Re: [ccp4bb] RES: [ccp4bb] About model building

[Sam Tang]

Dear all

Thanks again for more input to the question. And credits to Eleanor and Kay
for pointing out the high R-factor and possible issue with the space group.
Their advice prompted me to revisit the MR solution and it happens that
another solution, in P3121, gave a better map with R-factor after one round
of refinement being 0.33/0.36, which was a remarkable difference with the
P321 solution (R~0.5).

So the next step I would take would be to re-try Arp/warp and see if things
work out with poly-A. I shall update the community with the outcome.

Kind regards

Sam




On Tue, 7 Nov 2023 at 01:13, Rafael Marques 
wrote:

> Hi Sam.
>
>
>
> If you still have any of your crystals or any protein solution left in the
> well you harvested your crystals, I would run a MS/MS with them. Next step
> would be to run AF with your known chain A and your best Mass Spec hit (s),
> and use the resulting model for MR.
>
>
>
> Good luck
>
>
>
>
> Rafael Marques da Silva
>
> PhD Student – Structural Biology
>
> University of Leicester
>
>
>
> Mestre em Física Biomolecular
>
> Universidade de São Paulo
>
>
>
> Bacharel em Ciências Biológicas
>
> Universidade Federal de São Carlos
>
>
>
> phone: +55 16 99766-0021
>
>
>
> *   "A sorte acompanha uma mente bem treinada"*
>
> **
>
>
> --
> *De:* CCP4 bulletin board  em nome de Boaz Shaanan
> 
> *Enviado:* Monday, November 6, 2023 2:41:43 PM
> *Para:* CCP4BB@JISCMAIL.AC.UK 
> *Assunto:* Re: [ccp4bb] About model building
>
> Hi,
> If you still have crystals left, you could soak crystals with KI3 and
> collect data at Cu wavelength for SAD phasing, which could help you to
> resolve the missing piece. Maybe.
> Cheers,
> Boaz
>
> Boaz Shaanan, Ph.D.
> Dept. of Life Sciences
> Ben Gurion University
> Beer Sheva, Israel
>
> On Nov 4, 2023 10:04, Sam Tang  wrote:
> Dear community,
>
> I am solving the structure of a complex between proteins A and B, where A
> is a protein with known homologs and B is a novel protein isolated from
> plant. The diffraction data was at 1.9 Ang collected in-house, indexed to
> P321. Using A as the search model, we have got a reasonable solution where,
> after one round of refinement, the A chain fits the map pretty well. What's
> left was to extend the termini and fit a few rotamers.
>
> For protein B (B chain) I have tried the web version of ARP/wARP but the
> outcome was not really good. The model was not successfully built as
> indicated by low model completeness and score. The tricky thing may be that
> we do not have the complete sequence information of this protein B in-hand.
> (The other way round, we more or less wish to rely on the high resolution
> data to confirm its sequence.) What approach would you then recommend to
> build the B chain in this scenario?
>
> Thanks in advance and best regards,
>
> Sam
>
> --
>
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Re: [ccp4bb] About model building

[Sam Tang]

Dear all

Thanks for all the input. I will definitely try out in the coming couple of
days. To provide more information:
- the data was checked in Xtriage and no major pathology was found.
Completeness was 99.2% with multiplicity of >13. Mean I/sigma(I) 18.8,
CC1/2 for outer shell 0.902.
- After one round of refinement, the R-factor was around 0.5, which looked
reasonable given the incorrect B-chain was not removed and sequence
deviations in chain A not yet rectified.
- While I said Arp/warp failed to rebuild the model, it did return 3
helices in chain A and one in chain B when I ran in the QuickFold mode
(secondary structure tracing) later on. So I am going to start with the
helix in chain B and see how further manual rebuild goes.

Thanks again and I shall send an update for any progress.

Kind regards

Sam


On Sun, 5 Nov 2023 at 06:26, Firdous Tarique 
wrote:

> Do the mass spec of your crystal to identify the other protein. Once done
> solve your structure and build the complete model. This should be straight
> forward and quick.
>
> Best Wishes
>
> On Sat, 4 Nov 2023, 09:05 Sam Tang,  wrote:
>
>> Dear community,
>>
>> I am solving the structure of a complex between proteins A and B, where A
>> is a protein with known homologs and B is a novel protein isolated from
>> plant. The diffraction data was at 1.9 Ang collected in-house, indexed to
>> P321. Using A as the search model, we have got a reasonable solution where,
>> after one round of refinement, the A chain fits the map pretty well. What's
>> left was to extend the termini and fit a few rotamers.
>>
>> For protein B (B chain) I have tried the web version of ARP/wARP but the
>> outcome was not really good. The model was not successfully built as
>> indicated by low model completeness and score. The tricky thing may be that
>> we do not have the complete sequence information of this protein B in-hand.
>> (The other way round, we more or less wish to rely on the high resolution
>> data to confirm its sequence.) What approach would you then recommend to
>> build the B chain in this scenario?
>>
>> Thanks in advance and best regards,
>>
>> Sam
>>
>> --
>>
>> 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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[ccp4bb] About model building

[Sam Tang]

Dear community,

I am solving the structure of a complex between proteins A and B, where A
is a protein with known homologs and B is a novel protein isolated from
plant. The diffraction data was at 1.9 Ang collected in-house, indexed to
P321. Using A as the search model, we have got a reasonable solution where,
after one round of refinement, the A chain fits the map pretty well. What's
left was to extend the termini and fit a few rotamers.

For protein B (B chain) I have tried the web version of ARP/wARP but the
outcome was not really good. The model was not successfully built as
indicated by low model completeness and score. The tricky thing may be that
we do not have the complete sequence information of this protein B in-hand.
(The other way round, we more or less wish to rely on the high resolution
data to confirm its sequence.) What approach would you then recommend to
build the B chain in this scenario?

