Nicholls]

Hi Tony,

If you're happy to go outside of Coot, you can use ProSMART to do this for you. 
You can view the results in a table, or coloured by residue using PyMOL or 
CCP4mg. It will also account for any backbone flexibility between the 
NCS-related copies, and tell you which side chains may be flipped. It will be 
distributed as part of the CCP4 package imminently, but for now you can get it 
from here: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Let me know if you want more info.

Regards,
Rob


On 20 Jun 2012, at 10:22, Antony Oliver wrote:

> 
> 
> Can I second that please?  I am possibly in a similar situation -
> 2.8 Angstrom structure, 6 molecules in the asymmetric unit, refining with
> ncs torsion restraint.
> It would be very useful to identify which side-chains are in different
> rotamers (without having to look at each and every side-chain).
> 
> Tony. 
> 
> ---
> Dr Antony W Oliver
> 
> Senior Research Fellow
> CR-UK DNA Repair Enzymes Group
> Genome Damage and Stability Centre
> Science Park Road
> University of Sussex
> Falmer, Brighton, BN1 9RQ
> 
> email: antony.oli...@sussex.ac.uk
> tel (office): +44 (0)1273 678349
> tel (lab): +44 (0)1273 677512
> 
> 
> 
> 
> On 6/20/12 10:04 AM, "Luca Pellegrini"  wrote:
> 
>> Hello,
>> 
>> Is there a way to flag up residues that have been modelled with different
>> side chain rotamers in two NCS-related molecules? I can use the NCS Ghost
>> Control tool to check individual residues but it would be useful to be
>> able to produce a list, so that one can zoom in on possible outliers.
>> 
>> Thanks,
>> Luca  

Re: [ccp4bb] Changing HETATM to ATOM record

[Robert Nicholls]

Hi,

Something like this may do the trick:

sed 's/HETATM/ATOM  /' current.pdb > new.pdb

Cheers
Rob


On 20 Aug 2012, at 16:14, Theresa Hsu wrote:

> Dear all
> 
> Is there any tool in CCP4 that can change all HETATM records in PDB file to 
> ATOM? I need the metals to be defined as ATOM for calculation of its normal 
> mode with elNémo.
> 
> Thank you.

Re: [ccp4bb] Jelly body refinement?

[Robert Nicholls]

Hi Nathan,

Jelly body refinement is basically a way of stabilising refinement, helping to 
achieve a more robust estimate of the curvature of the likelihood function, 
thus leading to a more robust search direction. This is similar to other 
regularisers used in refinement, e.g. geometry restraints, B-value restraints, 
external restraints. The idea is that such restraints should make refinement 
should be more robust to effects such as noisy data.

There are two fundamental differences with jelly body restraints: (1) these 
"restraints" do not affect the likelihood function, or the gradient, but are 
only applied to the 2nd derivative, thus only affecting the curvature of space; 
(2) rather than being based on externally-acquired prior knowledge, the prior 
knowledge is the current state, i.e. the current interatomic distances between 
close atoms.

The sigma controls the effective weight of the jelly body "restraints" relative 
to the other terms, geometry, x-ray, etc. Low sigma = strong weight. Try sigma 
= 0.01 - that is the current favourite value. But play around with the sigma 
and use what works best! Hope that helps.

Regards
Rob


On 23 Aug 2012, at 18:27, Nathan Pollock wrote:

> Dear experts,
> 
> Could someone explain what it is exactly that jelly body refinement
> does? I think that I understand it intuitively but want to make sure.
> In the same vein, what does jelly body refinement sigma parameter
> control? I.e., in comparison to the default sigma = 0.02, does sigma =
> 0.1 make body more or less like a jelly fish?
> 
> Thanks!
> 
> - Nate

Re: [ccp4bb] Jelly body refinement?

[Robert Nicholls]

Hi Roger,

You are correct, that *conceptually* the contribution to the target function is 
sum(w (|d|-|dcurrent|)^2)… however this is not actually applied to the target 
function. The target function remains unchanged. Only the 2nd derivative is 
affected by the jelly-body restraints.

Also, note that the refmac5 ccp4i interface quotes: "use jelly body refinement 
with sigma 0.02". You mention that "bigger w, more rigid jellyfish". This is 
correct. However, note that w is inversely related to sigma, thus it should be 
acknowledged that "smaller sigma, more rigid jellyfish"…

Also, note that the utility of such regularisers is greater when the effective 
observation-to-parameter ratio is worse, i.e. at lower resolutions. At this 
stage, it is not certain exactly what the resolution threshold is such that 
jelly-body restraints are useful. I can envisage that it not only depends on 
the resolution, but also on the quality (or noisiness) of the data. I am sure 
that there are 2.9A datasets out there that would benefit from such 
regularisers.

Cheers
Rob



On 23 Aug 2012, at 19:31, Roger Rowlett wrote:

> Garib gave a nice description of jelly-body refinement at the ACA meeting. 
> IIRC from his talk, conceptually jelly-body refinment is the equivalent of 
> adding "springs" between atoms within a certain radius of each other that 
> restrain their movement during refinement. The restraints contribute to the 
> target function curvature. The weight factor describes the contribution of 
> the restraints to the overall target function. If w=1 and and the radius of 
> atoms considered was infinity, you would have rigid body refinment. If w=0 
> you have normal uncontrained refinment. The REFMAC defaults are 4.2 A for the 
> constraints radius, and 0.02 for the weighting factor. If I understand it 
> correctly, it's basically like a slightly flexible rigid body refinement. 
> Bigger w, more rigid jellyfish. (Someone will correct me if I have this 
> wrong.)
> 
> Mathematically, the contribution to the target function is sum(w 
> (|d|-|dcurrent|)^2)  where is d is a measure of the distances between atom 
> pairs within a certain radius. The value d is the new distances and dcurrent 
> is the old distances. The value w is the weighting factor.
> 
> I have a recently obtained 2.9A dataset for which this approach might be 
> interesting to try and see how it works compared to the usual unrestrained 
> refinement and/or TLS, etc.
> 
> Cheers,
> 
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
> 
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
> 
> On 8/23/2012 1:27 PM, Nathan Pollock wrote:
>> Dear experts,
>> 
>> Could someone explain what it is exactly that jelly body refinement
>> does? I think that I understand it intuitively but want to make sure.
>> In the same vein, what does jelly body refinement sigma parameter
>> control? I.e., in comparison to the default sigma = 0.02, does sigma =
>> 0.1 make body more or less like a jelly fish?
>> 
>> Thanks!
>> 
>> - Nate

Re: [ccp4bb] Jelly body refinement?