Thanks in advance and best regards,

Sam



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Re: [ccp4bb] Add hydrogens

[Sam Tang]

Have just tried out some of the options -- done within minutes! Many thanks
for the numerous input!

All the best

Sam


On Wed, 29 Sept 2021 at 19:03, Sam Tang  wrote:

> Dear community
>
> This may appear to be a silly question -- I am trying to add hydrogens to
> the structure in PDB 1CDW. My initial thought is to run a single run of
> refinement with a refinement program. It happens that I cannot locate the
> map coefficients under the entry (am I missing something?) So... is there
> an easy way to do what I want in this case?
>
> Warm regards
>
> Sam
>



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[ccp4bb] Add hydrogens

[Sam Tang]

Dear community

This may appear to be a silly question -- I am trying to add hydrogens to
the structure in PDB 1CDW. My initial thought is to run a single run of
refinement with a refinement program. It happens that I cannot locate the
map coefficients under the entry (am I missing something?) So... is there
an easy way to do what I want in this case?

Warm regards

Sam



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[ccp4bb] off-topic: structural motif / domain comparison

[Sam Tang]

Dear all

Sorry for an off-topic question here. I wonder if anyone may be aware of
any search program which allows one to 'blast' a protein domain just like
we 'blast' a protein sequence? For example I have an epitope in hand and
would like to find out whether this also exists in other proteins. Most
programs I accessed are based on sequence similarity but is there any
program which searches a structure against a database of structures?

BRs

Sam



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Re: [ccp4bb] off-topic: glycans

[Sam Tang]

Thanks David! This is exactly what I am looking for.

Sam


On Tue, 29 Jun 2021 at 19:35, David Briggs  wrote:

> Hi Sam,
>
> GlycoMod from Expasy sounds like it might do what you want to do.
>
> https://web.expasy.org/glycomod/
>
> D
>
> --
>
> *Dr David C. Briggs*
>
> Senior Laboratory Research Scientist
>
> Signalling and Structural Biology Lab
>
> The Francis Crick Institute
>
> London, UK
>
> ==
>
> Diamond User Committee (MX)
>
> CCP4 WG2
>
> ==
>
> about.me/david_briggs
> --
> *From:* CCP4 bulletin board  on behalf of Sam Tang
> 
> *Sent:* 29 June 2021 06:12
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] off-topic: glycans
>
>
> *External Sender:* Use caution.
>
> Dear community
>
> Sorry for an off-topic question here. I wonder if anyone may be aware of
> any glycan modification database where we can predict what is what. For
> example, if I got a mass difference of m/z X on LC-MS, and I would like to
> have a rough idea what it might be, where should I go for?
>
> Thanks!
>
> BRs
>
> Sam
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] off-topic: glycans

[Sam Tang]

Dear community

Sorry for an off-topic question here. I wonder if anyone may be aware of
any glycan modification database where we can predict what is what. For
example, if I got a mass difference of m/z X on LC-MS, and I would like to
have a rough idea what it might be, where should I go for?

Thanks!

BRs

Sam



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Re: [ccp4bb] High Rs

[Sam Tang]

Dear all

Thanks a lot for the numerous input. Will try the different strategies and
get back again soon.

BRs

Sam


On Thu, 1 Apr 2021 at 20:28, Sam Tang  wrote:

> Dear all
>
> I have a dataset processed to 2.2 A, P212121, with no major issues
> identified by Xtriage (no tNCS, no twinning, no ice ring, good
> completeness). Phaser-MR gave a good solution except some loop regions are
> shifted. There is only 1 molecule in the ASU (and seemingly no more
> molecule can be accommodated). I fitted the displaced loops, and confirmed
> the space group by Zanuda, but the R-factors stuck at 0.34/0.41 range.
> What other aspects should I look into? Twin-refinement? (I am a bit
> reluctant to do so because neither Xtriage nor Pointless report it is
> twinned) I should also point out that the crystal was grown with its ligand
> but I cannot see good density for it.
>
> BRs
>
> Sam
>



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[ccp4bb] High Rs

[Sam Tang]

Dear all

I have a dataset processed to 2.2 A, P212121, with no major issues
identified by Xtriage (no tNCS, no twinning, no ice ring, good
completeness). Phaser-MR gave a good solution except some loop regions are
shifted. There is only 1 molecule in the ASU (and seemingly no more
molecule can be accommodated). I fitted the displaced loops, and confirmed
the space group by Zanuda, but the R-factors stuck at 0.34/0.41 range.
What other aspects should I look into? Twin-refinement? (I am a bit
reluctant to do so because neither Xtriage nor Pointless report it is
twinned) I should also point out that the crystal was grown with its ligand
but I cannot see good density for it.

BRs

Sam



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[ccp4bb] unknown density

[Sam Tang]

Hello fellow colleagues

Hope you are all well while the pandemics persists. I just wonder if anyone
may have an idea what this density (looking like a pentagon) might be. The
data was collected to 1.8 A and crystal was grown in Bis-tris + PEG3350.
Imidazole residual? Nucleotide (the protein itself is nucleotide-binding,
but shouldn't be at this particular site)?

https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing

Thanks!

BRS

Sam



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[ccp4bb] Saving multi-state obj in Pymol

[Sam Tang]

Hi

Not sure if it is the right place to go but I'm sure I can get some help
here. I am trying to combine two multi-state objects and save it to a new
object. It happens that the molecule is not correctly displayed after Save.
Would anyone have an idea what may have gone wrong?

https://drive.google.com/file/d/1fz1oaSa4ahuEe1SRK1Nlvrmrb9KESEq_/view?usp=sharing

(PS - I checked in Chimera and found that both states have been saved and
the display in Chimera is OK. So I believe it is something to do with
Pymol?)

Thanks.

Sam



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Re: [ccp4bb] PISA

[Sam Tang]

Dear Jon

Thanks for the suggestion and that works for me to calculate the interface
area and other parameters I need. Many thanks!

Best regards

Sam


On Wed, 20 Jan 2021 at 22:38, Jon Cooper 
wrote:

> Dear Sam
>
> I can only suggest trying the old trick of editing the pdb file and giving
> all the DNA bases the same residue number. As you are using the old gui you
> might be able to do the same with pdbset or pdbcur, or maybe a graphics
> program.
>
> Cheers, Jon Cooper
>
>
> Sent from ProtonMail mobile
>
>
>
>  Original Message 
> On 20 Jan 2021, 13:27, Sam Tang < samtys0...@gmail.com> wrote:
>
>
> Dear community
>
> I ran PISA from CCP4i to analyze a protein-ssDNA complex with non-standard
> modified nucleotides. It turns out that PISA identifies each nucleotide in
> the DNA chain as a ligand (i.e. I have 7 DNA bases and PISA calls out 7
> ligands). May I solicit your experience if there is a way to force PISA to
> recognize the DNA as one single ligand? Thanks a lot.
>
> Best regards
> Sam
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] PISA

[Sam Tang]

Dear community

I ran PISA from CCP4i to analyze a protein-ssDNA complex with non-standard
modified nucleotides. It turns out that PISA identifies each nucleotide in
the DNA chain as a ligand (i.e. I have 7 DNA bases and PISA calls out 7
ligands). May I solicit your experience if there is a way to force PISA to
recognize the DNA as one single ligand? Thanks a lot.

Best regards
Sam



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[ccp4bb] over-fitting? over-refinement?

[Sam Tang]

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?

BRS

Sam



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Re: [ccp4bb] Element type N+1

[Sam Tang]

Hello,

When I further refine my structure the N+1 is gone and it becomes a
normal N again. So it is very much possible this is related to geometry.
Thanks everyone (Esp. to Jon) for the input!

Sam


On Fri, 16 Oct 2020 at 19:31, Jon Cooper 
wrote:

> Hello Sam
>
> thanks for the pictures. It looks like the guanidinium group of the first
> Arg is not very planar. I don't know the detailed nuts-and-bolts of how
> Coot and Refmac handle this but the problem may arise from the initial
> geometry at the affected nitrogen being off-target.
>
> Best wishes, Jon Cooper. jon.b.coo...@protonmail.com
>
>
>
>
>
>  Original Message 
> On 16 Oct 2020, 06:57, Sam Tang < samtys0...@gmail.com> wrote:
>
>
> Dear all
>
> I attach herewith the Arg concerned. I actually saw no issue in Coot when
> I checked residue info. The issue was repeatable using both Refmac from
> Coot and from CCP4i. I rectified the issue simply by changing "N+1" back to
> "N" using a word editor, but would surely be glad to know more about the
> cause of the problem.
>
>
> https://drive.google.com/file/d/137Q0CybNynOlI0R-b-3-OL2eWk7f6L2a/view?usp=sharing
>
> Best Regards
>
> Sam
>
>
> On Fri, 16 Oct 2020 at 00:51, Jon Cooper 
> wrote:
>
>> Hello, can you possibly show us a couple of screenshots with atom labels
>> of the affected side chain and a normal one?
>>
>> Best wishes, Jon Cooper. jon.b.coo...@protonmail.com
>>
>>
>>
>>
>>
>>  Original Message 
>> On 15 Oct 2020, 15:07, Sam Tang < samtys0...@gmail.com> wrote:
>>
>>
>> Dear colleagues
>>
>> I am trying to refine a structure with Refmac and the work completes
>> without any warning. However I am a bit puzzled for one single N atom on an
>> Arg residue the element type becomes N+1. This doesn't happen on my another
>> NCS chain and the input PDB seems fine. Could anyone kindly point me to the
>> possible cause?
>>
>> Thanks in advance!
>>
>> BRS
>>
>> Sam
>>
>> --
>>
>> 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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>
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Re: [ccp4bb] Element type N+1

[Sam Tang]

Dear all

I attach herewith the Arg concerned. I actually saw no issue in Coot when I
checked residue info. The issue was repeatable using both Refmac from Coot
and from CCP4i. I rectified the issue simply by changing "N+1" back to "N"
using a word editor, but would surely be glad to know more about the cause
of the problem.

https://drive.google.com/file/d/137Q0CybNynOlI0R-b-3-OL2eWk7f6L2a/view?usp=sharing

Best Regards

Sam


On Fri, 16 Oct 2020 at 00:51, Jon Cooper 
wrote:

> Hello, can you possibly show us a couple of screenshots with atom labels
> of the affected side chain and a normal one?
>
> Best wishes, Jon Cooper. jon.b.coo...@protonmail.com
>
>
>
>
>
>  Original Message 
> On 15 Oct 2020, 15:07, Sam Tang < samtys0...@gmail.com> wrote:
>
>
> Dear colleagues
>
> I am trying to refine a structure with Refmac and the work completes
> without any warning. However I am a bit puzzled for one single N atom on an
> Arg residue the element type becomes N+1. This doesn't happen on my another
> NCS chain and the input PDB seems fine. Could anyone kindly point me to the
> possible cause?
>
> Thanks in advance!
>
> BRS
>
> Sam
>
> --
>
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[ccp4bb] Element type N+1

[Sam Tang]

Dear colleagues

I am trying to refine a structure with Refmac and the work completes
without any warning. However I am a bit puzzled for one single N atom on an
Arg residue the element type becomes N+1. This doesn't happen on my another
NCS chain and the input PDB seems fine. Could anyone kindly point me to the
possible cause?

Thanks in advance!

BRS

Sam



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[ccp4bb] ccp4 and ccpEM on same workstation?