[Robert Nicholls]

Glad to clarify!

Also, note that whilst the "springs between atoms" analogy is nice for 
visualisation purposes, and certainly helps to initially explain the concept, 
it is not technically correct. Certainly, a similar analogy would be 
appropriate for external restraints and NCS local restraints, but not for 
jelly-body restraints.

In the case of jelly-body, applying "springs between atoms" (i.e. altering the 
likelihood function) would effectively slow refinement, thus requiring more 
cycles in order to reach convergence. Consequently, only the second derivative 
is altered, and the "springs between atoms" are not actually applied. This 
helps to stabilise refinement (purely because it is a regulariser and thus 
helps robustness to noise) without overly impeding speed of convergence. Still, 
many cycles may be required…

Cheers
Rob

On 23 Aug 2012, at 19:44, Robert Nicholls wrote:

> Hi Roger,
> 
> You are correct, that *conceptually* the contribution to the target function 
> is sum(w (|d|-|dcurrent|)^2)… however this is not actually applied to the 
> target function. The target function remains unchanged. Only the 2nd 
> derivative is affected by the jelly-body restraints.
> 
> Also, note that the refmac5 ccp4i interface quotes: "use jelly body 
> refinement with sigma 0.02". You mention that "bigger w, more rigid 
> jellyfish". This is correct. However, note that w is inversely related to 
> sigma, thus it should be acknowledged that "smaller sigma, more rigid 
> jellyfish"…
> 
> Also, note that the utility of such regularisers is greater when the 
> effective observation-to-parameter ratio is worse, i.e. at lower resolutions. 
> At this stage, it is not certain exactly what the resolution threshold is 
> such that jelly-body restraints are useful. I can envisage that it not only 
> depends on the resolution, but also on the quality (or noisiness) of the 
> data. I am sure that there are 2.9A datasets out there that would benefit 
> from such regularisers.
> 
> Cheers
> Rob
> 
> 
> 
> On 23 Aug 2012, at 19:31, Roger Rowlett wrote:
> 
>> Garib gave a nice description of jelly-body refinement at the ACA meeting. 
>> IIRC from his talk, conceptually jelly-body refinment is the equivalent of 
>> adding "springs" between atoms within a certain radius of each other that 
>> restrain their movement during refinement. The restraints contribute to the 
>> target function curvature. The weight factor describes the contribution of 
>> the restraints to the overall target function. If w=1 and and the radius of 
>> atoms considered was infinity, you would have rigid body refinment. If w=0 
>> you have normal uncontrained refinment. The REFMAC defaults are 4.2 A for 
>> the constraints radius, and 0.02 for the weighting factor. If I understand 
>> it correctly, it's basically like a slightly flexible rigid body refinement. 
>> Bigger w, more rigid jellyfish. (Someone will correct me if I have this 
>> wrong.)
>> 
>> Mathematically, the contribution to the target function is sum(w 
>> (|d|-|dcurrent|)^2)  where is d is a measure of the distances between atom 
>> pairs within a certain radius. The value d is the new distances and dcurrent 
>> is the old distances. The value w is the weighting factor.
>> 
>> I have a recently obtained 2.9A dataset for which this approach might be 
>> interesting to try and see how it works compared to the usual unrestrained 
>> refinement and/or TLS, etc.
>> 
>> Cheers,
>> 
>> ___
>> Roger S. Rowlett
>> Gordon & Dorothy Kline Professor
>> Department of Chemistry
>> Colgate University
>> 13 Oak Drive
>> Hamilton, NY 13346
>> 
>> tel: (315)-228-7245
>> ofc: (315)-228-7395
>> fax: (315)-228-7935
>> email: rrowl...@colgate.edu
>> 
>> On 8/23/2012 1:27 PM, Nathan Pollock wrote:
>>> Dear experts,
>>> 
>>> Could someone explain what it is exactly that jelly body refinement
>>> does? I think that I understand it intuitively but want to make sure.
>>> In the same vein, what does jelly body refinement sigma parameter
>>> control? I.e., in comparison to the default sigma = 0.02, does sigma =
>>> 0.1 make body more or less like a jelly fish?
>>> 
>>> Thanks!
>>> 
>>> - Nate
> 

Re: [ccp4bb] Jelly body refinement?

[Robert Nicholls]

Hi Gunnar,

A couple of comments, to clarify a few of the similarities and dissimilarities 
between DEN and analogous technologies:

According to your very nice paper from 2010, DEN refinement with gamma=0 gives 
a higher weight to external information, whilst gamma=1 ignores external 
information in favour of self-restraints. Thus, unless I am mistaken, isn't it 
gamma=1 that would be more analogous to jelly-body refinement? 

Both jelly-body and DEN with gamma=1 are similar in that they are both 
independent of explicit externally-derived information. Indeed, DEN with gamma 
in [0,1] is analogous, but not equivalent, to a combination of jelly-body (or 
self-restraints) and external reference structure restraints as implemented in 
REFMAC5. In fact, jelly-body is actually quite different to DEN with gamma=1.

Since jelly-body "restraints" are not applied to the target function (or 1st 
derivative), the "restrained" atoms are allowed to move easily if there is 
evidence to suggested that they should, e.g. from the electron density, or from 
other (external) restraints. The principal purpose of jelly-body restraints is 
simply to act as a regulariser thus stabilise refinement, not to inhibit 
deformation of interatomic distances where appropriate.

Jelly-body is only applied to the 2nd derivative simply due to the form of the 
function: X=(d-d_current)^2. Note that d_current is updated at each step, thus 
we always have d=d_current. Thus, X=0, X'=0, but X''!=0. This formulation makes 
sense - in the absence of any external prior knowledge, we shouldn't change the 
likelihood function or the gradient, as we want the minima to remain in the 
same place. However, we can reasonably change the 2nd derivative, and we would 
like to benefit from the decreased effective parameter-to-observation ratio 
from this regulariser. Hopefully, that explains why jelly-body is actually 
quite different to DEN with gamma=1.

Importantly, note that with jelly-body d_current is updated/reset at each step, 
which means that the structure is indeed very deformable. The structure is 
allowed to move away from the start values - in fact, d_current at cycle n is 
not dependent on d_current at cycle 0. I believe this contrasts with DEN, 
unless kappa=1.