[Sam Tang]

Hi,

I'm going to install ccpEM on a same fedora 30 workstation that already has
ccp4 will they touch on my pre-existing Coot and Refmac setup?

Maybe I'm just worrying too much..?

Cheers

Sam



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[ccp4bb] disulfides in coot

[Sam Tang]

Dear all

A very technical question which I believe a few simple mouse clicks would
solve. Is there a way I can ask Coot not to build the disulfide linkage
automatically (which lies within a strong red density)?

My WinCoot is version 0.8.9.2

Many thanks!

Regards

Sam



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Re: [ccp4bb] resolution

[Sam Tang]

Dear all

Hello again

Thanks a lot for the numerous input.

I received a dataset which was processed to 2.4A but refined to 3A -- this
was the background I raised this question in the first place. Then I looked
at the aimless statistics. At 2.4A the high resolution bin CC1/2 0.626,
I/sigI 2.0, Completeness 84.6, Multiplicity 1.7 (P1 spacegroup).  I suspect
the reason for the refinement resolution limit to be set at 3 A was simply
due to better Rw/Rf (0.236/0.294 at 3A; 0.284/0.341 at 2.4A).

Based on these information am I justified to say that data quality at 2.4 A
was suboptimal? In this case do you think refining at a (much) lower
resolution is acceptable?

Best regards

Sam

On Fri, 5 Jul 2019 at 13:43, Sam Tang  wrote:

> Hello everyone
>
> 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?
>
> Thanks!
>
> Sam Tang
>



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[ccp4bb] resolution

[Sam Tang]

Hello everyone

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?

Thanks!

Sam Tang



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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] High Rfree - ice ring

[Sam Tang]

Hello again.

I agree the get-around strategy we took is not a good practice at all.

For our initial imosflm run we actually turned on 'exclude ice ring'
button. The following was reported in the log:


ICE RING SUMMARY:

 reso  ice_ring  mean_I mean_Sigma Estimated_I   Ratio Zscore Completeness
Ave_Completeness
 3.88   yes39933.043690.30 1398.21   28.56  10.440.48
nan
 3.67   yes44809.764257.56  778.04   57.59  10.340.58
nan
 3.43   yes 7270.25 885.61  532.75   13.65   7.610.54
nan
 2.66   yes 2070.19 488.66  156.09   13.26   3.920.46
nan


A total of >2200 reflections were already omited.

The range of poor R seems to correlate to the range 3.8-3.6A. I am thus
also thinking there may be other issues (not visibly identified on images)
other than ice rings.

We actually first merged the two datasets (high and low resolutions) in
pointless before presenting to aimless.

We are trying over other different strategies to see if we can get a better
tackle. Will report again soon.

Sam


On Tue, 9 Apr 2019 at 18:47, Johan Turkenburg <
2a539df422fe-dmarc-requ...@jiscmail.ac.uk> wrote:

>
> I agree with Harry that an ice ring should never require you to process
> the data in two separate runs, and hopefully this does not become a
> standard approach..
>
> How did you present those data to aimless so it could scale the two
> datasets that have no overlap at all?
>
> Johan
>
> On Tue, 9 Apr 2019 at 10:07, Harry Powell <
> 193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> Hi Sam
>>
>> Did you use the ice-ring exclusion option in iMosflm (a button that has
>> an image like a snowflake)? It should exclude data in _narrow_ resolution
>> rings (substantially less than 0.2Å!) around the ice rings, and can be set
>> for any combination of indexing, refinement and integration. There should
>> not be any need to process the data twice, once for the low resolution data
>> and once for the high.
>>
>> Harry
>> --
>> Dr Harry Powell
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>> On 8 Apr 2019, at 19:50, Sam Tang wrote:
>>
>> Hello everyone
>>
>> Thanks a lot for your input and advices. To report on how we tackled the
>> issue -
>>
>> (1) We used imosflm to integrate the data.
>> (2) We eventually integrated the data in two resolution ranges, say
>> 45A-3.5A, and 3.3A-3A, and merge them by Aimless. I must add that indeed
>> from the log file for our initial round the program had already identified
>> some ice ring regions.
>>
>> Aimless statistics looked fine and we were able to get a MR solution
>> which was refined to much better Rf/Rw.
>>
>> This is definitely not a smart solution because we effectively 'throw
>> away' useful data between 3.5A-3.3A, but for the purpose of MR and
>> refinement, it seems we have solved (or simply bypassed?) the problem.
>>
>> Suggestions on XDS/DIALS are appreciated. We are actually using this
>> dataset as a test set for XDS/DIALS to deal with ice rings. Will further
>> report if we've got anything interesting.
>>
>> Thanks again!
>>
>> Sam
>>
>>
>>
>>
>> On Thu, 4 Apr 2019 at 20:54, Clemens Vonrhein 
>> wrote:
>>
>>> Dear all,
>>>
>>> And if you want to process with XDS: autoPROC [1] will try to detect
>>> and exclude ice-rings automatically - if present [2].
>>>
>>> If you know that you have ice-rings you can force it [3] to exclude
>>> all known ice-rings ranges - but this might not be the best solution
>>> if you have "just" diffuse ice-rings (where the special treatment of
>>> background within DIALS might be better). Something to test and
>>> compare maybe?
>>>
>>> Cheers
>>>
>>> Clemens
>>>
>>> [1] https://www.globalphasing.com/autoproc/
>>>
>>> https://www.globalphasing.com/autoproc/wiki/index.cgi?IceRingHandling
>>> [2]
>>> https://www.globalphasing.com/autoproc/manual/autoPROC7.html#step1_spotnohkl
>>>
>>> https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Ice_rings
>>> [3]
>>> https://www.globalphasing.com/autoproc/manual/appendix1.html#SetvarParameter_XdsExcludeIceRingsAutomatically
>>>
>>> On Thu, Apr 04, 2019 at 10:51:19AM +, melanie.voll...@diamond.ac.uk
>>> wrote:
>>> > Dea

Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] High Rfree - ice ring

[Sam Tang]

Hello everyone

Thanks a lot for your input and advices. To report on how we tackled the
issue -

(1) We used imosflm to integrate the data.
(2) We eventually integrated the data in two resolution ranges, say
45A-3.5A, and 3.3A-3A, and merge them by Aimless. I must add that indeed
from the log file for our initial round the program had already identified
some ice ring regions.

Aimless statistics looked fine and we were able to get a MR solution which
was refined to much better Rf/Rw.

This is definitely not a smart solution because we effectively 'throw away'
useful data between 3.5A-3.3A, but for the purpose of MR and refinement, it
seems we have solved (or simply bypassed?) the problem.

Suggestions on XDS/DIALS are appreciated. We are actually using this
dataset as a test set for XDS/DIALS to deal with ice rings. Will further
report if we've got anything interesting.

Thanks again!

Sam




On Thu, 4 Apr 2019 at 20:54, Clemens Vonrhein 
wrote:

> Dear all,
>
> And if you want to process with XDS: autoPROC [1] will try to detect
> and exclude ice-rings automatically - if present [2].
>
> If you know that you have ice-rings you can force it [3] to exclude
> all known ice-rings ranges - but this might not be the best solution
> if you have "just" diffuse ice-rings (where the special treatment of
> background within DIALS might be better). Something to test and
> compare maybe?
>
> Cheers
>
> Clemens
>
> [1] https://www.globalphasing.com/autoproc/
> https://www.globalphasing.com/autoproc/wiki/index.cgi?IceRingHandling
> [2]
> https://www.globalphasing.com/autoproc/manual/autoPROC7.html#step1_spotnohkl
> https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Ice_rings
> [3]
> https://www.globalphasing.com/autoproc/manual/appendix1.html#SetvarParameter_XdsExcludeIceRingsAutomatically
>
> On Thu, Apr 04, 2019 at 10:51:19AM +, melanie.voll...@diamond.ac.uk
> wrote:
> > Dear Sam,
> >
> >
> > to continue from James Parkhurst's email...
> >
> >
> > You can do more analysis regarding ice rings using Auspex (
> https://www.auspex.de/) if you already have some integrated file.
> >
> > Regarding re-integrating images, what did you use the first time round?
> I think if you use DIALS it got some clever implementation in it that is
> better in estimating the errors of reflections in proximity to ice rings.
> Hence you should get better Rfactors without having to remove the effected
> resolution range and the data it covers (
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5619854/).
> >
> >
> > HTH
> >
> >
> > M
> >
> >
> > 
> > From: CCP4 bulletin board  on behalf of
> herman.schreu...@sanofi.com 
> > Sent: 04 April 2019 10:26:09
> > To: ccp4bb
> > Subject: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] High Rfree - ice ring
> >
> >
> > Dear Sam,
> >
> >
> >
> > I would remove the ice ring and reprocess the data. Ice rings may wreak
> havoc with scaling so at minimum you have to redo the scaling.
> >
> >
> >
> > Best,
> >
> > Herman
> >
> >
> >
> > Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
> Sam Tang
> > Gesendet: Donnerstag, 4. April 2019 11:01
> > An: CCP4BB@JISCMAIL.AC.UK
> > Betreff: [EXTERNAL] Re: [ccp4bb] High Rfree - ice ring
> >
> >
> >
> >
> > Dear Eleanor and Eric
> >
> >
> >
> > Thanks for your replies.
> >
> >
> >
> > Yes indeed when we looked at the plots e.g. R factor vs resln there was
> a sharp peak near 3.6 - 3.8 A which is where we visibly saw an ice ring on
> the image. Thus our first thought was to remove the ic ring. (either
> reprocess or can we bypass this resolution range during refinement?)
> >
> >
> >
> > The protein is 50 kDa, two molecule in the ASU, seemingly no obvious
> density was unassigned. We got ~3 total observations, ~15000 unique
> observations. NCS restraints was applied.
> >
> >
> >
> > Best regards
> >
> > Sam
> >
> >
> >
> >
> >
> >
> >
> > On Thu, 4 Apr 2019 at 08:57, Eric Montemayor  <mailto:montemayor.e...@gmail.com>> wrote:
> >
> > That’s a rather large gap between Rwork and Rfree.  I suspect you have
> mis-assigned your space group and as a result have a large number of copies
> in your asymmetric unit.  Any structure can be solved in P1, but that does
> not mean the true space group is indeed P1. If you use P1 when it’s not
> actually P1, you will have an unnecessarily overparamerized model, hence
> the lar

Re: [ccp4bb] High Rfree - ice ring

[Sam Tang]

Dear Eleanor and Eric

Thanks for your replies.

Yes indeed when we looked at the plots e.g. R factor vs resln there was a
sharp peak near 3.6 - 3.8 A which is where we visibly saw an ice ring on
the image. Thus our first thought was to remove the ic ring. (either
reprocess or can we bypass this resolution range during refinement?)

The protein is 50 kDa, two molecule in the ASU, seemingly no obvious
density was unassigned. We got ~3 total observations, ~15000 unique
observations. NCS restraints was applied.