In contrast with jelly-body, external restraints and local NCS restraints are 
applied to the target function. In order to allow the inter-atomic distances to 
exhibit large deviations from the prior information, the Geman-McClure robust 
estimator function is used instead of assuming least squares residuals (i.e. 
parameters are estimated using generalised M-estimators instead of the 
traditional maximum likelihood method). Consequently, when using jelly-body and 
external restraints, regions of structure that need to move far should be able 
to do so, whilst the regions that are happy should remain where they are 
(ideally with more stable refinement and less overfitting) .

Hopefully that helps to clarify a few of the similarities and dissimilarities 
between DEN and the analogous technologies implemented in REFMAC5 to anyone who 
may find it useful!

Regards
Rob



On 28 Aug 2012, at 20:23, Gunnar Schroeder wrote:

> Just a quick comment on low resolution refinement: 
> 
> The concept of Deformable Elastic Network (DEN) refinement
> is quite similar to jelly-body refinement in the special case of 
> gamma=0, for which the network is not deformable.
> In contrast to jelly-body refinement, the DEN restraints are 
> however actually applied to the target function (and the first 
> derivative).
> 
> For gamma>0 the minimum of the elastic network potential 
> is allowed to move and, thus, to deform the restraints (which 
> changes their equilibrium distances).  Some individual distances 
> can deform more than others depending on the force they feel 
> from the target function. 
> 
> This automatically discriminates between those regions in the 
> structure that need to move far (and are allowed to do so) and 
> those regions that are happy where they are (and remain 
> restrained). 
> 
> DEN refinement is implemented in CNS (1.3) and 
> now also in Phenix (>=1.7.3).
> 
> Cheers,
>   Gunnar

Re: [ccp4bb] Jelly body refinement?

[Robert Nicholls]

Hi Gunnar,

I generally agree with your comments. However, I'd like to clarify a couple of 
points:

> For gamma=1 the DEN potential can follow anywhere, the entire conformational 
> space is accessible and  dij(t+1) depends only on Dij(t) and dij(t).
...
> But, again, the starting (or reference) 
> model is completely forgotten and never used after the first iteration. 


Certainly, the entire conformational space is accessible. However, I'm not so 
sure about the starting model being completely forgotten and never used after 
the first iteration. Here are my thoughts: since the DEN update formula is 
recursive, the equilibrium distance can also be written in terms of the Dij 
alone (still assuming gamma=1):
dij(t+1) = Dij(0)*(1-kappa)^(t+1) + kappa*sum_n=0^t{Dij(t+1-n)*(1-kappa)^n} 
This means that the equilibrium distance is indeed dependent on the initial 
distance Dij(0) for all times t. For values of kappa in (0,1), this dependency 
will diminish with time t, but will always exist. In fact, the equilibrium 
distance dij(t) is dependent on the whole history of the distance throughout 
the procedure, i.e. Dij(n) for n=0…t. Of course, the degree of influence of the 
historical information is controlled by kappa. Values of kappa~=0 would mean 
that the initial distance has very high weight (equilibrium distance dij(t) = 
Dij(0) in the limit kappa=0), and kappa~=1 would mean that the most recent 
distances have very high weight (equilibrium distance dij(t) = Dij(t) in the 
limit kappa=1, as you have already stated). Intermediate values of kappa will 
give various non-zero weights to the historical values of Dij.

> This also means that the position of the minima of the target function 
> are not changed by the DEN (gamma=1) restraints.


I would have thought that changing the value and gradient of the target 
function had the potential to alter the minima?

>  It is therefore usually useful to run a final minimization without 
> restraints to test whether the refinement reached a stable minimum of the 
> target function.

I agree. In the context of REFMAC5, my current favourite strategy at low 
resolution is to first use external restraints in order to aid the structure to 
adopt a more sensible conformation, but then subsequently release the external 
restraints and replace them with jelly-body restraints towards the final 
refinement stages.

> From the user perspective, I think the main difference is that DEN is 
> designed 
> to be used in simulated annealing MD refinement,  whereas jelly-body is 
> designed 
> to be used in minimization (and cannot be used for MD refinement as there are 
> no second derivatives).

I agree. Since the second derivative is utilised in ML refinement, it is 
possible to design a regulariser that has the desirable properties X=0 and X'=0 
(e.g. jelly-body refinement) in the absence of any externally-derived prior 
information. Since this is not possible in simulated annealing MD refinement, 
the analogous solution will undoubtedly have to alter X and/or X'. Either way, 
all of these 'tricks' are just designed to aid robustness and combat 
overfitting! Certainly, both approaches can give positive results when refining 
at low resolution.

Cheers
Rob



On 30 Aug 2012, at 19:43, Gunnar Schroeder wrote:

> Hi Rob, 
> 
> thank you, your comments helped a lot. 
> 
> From the Refmac5 paper I did not get the fact that d is set to d_current 
> after each step. In that case you are right, jelly-body corresponds rather to 
> DEN with gamma=1 than to gamma=0. 
> 
> And of course, a very important difference is, as you said, the fact that 
> jelly-body is applied only to the second derivative.  
> 
> However,  I would like to clarify this one point you made:
> For gamma=1 the DEN potential can follow anywhere, the entire conformational 
> space is accessible and  dij(t+1) depends only on Dij(t) and dij(t).
> The update formula is (again, for gamma=1):
> dij(t+1) = (1-kappa)*dij(t) + kappa * Dij(t+1) 
> 
> Dij(t) : distance between atom i and j and time t. 
> dij_ref : distance between atom i and j in the reference structure.
> dij(t)  : equilibrium distance of restraint between atom i and j at time t.
> 
> The parameter kappa just defines how quickly dij(t) changes, 
> i.e. kappa=1 sets  dij(t+1)= Dij(t+1)  at each time step.
> 
> The parameter kappa is usually set to 0.1, which means the restraints 
> slowly follow the atomic coordinates.  But, again, the starting (or 
> reference) 
> model is completely forgotten and never used after the first iteration. 
> This also means that the position of the minima of the target function 
> are not changed by the DEN (gamma=1) restraints. It could just take longer 
> to get there as the restraints need to be dragged along. 
> 
> For gamma<1, the situation is different, there are additional forces toward  
> the reference (could be the starting) model, in which case dij(t+1) 
> additionally 
> depends on dij_ref.   This also changes the positio

Re: [ccp4bb] Jelly body refinement?

[Robert Nicholls]

s not robust. 
I was merely investigating the exact nature of the technique.