Best regards
Sam



On Thu, 4 Apr 2019 at 08:57, Eric Montemayor 
wrote:

> That’s a rather large gap between Rwork and Rfree.  I suspect you have
> mis-assigned your space group and as a result have a large number of copies
> in your asymmetric unit.  Any structure can be solved in P1, but that does
> not mean the true space group is indeed P1. If you use P1 when it’s not
> actually P1, you will have an unnecessarily overparamerized model, hence
> the large gap between Rwork and Rfree.
>
> Questions:
> 1- how many copies in your asymmetric unit in P1?
> 2- how many atoms in your model vs number of unique reflections?
> 3- if more than one copy per asymmetric unit, are you imposing NCS
> restraints during refinement?
>
> -Eric
>
>
>
> On Wed, Apr 3, 2019 at 1:41 PM Sam Tang  wrote:
>
>> Hi everyone again
>>
>> Hmmm I think we have solved a structure in P1 space, to 2.5 A. However
>> after refinement the Rfree stuck at 33%-35% with Rwork around 26%. The
>> structure was solved by MR and current model seems to fit density well. In
>> Refmac log I found that at the resolution corresponding to high R there may
>> be a solvent/ice ring. Since imosflm should be able to exclude ice rings, I
>> am not 100% sure whether it's the cause to high R. But if this is actually
>> the case, is there a way I can exclude certain resolution bins during
>> Refmac (and is it an appropriate way to do so?)
>>
>> PS - the data is not affected by twining or pseudosymmetry as checked by
>> Xtriage.
>>
>> Many thanks!
>>
>> Sam
>>
>> --
>>
>> 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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[ccp4bb] High Rfree - ice ring

[Sam Tang]

Hi everyone again

Hmmm I think we have solved a structure in P1 space, to 2.5 A. However
after refinement the Rfree stuck at 33%-35% with Rwork around 26%. The
structure was solved by MR and current model seems to fit density well. In
Refmac log I found that at the resolution corresponding to high R there may
be a solvent/ice ring. Since imosflm should be able to exclude ice rings, I
am not 100% sure whether it's the cause to high R. But if this is actually
the case, is there a way I can exclude certain resolution bins during
Refmac (and is it an appropriate way to do so?)

PS - the data is not affected by twining or pseudosymmetry as checked by
Xtriage.

Many thanks!

Sam



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Re: [ccp4bb] Refmac dictionary

[Sam Tang]

Hi

My apologies. This is indeed a silly question. It appears I forgot to
remove the extra O when linking them together!

Sam


On Wed, 3 Apr 2019 at 07:21, Sam Tang  wrote:

> Dear all
>
> Hello again.
>
> We have another protein-RNA dataset which we are trying to refine. For
> this dataset we have three OMU nucleotides modelled. We got the monomer
> from Coot 'Get Monomer'. Refmac returned the following error:
> ERROR : atom :OP3  OMU 2  CCC  is absent in the library
> ERROR : atom :OP3  OMU 3  CCC  is absent in the library
>
> The library version is 5.44. I tried to download a OMU.cif (from this link
> https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/Dictionary/dictionary.html)
> for use in Refmac but the same error occurs.
>
> So... what should I try now?
>
> Many thanks in advance!
>
> Sam
>



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[ccp4bb] Refmac dictionary

[Sam Tang]

Dear all

Hello again.

We have another protein-RNA dataset which we are trying to refine. For this
dataset we have three OMU nucleotides modelled. We got the monomer from
Coot 'Get Monomer'. Refmac returned the following error:
ERROR : atom :OP3  OMU 2  CCC  is absent in the library
ERROR : atom :OP3  OMU 3  CCC  is absent in the library

The library version is 5.44. I tried to download a OMU.cif (from this link
https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/Dictionary/dictionary.html)
for use in Refmac but the same error occurs.

So... what should I try now?

Many thanks in advance!

Sam



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[ccp4bb] modelling of modified RNA

[Sam Tang]

Hello

I have been refining a protein-RNA complex with Refmac. The RNA we used was
tagged with a Cy label. I modelled the RNA in Coot using the RCrane add-on
and manually built the Cy tag. However after Refmac (also via Coot) the
linkage between Cy and the RNA was always broken.

I guess I must have missed something obvious. Any suggestions would be
appreciated..

Sam



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Re: [ccp4bb] Weird diffraction pattern

[Sam Tang]

Dear all

Thanks for all the input both on- and off- the list. We shall definitely
look into these suggestions further and report again here in due course.

Kind regards

Sam


On Tue, 9 Oct 2018 at 19:12, Sam Tang  wrote:

> Dear all
>
> Hello. We recently shot a crystal (a protein with small molecule as
> ligand) at a synchrotron source and see a weird pattern. (
> https://drive.google.com/file/d/11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV/view?usp=sharing
> )
>
> Crystal was grown in Citric acid and ammonium sulfate, cryoprotected with
> glycerol.
>
> At first we thought it was a protein crystal contaminated with salt but on
> second thought, the lowest resolution spot was at around 7 A, which doesn't
> make sense for a protein. So we would like to solicit your experience and
> perhaps someone may have encountered similar pattern before?
>
> Many thanks.
>
> Kind regards
>
> Sam
>
>



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Re: [ccp4bb] Weird diffraction pattern

[Sam Tang]

Hello Colin

Although the unit cell dimensions from mosflm should be largely unreliable
in this case, the software actually returned a P2 space group with a=24.6,
b=7.5, c=69.5  where b is so short that it resembles a small molecule
crystal.

Regards

Sam
Sam


On Tue, 9 Oct 2018 at 20:53, colin.n...@diamond.ac.uk <
colin.n...@diamond.ac.uk> wrote:

> Sam
>
> Would this unit cell index some of the spots?
>
> a = 7.00 ± 0.04 A, b = 9.96 ± 0.05 A, c = 6.29 ± 0.04 A.
>
>   Colin
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Sam
> Tang
> *Sent:* 09 October 2018 12:13
> *To:* ccp4bb 
> *Subject:* [ccp4bb] Weird diffraction pattern
>
>
>
> Dear all
>
>
>
> Hello. We recently shot a crystal (a protein with small molecule as
> ligand) at a synchrotron source and see a weird pattern. (
> https://drive.google.com/file/d/11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV/view?usp=sharing
> )
>
>
>
> Crystal was grown in Citric acid and ammonium sulfate, cryoprotected with
> glycerol.
>
>
>
> At first we thought it was a protein crystal contaminated with salt but on
> second thought, the lowest resolution spot was at around 7 A, which doesn't
> make sense for a protein. So we would like to solicit your experience and
> perhaps someone may have encountered similar pattern before?
>
>
>
> Many thanks.
>
>
>
> Kind regards
>
>
> Sam
>
>
>
>
> --
>
> 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] Electrostatic Potential: Poisson-Boltzmann

[Sam Tang]

To add to the discussion, could I raise a relevant question about
generating ESP (Apologies to Jiri if this distracts too much from your
initial thread).