Interestingly, note that DEN requires both the atomic coordinates to be at the 
same position AND the DEN minimum to be at the same position in order for this 
argument to hold. This is a much stronger condition than the analogous 
requirement without DEN, which would require only that the atomic coordinates 
be near the same positions in order to achieve in the same final result.

If two paths from different starting values cross such that their atomic 
coordinates are within some neighbourhood of each other, then one would hope 
that the DEN potentials would adjust themselves (without taking too many 
refinement cycles, which would depend on kappa) so that they too become within 
some neighbourhood of each other, hopefully resulting in them both converging 
to the same local minima.


> This shows that the DEN update does not depend on the starting point.


No, I believe that the DEN update does depend on the starting point. However, 
kappa can be set sufficiently large so that the dependency on the starting 
point is very low. The higher dependency on more recent historical distances 
does indeed allow the entire conformational space to be accessible, but there 
is still a dependency on the starting point, technically speaking. True, upon 
convergence, this dependency on the starting point should be negligible. 
Logically, this will result in a reduced rate of convergence during refinement, 
but hopefully with the implicit benefit of increased robustness from 
regularisation. 


Thanks for the interesting discussion! It is good to dissect these techniques, 
and hopefully it will be useful for some users out there who want to know 
exactly what is going on!
Cheers,
Rob




On 2 Sep 2012, at 20:15, Gunnar Schroeder wrote:

> Hi Rob, 
> 
>>> This also means that the position of the minima of the target function 
>>> are not changed by the DEN (gamma=1) restraints.
> 
>> I would have thought that changing the value and gradient of the 
>> target function had the potential to alter the minima?
> 
> Indeed, the target function is changed during the search, but
> once a stable minimum is found, the DEN potential needs to 
> be zero by definition and the coordinates have to sit in a minimum 
> of the original target function.  
> 
> First we note that if the DEN potential minimum is at the same position 
> as the atomic coordinates, the potential and the first derivative are zero. 
> 
> Assume the atoms are at a stable minimum of the combined energy function 
> (original target function + DEN potential with gamma=1) AND the DEN potential 
> minimum is different from the atomic positions.  Then the DEN potential 
> minimum would move towards the atomic positions, which would change the 
> combined energy function and its derivative. The atoms would not be in a 
> stable minimum anymore, which contradicts the assumption and proofs that 
> the DEN potential is always zero if the atoms are in a stable minimium of the 
> combined energy function.
> 
> 
>> Here are my thoughts: since the DEN update formula is recursive, the 
>> equilibrium distance can also be written in > terms of the Dij alone (still 
>> assuming gamma=1):
>> dij(t+1) = Dij(0)*(1-kappa)^(t+1) + kappa*sum_n=0^t{Dij(t+1-n)*(1-kappa)^n} 
>> This means that the equilibrium distance is indeed dependent on the initial 
>> distance Dij(0) for all times t. …
> 
> I hope I do not get you wrong, but with this argument 
> aren't you just saying that the path/trajectory (of both the atomic 
> coordinates 
> and the DEN potential) depends on the starting point?
> Every simulation/minimization depends on the starting point.
> In a steepest descent minimization the step size determines
> how long it takes to move away from the starting point, just like
> the parameter kappa determines how long it takes for the DEN potential
> and the atomic coordinates to move away from the starting 
> model.  I do not see the difference? Am I missing something here?
> 
> The important point is that the decision on how to move the DEN 
> minimum from one iteration (at time t) to the next (at time t+1)
> depends only on where the atoms are at t+1 and where the DEN minimum was 
> at time t.  If we assume that there is a second starting point which results
> in a minimization path that happens to cross exactly the path from the first 
> starting point (same atomic coordinates and same position of DEN minimum)
> at some time t'.  Then the new position of the DEN minimum at time t'+1 would 
> be 
> exactly at the same position that you get from the first path at time t+1.  
> This 
> shows that the DEN update does not depend on the starting point.
> 
> Cheers,
>Gunnar
> 
>

Re: [ccp4bb] Calculating water accessible volume in active site

[Robert Nicholls]

Hi Yarrow,

You can use the likes of areaimol to calculate the surface accessible area, 
with or without accounting for crystal symmetry: 
http://www.ccp4.ac.uk/html/areaimol.html

There are other alternatives, but I hope that helps,
Rob


On 8 Sep 2012, at 00:37, Yarrow Madrona wrote:

> I have a buried active site and would like to determine if there is room
> for a water molecule in various mutants. Does anyone know of a good
> program to calculate this? I have heard of GRID and VOID but have never
> used them.
> 
> Thanks.
> 
> 
> -- 
> Yarrow Madrona
> 
> Graduate Student
> Molecular Biology and Biochemistry Dept.
> University of California, Irvine
> Natural Sciences I, Rm 2403
> Irvine, CA 92697

Re: [ccp4bb] Ligand geometry obs. vs. ideal

[Robert Nicholls]

In case it helps… After you've done unrestrained refinement, you can use 
prosmart to generate external self-restraints to the current conformation 
(using the -self_restrain keyword). This is flexible - you can specify residue 
ranges, and it works for protein, ligand, DNA/RNA, waters, etc. These external 
restraints will attempt to maintain the original relative conformation 
throughout refinement. If you want any help doing this, feel free to email me 
off-board.

Cheers,
Rob


On 12 Sep 2012, at 21:11, Edwin Pozharski wrote:

> You can do unrestrained refinement in refmac, at your resolution it may be 
> OK.   If you want to keep protein restrained, you can either use harmonic 
> restraints or come up with a special cif-file for your ligand with large esd 
> targets.  There is no direct way to tell refmac to exclude specific residue 
> from restraints, at least to my knowledge.
> 
> Cheers,
> 
> Ed.
> 
> On 09/12/2012 02:44 PM, Yuri Pompeu wrote:
>> What is the best way to refine the ligand unrestrained and then generate 
>> measurements?

Re: [ccp4bb] low-resolution refinement

[Robert Nicholls]

Hi Andreas,

In your case, it sounds like a reasonable strategy would be to use external 
restraints for a few rounds of refinement (as you have done), but then release 
them and instead use jelly-body restraints. This two-stage process will help to 
initially hold your model in a sensible conformation using external restraints, 
but then gently release the structure in order to reduce further bias in later 
rounds. The immediate subsequent use of jelly-body restraints after external 
restraints will ensure that the model won't deviate too far from that sensible 
conformation, unless the data suggests otherwise.

Of course, if certain regions lose their sensible conformations in subsequent 
rounds of refinement, you can continue to use external restraints just in these 
regions.