In our structure in hand, the density for two conformations of the side
chain are clearly seen and they could be modeled. This brings a bit of
problem because the positive charge becomes more prominent with two
conformations there than with one. So what do we usually do when generating
ESP for such structures with alternate conformations? Do we remove one
before the calculation?

PS - I use online PDB2PQR server to do my calculation with PARSE field. I
did notice from some old archived discussion on the Web that it ignores one
conformation by default. But this seemingly is not the case in newer
versions?

Regards

Sam

School of Life Sciences, CUHK

On 2 December 2017 at 02:59, Robbie Joosten 
wrote:

> If you cannot trust the surface of your protein, perhaps you should not
> look at the the potential on the surface. Instead you can look at the field
> around your protein. This is less precise, but also less sensitive to local
> errors. If you want to know how your peptide finds your protein, this is
> actually more informative anyway.
>
> There must be several programs that do this. I have done this for MHC in
> the past with YASARA. It really explained nicely how he peptide moved in.
>
>
>
> Cheers,
>
> Robbie
>
>
>
> Sent from my Windows 10 phone
>
>
> --
> *From:* CCP4 bulletin board  on behalf of Dale
> Tronrud 
> *Sent:* Friday, December 1, 2017 7:29:01 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Electrostatic Potential: Poisson-Boltzmann
>
>These are not easy questions to answer.  Certainly atoms,
> particularly ones that are charged, even with fractional charges, have a
> strong effect on the ESP.  If you delete them because you don't know
> exactly where they are you will get a different answer than if you put
> them in in some reasonable but unsupported location (as you have found).
>  This result indicates that the peptide does affect the ESP
> significantly and you have to consider it.
>
>You could build lots of models with the peptide in different
> conformations and average all the maps.  This misses the point.  You
> have uncertainty in your model which means that you have uncertainty in
> your electrostatic potential.  Any particular ESP that you calculate and
> draw conclusions from will have a large uncertainty and you must
> consider that uncertainty when deciding between your potential
> conclusions.  (I'm not sure if the pun is intended or not!)
>
>I suppose you could believe that each possible conformation exists to
> some extent in reality which means that all the ESP's you calculate
> exist in some fraction of the molecules in the cell.  It is possible
> that only the molecules with a particular conformation of this peptide
> have the ESP that allows the molecule to function.  Life is hard.
>
>Another issue that you must consider: If the exact conformation of
> this loop causes changes to the ESP that you consider significant to
> your understanding of the function of this protein, the presence and
> conformation of neighboring proteins and solvent will also cause
> significant changes to the ESP.  The biological context of the protein
> becomes important.  If your interpretation depends critically on the
> value and distribution of ESP then I'm not sure you can work this out
> based on calculated ESP, considering the large uncertainty.
>
> Dale Tronrud
>
>
> On 12/1/2017 10:02 AM, chemocev marker wrote:
> > Hi
> >
> > I am calculating the Electrostatic Potential of my protein. But there
> > were few flexible region with high B-factor and I deleted that part of
> > the protein and then recalculated it. But there I can see a big
> > change.As I have a structure in the presence and the absence of the
> > peptide and the these flexible regions have a better map in the
> > structure without peptide and with peptide I have to delete them.
> > I have a question, should I model these missing regions
> >
> > or
> >
> > I should ignor them
> >
> > best
> >
> > Jiri
>

[ccp4bb] Different loop conformations

[Sam Tang]

Dear all

Sorry for the slightly off-topic thread.

We are lucky enough to have recently solved a protein-ligand structure in
P1 space to 2.4 A by molecular replacement using the apo-protein as model.
The protein is known to have a flexible loop region of about 20 amino acid
long. In the apo-protein (model), density for > half of this loop are not
seen.

It seems interesting to us that, in the complex structure, although still
incomplete, there seems to be loop densities at two distinct conformations
(named up and down, which point to opposite directions almost 120 degree)
-- when we fit the loop to the up conformation, difference map shows green
blots at the down conformation, and vice versa.

What we feel puzzled are:
(1) Is it possible, at this resolution, for the two conformations be
observed? Or is there any similar case we can make reference to?

(2) If the densities below to two conformations of the loop, how should we
model it?  Add alternate conformation function in Coot doesn't seem
suitable. Or should we make up (and eventually deposit) two models
showcasing the two conformations?

Many thanks in advance for your attention!

Regards

Sam

Re: [ccp4bb] Modelling of ligand and Refmac5

[Sam Tang]

Dear Dr Emsley

Many thanks for your reply.

I have now updated my Coot and am able to run refmac refinement using my
ligand PDB and CIF generated from elbow2 under Phenix.

This is indeed a lesson on the importance of keeping all softwares updated!