> I substantially rebuilt a surface loop that I don't want to restrain by the 
> model.


In this case, I would recommend re-generating the external restraints, this 
time telling ProSMART not to generate restraints for these particular 
residues/regions. This can be done using the -restrain and -restrain_rm 
keywords, as described in the documentation (let me know off-board if you want 
help with this).

If you enable map sharpening then the single output MTZ file should be the 
sharpened map… I'm not sure why you are finding that the map is not displayed… 
do you see any difference between enabling/disabling map sharpening?

Cheers,
Rob


On 25 Sep 2012, at 11:19, Andreas Förster wrote:

> Dear all,
> 
> I'm making first steps in the desolate world of low-resolution refinement.  
> With dodgy 3.8A data, the magic of Phaser was able to solve the structure of 
> a complex by MR with its components as MR models. Jelly-body refinement does 
> wonders for R free.  There are three issues that I would like to get some 
> advice on:
> 
> 1)  Using external restraints calculated with ProSMART improved the structure 
> further, but I'm worried that using restraints derived from the structures 
> used for MR gets me into a sinkhole of model bias. Should it be either 
> molecular replacement or homology restraints?
> 
> 2)  Do I recalculate restraints at each round of refinement?  In particular, 
> I substantially rebuilt a surface loop that I don't want to restrain by the 
> model.
> 
> 3)  Activating map sharpening results in mtz files that look just normal and 
> open in coot after the typical map calculation break, but no maps are 
> displayed.  This is independent of the sharpening factor I choose (between 5 
> and 60).
> 
> Thanks for your help.
> 
> 
> Andreas
> 
> 
> -- 
>Andreas Förster, Research Associate
>Paul Freemont & Xiaodong Zhang Labs
> Department of Biochemistry, Imperial College London
>http://www.msf.bio.ic.ac.uk

Re: [ccp4bb] Planar restraints in Refmac

[Robert Nicholls]

In the meantime, you can compact the line by replacing "external" with "exte" 
and "residue" with "resi". Not a general solution, but may help in the short 
term.

Regards,
Rob


On 29 Nov 2012, at 11:10, Garib N Murshudov wrote:

> Yes, it can be done. I may be able to do it soon. I have to find a better 
> delimiter, + may not be good becuase it may be part of atom names.
> Another way would be: if you have more than 10-15 atoms then you can divide 
> planes into several overlapping planes
> And yet another way (perhaps better way for large planes): describe planes 
> with all possible local torsion angles with 0 or 180 degree target.
> 
> 
> regards
> Garib
> 
> 
> On 29 Nov 2012, at 10:36, andrea.p...@unina.it wrote:
> 
>> Thank you very much to all of you for your replies.
>> The external restraints mechanism works perfectly as far as you do not 
>> exceed 468 characters in a string. If one wants to restraint more than let's 
>> say 15 atoms it is not feasible!
>> 
>> Is it possible to list the atoms in a more compact way? I have in mind 
>> something like:
>> first atom CA chain A residue 25 next atom CB+C+O+X1+X2 chain B residue 50
>> or something similar.
>> 
>> Thank you again for your help.
>> 
>> Andrea
>> 
>> 
>> 
>> Quoting Garib N Murshudov :
>> 
>>> Dear Andrea
>>> 
>>> There are two ways as they were mentioned by Tim and Ian:
>>> 1) Using external restraints mechanism. You define something like (it is an 
>>> example):
>>> 
>>> external plane first atom CA chain A residue 25 next atom CB chain B 
>>> residue 50 next atom OG chain B residue 100 next atom CC chain C residue 2 
>>> sigma 0.02
>>> 
>>> You need to add one line per plane. It is better to define these restraints 
>>> in a file and then read in external keywords part of the refmac5 interface 
>>> of ccp4i
>>> 
>>> 2) Define link and use planes there. Links are defined for pairs of 
>>> residues. The best way of defining links is using JLigand that can be 
>>> downloaded from (it should be available from ccp4 6.3 also):
>>> 
>>> http://www.ysbl.york.ac.uk/mxstat/JLigand/index.html
>>> 
>>> there are very good tutorials written by Andrey Lebedev for new ligands as 
>>> well as how to define links and use them in refmac
>>> 
>>> regards
>>> Garib
>>> 
>>> 
>>> On 28 Nov 2012, at 07:03, Andrea Pica wrote:
>>> 
 Hi everybody!
 
 Is there a simple way in REFMAC to restraints a group of atoms belonging 
 to different residues to lay on a plane? Of course I would like to set the 
 sigma!
 
 Do I have to add any line in the PDB header? Is it that simple?
 
 Thank you very much!
 
 Andrea
 
 
 Andrea Pica, Ph.D. student
 University of Naples "Federico II"
 Department of Chemical Sciences - Room 1N-04
 Complesso Universitario Monte S. Angelo
 Via Cintia
 I-80126 Naples - Italy
 Phone 39-081 674269
 Fax 39-081 674090
>>> 
>>> Dr Garib N Murshudov
>>> Group Leader, MRC Laboratory of Molecular Biology
>>> Hills Road
>>> Cambridge
>>> CB2 0QH UK
>>> Email: ga...@mrc-lmb.cam.ac.uk
>>> Web http://www.mrc-lmb.cam.ac.uk
>>> 
>>> 
>>> 
>>> 
>>> 
>> 
>> 
>> 
>> Andrea Pica, Ph.D. student
>> University of Naples "Federico II"
>> Department of Chemical Sciences - Room 1N-04
>> Complesso Universitario Monte S. Angelo
>> Via Cintia
>> I-80126 Naples - Italy
>> Phone 39-081 674269
>> Fax 39-081 674090
>> 
> 
> Dr Garib N Murshudov
> Group Leader, MRC Laboratory of Molecular Biology
> Hills Road 
> Cambridge 
> CB2 0QH UK
> Email: ga...@mrc-lmb.cam.ac.uk 
> Web http://www.mrc-lmb.cam.ac.uk
> 
> 
> 
> 

[ccp4bb]

[Robert Nicholls]

Or you can use ViewHKL if you want a graphical interface.

Rob


> On 7 Mar 2019, at 12:58, Eleanor Dodson 
> <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> mtzdump hklin my.mtz
> 
> That reads the header and the first 10 lines
> 
> or if you want all as ASCII
> mtzdump hklin my.mtz > mine.hkl
> nref 1
> end
> 
> and that will list the first 1 reflections to an ascii file..
> 
> 
> On Thu, 7 Mar 2019 at 12:52, Sanaz Asadollahpour 
>  > wrote:
> Dears,
> with which program in CCP4 I can read MTZ file?
> 
> regards,
> S.A.
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 
> 



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Ramachandran outliers

[Robert Nicholls]

Dear Tereza,

It is highly recommended that you do not attempt to directly optimise the 
Ramachandran plot during refinement. Doing so would not guarantee you a better 
model, and would mean that you could no longer use the Ramachandran plot for 
validation purposes.

I suggest that you inspect each of the residues corresponding to the outliers, 
and assess the conformation, geometry and density fit of that residue and 
residues in the surrounding region. There are various tools in Coot to help you 
with this. It should be clear which regions are in need of attention (outliers 
that should be fixed) and which "outliers" should be considered acceptable. 
Indeed, ensuring that there are no Ramachandran outliers is not an 
objective/requirement for a good model.

> I refine in Refmac, using h-bond based Prosmart restraints based on PDB 
> structures (identical molecules with high resolution)

Prosmart h-bond based restraints, and Prosmart restraints based on PDB 
structures are two different things. Are you using Prosmart h-bond restraints, 
or Prosmart restraints to a high-resolution homologous model? If you're using 
restraints to a high-resolution homologue, are you generating restraints for 
all of your chains, or just some of them? If you're just generating restraints 
for some of them, then you should ensure that the others are appropriately 
restrained also.

> I use NCS, medium between AB (protein 1) and loose between CDE (protein 2).


From your mention of "medium" and "loose" NCS restraints, I'm guessing you're 
using CCP4i. Why not try using CCP4i2? This is the currently recommended and 
supported interface for CCP4 software. Don't use manual NCS restraints (medium, 
loose, etc.). Try using automatically generated local NCS restraints - we find 
they work better.

How are your R/Rfree behaving in refinement? If you're using Prosmart 
restraints to homologous models then do you need/benefit from the use of NCS 
restraints too, or are they working against each other?

Best regards,
Rob



> On 8 Mar 2019, at 09:08, Tereza Skalova  wrote:
> 
> Dear all,
> 
> I have structure at 3.3A resolution and I have ca. 35 Ramachandran outliers.
> Do you have any idea how to reduce the number?
> I refine in Refmac, using h-bond based Prosmart restraints based on PDB 
> structures (identical molecules with high resolution) and I use NCS, medium 
> between AB (protein 1) and loose between CDE (protein 2). I use overall 
> B-factor and 8 TLS groups.
> Is it possible to optimize Ramachandran plot directly in Refmac?
> 
> Thank you
> 
> Tereza Skalova
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 
> 



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] how to insert a ligand into a possible binding pocket of one protein strucure model?

[Robert Nicholls]

If you're talking about fitting into density, I suggest having a look at this 
tutorial:

https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/tutorials/ligand_tutorial/index.html
 


Regards,
Rob


> On 30 Mar 2019, at 08:49, Xavier Brazzolotto  
> wrote:
> 
> Do you mean docking ?
> 
>> Le 30 mars 2019 à 00:11, Zing > > a écrit :
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 
>> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 
> 



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Robert Nicholls]

Dear HK,

No that's not quite correct - 'occupancy group alts complete' means that both 
R120 and the ligand are constrained so that their occupancies sum to unity. In 
contrast, 'occupancy group alts incomplete' means that the occupancies of R120 
and the ligand will not be constrained to sum to unity (but the sum of their 
occupancies must be less than one). In both cases, R120 and the ligand will 
"see" each other in a certain sense. But, because they are assigned to 
different groups, it is assumed that they are present in different parts of the 
crystal. This means that they can overlap.

Assuming that the ligand is in the correct conformation, I suspect the source 
of your problem is that you are modelling the side chain of R120 as only one 
conformation. And I would also include the other atoms in the side chain - CB 
and CG.

If you are modelling the sidechain of R120 with partial occupancies, then you 
should model those side chain atoms in two alternative positions (i.e. 
representing the portions of the crystal that do/don't have the ligand bound). 
This will help to ensure that your model makes physical sense. So the ligand 
plus the alt of R120 in the portion of the crystal that contains the ligand 
would be assigned to one occupancy group, and the alt of R120 in the portion of 
the crystal that does not contain the ligand would be assigned to the second 
group. In this case it would be appropriate to specify 'occupancy group alts 
complete', because the occupancies for the two alternative conformers of R120 
should sum to unity. Although no doubt the estimation of the occupancies would 
be dominated by the ligand in this case.

Be sure to check your B-factors after occupancy refinement to make sure the 
whole picture makes sense. Assuming your current model is essentially correct, 
from visual inspection it looks like R120 will have low occupancy and low 
B-factors when the ligand is not present (or at least B-factors consistent with 
the environment), but will have high occupancy and high B-factors when the 
ligand is present.

On another note, have you considered whether that part of the ligand could be 
modelled in a slightly different conformation, or whether there could be a 
post-translation modification?

I hope that helps,
Rob


Dr Rob Nicholls
Senior Investigator Scientist
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH



> On 5 Nov 2019, at 13:36, HK  wrote:
> 
> Dear all,
> 
> I have problem with occupancy refinement by Refmac5 for an overlapping 
> electron density of part of residue (an arginine) and part of ligand 
> (tetracyclic compound) (attached figures in Dropbox with a link as shown 
> below).
> 
> https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?dl=0
> 
> I refined part of the side chain of residue 120 and ligand (chain J residue 
> 1105) with Refmac keyword as shown below. Unfortunately, the side chain of 
> arginine moves away from the density but the ligand stays in the density. As 
> far as I understood, occupancy refinement with keyword 'occupancy group alts 
> complete' means both R120 and the ligand do not meet each other.  Did I miss 
> something from the occupancy refinement keyword?
> 
> occupancy group id 1 chain A residue 120 atom NE
> occupancy group id 1 chain A residue 120 atom CZ
> occupancy group id 1 chain A residue 120 atom NH2
> occupancy group id 1 chain A residue 120 atom NH1
> occupancy group id 1 chain A residue 120 atom CD
> occupancy group id 2 chain J residue 1105
> occupancy group alts complete 1 2
> occupancy refine
> 
> Thank you for the advice.
> 
> Best regards
> HK
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Robert Nicholls]

Dear HK,

If you believe that R120 only adopts one conformation then you should not 
refine the occupancies of the side chain atoms. It makes no physical sense to 
have occupancies less than one for these atoms, but occupancy equal to one for 
the rest of the protein (unless you believe the side chain to be cleaved, or 
otherwise chemically modified). However, occupancy refinement of the ligand 
itself can be justified by partial binding.