Kind regards

Sam

On 24 January 2017 at 18:29, Paul Emsley <pems...@mrc-lmb.cam.ac.uk> wrote:

> On 24/01/2017 08:04, Sam Tang wrote:
>
>>
>> I am trying to fit a small molecule ligand into a protein complex using
>> Coot. The data was
>> processed to P212121, at 2.6 A.  What I did was to input a SMILES string,
>> fit the ligand,
>> merge the ligand into the protein molecule using 'Merge Molecules' and
>> save coordinates.
>>
>> After fitting the ligand (now as chain O) I ran restrained refinement in
>> Refmac5 and the
>> following error returns:
>>
>>
> Into what did you input a SMILES? If the answer is Coot, then you have an
> Old Coot and will be lead down the garden path.
>
> > I believe it is due to the nomenclature of the ligand wherein Refmac
> mistook atoms as DUM
>
> I think that you're more or less right.
>
> The modern approach is to use Acedrg to generate the ligand either via a
> GUI or the command line. That will give you a PDB file which you can fit,
> and a dictionary that you can use in Refmac, Coot (and, I believe, Phenix).
>
> Paul.
>

[ccp4bb] Modelling of ligand and Refmac5

[Sam Tang]

Dear all

I am trying to fit a small molecule ligand into a protein complex using
Coot. The data was processed to P212121, at 2.6 A.  What I did was to input
a SMILES string, fit the ligand, merge the ligand into the protein molecule
using 'Merge Molecules' and save coordinates.

After fitting the ligand (now as chain O) I ran restrained refinement in
Refmac5 and the following error returns:

Input file :C:/20161226_1.1_refmac1-coot-1.pdb

  --

  ---  LIBRARY OF MONOMERS   ---

 _lib_name mon_lib

 _lib_version  5.44

 _lib_update   30/05/14

  --

  NUMBER OF MONOMERS IN THE LIBRARY  : 13409

with complete description: 13409

  NUMBER OF MODIFICATIONS:63

  NUMBER OF LINKS:73

  I am reading libraries. Please wait.

  - energy parameters

  - monomer"s description (links & mod )


FORMATTED  OLD file opened on unit  45



Logical name: ATOMSF, Filename: C:\CCP4-7\7.0\lib\data\atomsf.lib




  Number of atoms:   19934

  Number of residues :2577

  Number of chains   :  15

  I am reading library. Please wait.

mon_lib.cif


ERROR : DUM : duplicated atom_name : "DUM ".

  chain: OO   residue:1


(And the error repeats itself for >100 times)


I believe it is due to the nomenclature of the ligand wherein Refmac
mistook atoms as DUM.  (Also, the chain ID O was identified as OO?)  In a
test run I carried out rigid body refinement and the programme finished
without issues.

Is there a way I could rectify the above problem? Thanks in advance for
your attention and input.

Kind regards


Sam Tang
Biochemistry Programme, School of Life Sciences, CUHK

Re: [ccp4bb] Two SGs in one droplet?

[Sam Tang]

Dear all

Thanks a lot for the numerous input which is highly appreciated.

I should provide some updates as to what I have done on these two crystals
/ datasets.

I processed both datasets with HKL2000 as well as imosflm. Both softwares
give (essentially) the same unit cells as in the first email. The C2 cell
doesn't look like a transformation of the P1 cell.

However Phaser (and in fact Xtriage) identified that there is translational
pseudosymmetry in the C2 dataset but not the P1 dataset. We are lucky
enough to obtain MR solutions for the dataset but we remain a bit cautious
about the C2 we had. Currently we are looking at Zanuda to see if this is
what it should be.

Thanks again!

Kind regards

Sam




On 30 October 2016 at 14:54, Kevin Jude <kevinmj...@gmail.com> wrote:

> 3ICE had two space groups in the same /crystal/ - P6 at one end, P1 with
> pseudo-6-fold NCS at the other. In the P6 case, the RNA ligand (in the
> center of a hexameric protein) was rotationally averaged, but in the P1
> case it could be resolved.
>
> Best wishes
> Kevin
>
> On Fri, Oct 28, 2016 at 6:13 AM, Sam Tang <samtys0...@gmail.com> wrote:
>
>> Dear all
>>
>> Sorry for going a bit off-topic in this thread.
>> May I seek your advice as on whether you have experienced that crystals
>> being obtained from the same droplet, looking alike under microscope (rod
>> shape) and in fact growing possibly from a same nuclei, give two space
>> groups after indexing?
>>
>> I recently obtain crystals for a protein (co-crystallized with a nucleic
>> acid ligand) and collected two datasets from synchrotron. Although these
>> two crystals are from the same drop, the SG and unit cell dimensions are
>> very different:
>>
>> Xtal1: C121 (156 60 105 90 111 90) (L-test, Pointless shows that there is
>> no twinning), ~2.5 Angstrom
>> Xtal2: P1 (53 60 79 106 105 98), ~3 Angstorm
>>
>> Would it be possible that the ligand changes the SG of the crystal so
>> that only one of the forms contains the ligand?
>>
>> Any advice is appreciated and thanks a lot in advance for your input.
>>
>> Regards
>>
>> Sam Tang
>> Biochemistry Programme, School of Life Sciences, CUHK
>>
>>
>

[ccp4bb] Two SGs in one droplet?

[Sam Tang]

Dear all

Sorry for going a bit off-topic in this thread.
May I seek your advice as on whether you have experienced that crystals
being obtained from the same droplet, looking alike under microscope (rod
shape) and in fact growing possibly from a same nuclei, give two space
groups after indexing?

I recently obtain crystals for a protein (co-crystallized with a nucleic
acid ligand) and collected two datasets from synchrotron. Although these
two crystals are from the same drop, the SG and unit cell dimensions are
very different:

Xtal1: C121 (156 60 105 90 111 90) (L-test, Pointless shows that there is
no twinning), ~2.5 Angstrom
Xtal2: P1 (53 60 79 106 105 98), ~3 Angstorm

Would it be possible that the ligand changes the SG of the crystal so that
only one of the forms contains the ligand?

Any advice is appreciated and thanks a lot in advance for your input.

Regards

Sam Tang
Biochemistry Programme, School of Life Sciences, CUHK