Best regards,
Rob


> On 5 Nov 2019, at 15:55, t...@em.uni-frankfurt.de wrote:
> 
> Dear Rob,
> 
> I would like to model the alternative position for the side chain of R120 but 
> I don't really know whether the alternative conformation exist as shown in 
> the attached figure - density without the ligand and R120 overlaid with the 
> refined structures of modeled ligand and R120. The ligand was modeled in two 
> different conformations. From the density, it seems to me that the density is 
> connected or overlapped.
> 
> It should not be a post-translation modification as it is well-known that 
> there is no post-translation modification for this protein. Furthermore, the 
> crystal was obtained by co-crystallization of protein with the ligand itself. 
> The density seems to be the expected ligand.
> 
> Thanks for the advice.
> 
> Best regards
> HK
> 
> 
> Quoting Robert Nicholls :
> 
>> Dear HK,
>> 
>> No that's not quite correct - 'occupancy group alts complete' means that 
>> both R120 and the ligand are constrained so that their occupancies sum to 
>> unity. In contrast, 'occupancy group alts incomplete' means that the 
>> occupancies of R120 and the ligand will not be constrained to sum to unity 
>> (but the sum of their occupancies must be less than one). In both cases, 
>> R120 and the ligand will "see" each other in a certain sense. But, because 
>> they are assigned to different groups, it is assumed that they are present 
>> in different parts of the crystal. This means that they can overlap.
>> 
>> Assuming that the ligand is in the correct conformation, I suspect the 
>> source of your problem is that you are modelling the side chain of R120 as 
>> only one conformation. And I would also include the other atoms in the side 
>> chain - CB and CG.
>> 
>> If you are modelling the sidechain of R120 with partial occupancies, then 
>> you should model those side chain atoms in two alternative positions (i.e. 
>> representing the portions of the crystal that do/don't have the ligand 
>> bound). This will help to ensure that your model makes physical sense. So 
>> the ligand plus the alt of R120 in the portion of the crystal that contains 
>> the ligand would be assigned to one occupancy group, and the alt of R120 in 
>> the portion of the crystal that does not contain the ligand would be 
>> assigned to the second group. In this case it would be appropriate to 
>> specify 'occupancy group alts complete', because the occupancies for the two 
>> alternative conformers of R120 should sum to unity. Although no doubt the 
>> estimation of the occupancies would be dominated by the ligand in this case.
>> 
>> Be sure to check your B-factors after occupancy refinement to make sure the 
>> whole picture makes sense. Assuming your current model is essentially 
>> correct, from visual inspection it looks like R120 will have low occupancy 
>> and low B-factors when the ligand is not present (or at least B-factors 
>> consistent with the environment), but will have high occupancy and high 
>> B-factors when the ligand is present.
>> 
>> On another note, have you considered whether that part of the ligand could 
>> be modelled in a slightly different conformation, or whether there could be 
>> a post-translation modification?
>> 
>> I hope that helps,
>> Rob
>> 
>> 
>> Dr Rob Nicholls
>> Senior Investigator Scientist
>> MRC Laboratory of Molecular Biology
>> Francis Crick Avenue
>> Cambridge Biomedical Campus
>> Cambridge CB2 0QH
>> 
>> 
>> 
>>> On 5 Nov 2019, at 13:36, HK  wrote:
>>> 
>>> Dear all,
>>> 
>>> I have problem with occupancy refinement by Refmac5 for an overlapping 
>>> electron density of part of residue (an arginine) and part of ligand 
>>> (tetracyclic compound) (attached figures in Dropbox with a link as shown 
>>> below).
>>> 
>>> https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?dl=0
>>> 
>>> I refined part of the side chain of residue 120 and ligand (chain J residue 
>>> 1105) with Refma

Re: [ccp4bb] Macromolecular Crystallography workshop in South America 2020

[Robert Nicholls]

As a Caucasian male I hesitate to post; I know that there are a lot of people 
who are sensitive to this subject, so feel that I'm treading on eggshells in 
responding... Nevertheless I feel obliged to respond, having a certain amount 
of insight into the topic in the context of these workshops. 

Personally I don't find posts such as this - which simply incite (positive) 
discrimination - to be very constructive.

From speaking to organisers and being involved in committees over numerous 
years I know that gender bias is always very much at the forefront of the 
organisers' minds when deciding whom to invite. Especially where funding 
requirements and committees are involved, the issue of gender bias is always 
raised with a heavy hand. Consequently, in modern times whenever there is a 
gender bias in our field it is not a result of naivety or discriminative 
cliqueness, but rather necessity due to the availability of appropriate tutors 
(as correctly indicated by Rasmus). And as Andrew points out, organising these 
workshops takes a lot of effort, and as such it is inappropriate to treat the 
organisers with undue disrespect.

Ultimately, the objective is to teach the topics intended to be covered in the 
workshop. For sure, there are a number of very good females who are appropriate 
for teaching on these courses, despite being in the relative minority. I know a 
number of the females who are very able to tutor on such courses, and by 
personal communication I also know why some of them might not be able to attend 
this particular one this year. As Eleanor has pointed out, sometimes life gets 
in the way.

There is a very good gender balance of participants in these workshops, as is 
indicative of the demographic of practicing structural biologists wishing to 
utilise these software tools. But clearly the gender balance of tutors is 
heavily in favour of males - this represents the difference in gender ratios of 
people engaging with practical structural biology versus computational methods 
development.

I understand the argument that it is good for female tutors to be present as 
role models in such workshops, but it is worth noting that the people attending 
these workshops do not intend to become methods developers - they simply want 
to know how to best use the tools available. Consequently there is a 
qualitative difference between tutors and participants. Therefore, there is 
limited capability for the participants to see the tutors as role models in 
this context.

I feel that the junior vs senior argument isn't at all relevant. More senior 
developers will of course be generally preferred - it is necessary for 
individuals with sufficient experience to be available to help in these 
workshops (again as indicated by Rasmus). But I have seen many junior software 
developers sent to teach at CCP4 workshops (irrespective of gender). Indeed, it 
is necessary for junior developers to gain such experience in order to grow. 
This is very necessary in order to meet the demands of an expanding and 
evolving community.

From a purely statistical point of view it seems reasonable to me for the 
gender bias of tutors in such workshops to realistically reflect the 
demographic of crystallographic software developers. Consequently, I feel that 
the real problem lies in the proportion of females who want to become methods 
developers. My personal experience is that there is absolutely no 
discrimination whatsoever when interviewing or encouraging potential scientists 
to enter the field. Perhaps I have been fortunate enough to not be exposed to 
an environment where there is any such discrimination.

For me, the real problem lies in the proportion of females who are interested 
in a career in computational methods development; I believe this issue stems 
back to the proportion of females who choose theoretical/computational degrees 
at undergraduate level. I do not know how to encourage more females to engage 
with the field at earlier stages in their career - perhaps this is a problem 
for those teaching in universities to attempt to address. Fortunately I have 
seen a rise in the number of females interested in computational methods 
development in recent years (particularly in bioinformatics), which is very 
encouraging - perhaps this represents changing times...

Ultimately, positive discrimination is nevertheless discrimination. And as such 
is unethical, and should not be supported by a conscientious scientific 
community. I would prefer to see a system whereby gender, race and creed are 
not seen as a factor when deciding who should be chosen for a particular role. 
Personal merit should be the only consideration. 

If this results in a gender bias then the problem should be addressed at the 
source, not downstream. A very vague scientific analogy would be: attempting to 
correct for an experimental error, versus trying to improve the experiment so 
that the error is not such an issue.

I feel that this

Re: [ccp4bb] Macromolecular Crystallography workshop in South America 2020

[Robert Nicholls]

Dear Susan,
For one I'm already committed to 7 workshops this year so far, with more to be 
confirmed. And for sure I'm by no means an anomoly - no doubt others within the 
community would tell a similar tale. The commitment to workshops for the CCP4 
core team and various software deveopers is a burden. It's no secret that 
there's room for expansion in the computational crystallography community, and 
that CCP4 is enthusiastic to support such expansion - please contact CCP4 if 
you are enthusiastic to actively work in this direction.
Rob


> On 6 Feb 2020, at 01:10, Susan Lea  wrote:
> 
> Dear Tim
> I’m already teaching at 3 workshops this year - there are many others who 
> could do a better job (and who are also women)
> Susan
> 
> Sent from my iPhone
> 
>> On 5 Feb 2020, at 12:21, Roversi, Pietro (Dr.)  wrote:
>> 
>> 
>> Dear all,
>> 
>> my twopenny worth - when Helen Walden and I put together the 2013 CCP4 
>> Weekend list of speakers,
>> we were delighted to end up with a not-so-gender-imbalanced list of speakers,
>> but we did achieve that balance *intentionally*. 
>> 
>> As we attempt to redress a de facto longstanding bias against women in the 
>> workplace,
>> everybody in charge of this kind of decision is expected to *intentionally* 
>> look for excellent women scientists.
>> 
>> They will find them if they look - and the event will be better because of 
>> participation of women as well as
>> male scientists.
>> 
>> with best wishes,
>> 
>> Pietro
>> 
>> 
>> 
>> Pietro Roversi
>> Lecturer (Teaching and Research) https://le.ac.uk/natural-sciences/ 
>> 
>> LISCB Wellcome Trust ISSF Fellow
>>  
>> https://le.ac.uk/liscb/research-groups/pietro-roversi
>>  
>> 
>> Leicester Institute of Structural and Chemical Biology
>> Department of Molecular and Cell Biology, University of Leicester
>> Henry Wellcome Building
>> Lancaster Road, Leicester, LE1 7HB
>> England, United Kingdom
>> 
>> Tel. +44 (0)116 2297237
>> 
>> 
>>  From: CCP4 bulletin board on behalf of Tim Gruene
>> Sent: Wednesday, 05 February 2020 16:18
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Macromolecular Crystallography workshop in South 
>> America 2020
>> 
>> Dear Susan, dear Silvia,
>> 
>> why don't both of you offer to teach as tutors at this CCP4 workshop?
>> 
>> I do not believe that this 'imbalance' has been done intentionally by any of 
>> the people involved. 
>> 
>> Both of you should take a step forward, tutor in South America. In case your 
>> offer is rejected, you may start to argue.
>> 
>> Best regards,
>> Tim
>> 
>> On Wednesday, February 5, 2020 1:48:32 PM CET Susan Lea wrote:
>> > ‘Role’ models are not what are needed - just a better representation of our
>> > community. This is lazy scheduling and in a community where there have
>> > always been influential women we should do better - CCP4 has let the whole
>> > community down by allowing their name to be associated with this.
>>  Perhaps
>> > we could encourage the speakers to do as many of my colleagues have started
>> > to do - ask who else is speaking at a meeting and refuse to participate if
>> > the list does not reflect the community. 
>> > Susan
>> > 
>> > Sent from my iPhone
>> > 
>> > On 5 Feb 2020, at 07:38, Silvia Onesti  wrote:
>> > 
>> >  Maybe CCP4 should explicitely ask for gender balance before sponsoring a
>> > workshop?
>>  
>> > Silvia
>> > 
>> > -
>> > Silvia Onesti - Head of Structural Biology
>> > 
>> > Elettra - Sincrotrone Trieste S.C.p.A.
>> > AREA Science Park, 34149 Basovizza, Trieste ITALY
>> > 
>> > Email: silvia.one...@elettra.eu
>> > Tel. +39 040 3758451  Mob +39 366 6878001
>> > 
>> > http://www.elettra.trieste.it/People/SilviaOnesti 
>> > 
>> > http://www.elettra.trieste.it/labs/structural-biology 
>> > 
>> > ---
>> > 
>> > 
>> > On 5 Feb 2020, at 03:09, Alejandro Buschiazzo
>> > mailto:ale...@pasteur.edu.uy>> wrote:
>>  
>> > Thank you for your comment, we cannot agree more.
>> > 
>> > We are aware of this, and still are in the process of inviting a few more
>> > speakers.
>>  We shall  thus bring more  women on board, and achieve a
>> > balanced group! 
>> > Cheers,
>> > Alejandro (on behalf of the organizers)
>> > 
>> > 
>> > El 4 feb. 2020, a la(s) 22:02, Edward Snell
>> > mailto:esn...@hwi.buffalo.edu>> escribió:
>>  
>> > 
>> > ​It is great that this workshop is occurring but I couldn't help but notice
>> > that there seem to be a lot of male speakers and tutors. I was wondering if
>> > it might be appropriate to add some female role models. There are some
>> > great candidates?
>>  
>> > 
>> > 
>> >