Re: [ccp4bb] Best protocols to advance a low resolution twin

[Ethan A Merritt]

On Monday, 14 September 2020 22:22:34 PDT Rezaul Karim wrote:
> Hi Eleanor,Thanks for asking.
> Yes, 2nd domain refine alright with Rfree ~25%, inhibitor at the binding site 
> and no major issues at all except that I don't see any trace of 1st domain 
> and the protein from dissolved crystals are on SDS gel at right MW size = 
> 1st+2nd domain.I tried all other possible space group of P3. Only P31 works 
> with ~26% Rfree but with 2 monomers of BD2. Strangely, I find this phenomenon 
> only in presence of a specific inhibitor. Unliganded protein structure ~2A 
> and with few other inhibitors (1.5-2.2A) all have two domains. Just can't 
> wrap my head around the mystery.

Rfree at 0.25 sounds pretty good to me.  "Good" as in "good enough to
indicate that you have already described most of the ordered cell content".

Does the lattice packing make sense with only that one domain placed?
I.e., are there enough contacts to make a crystal hold together as a crystal
in the absence an ordered seconds domain?

Ethan



> Thanks,Reza
> 
> Md Rezaul Karim
> PhD candidate
> PhD Program in Integrated Biomedical Sciences
> Dept. of Molecular Medicine, Morsani College of Medicine, USF,Tampa
> Schonbrunn lab, Moffitt Cancer Center, Tampa
> Email: rez...@health.usf.edu, md.ka...@moffitt.org
> Phone: (813) 745 4673 ext. 5462
>  
>  
>   On Mon, Sep 14, 2020 at 5:50 AM, Eleanor Dodson 
> wrote:   Rezaul: You sayBut I only see the 2nd domain with exact same unit 
> cell that I have solved for 2nd domain's  with same space group. 
> 
> I am afraid if that is so that you probably have only crystallised the 2nd 
> domain. Does that domain refine all right?Eleanor
> On Mon, 14 Sep 2020 at 09:02, Rezaul Karim 
> <1e364c8f16de-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Eagerly following >I have a similar case. A 3A data with P3(1)21 from two 
> domains (80+% sequence identity) protein. No major twinning issue. But I only 
> see the 2nd domain with exact same unit cell that I have solved for 2nd 
> domain's  with same space group. No proteolysis- crystals give protein band 
> on SDS at right MW. Data from 20+ crystals from various condition & additive 
> screens came out all the same.Resorted to changing the construct to make 
> variable N/C-term for future.
> Thanks,Reza
> 
> Md Rezaul Karim
> Postdoctoral Research FellowH. Lee Moffitt Cancer Center and Research 
> InstituteTampa, FL
> Email: rez...@health.usf.edu, reza.ka...@moffitt.org
> Phone: (813) 745 4673 ext. 5462
>  
>  
>   On Sun, Sep 13, 2020 at 7:33 PM, Jon 
> Cooper<488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:   Hello, a 
> couple of thoughts. 
> 
> If your best dataset has a twinning fraction of 40% (i.e. almost 50:50 ;-?) 
> and the twin operator corresponds to a 2-fold rotation parallel to the 
> z-axis, is it definitely trigonal, rather than hexagonal? The 2-fold NCS 
> operator that you found for the d3 domains, is it parallel to an axis, e.g. 
> z? Also, how long did the crystals take to grow and do you have the original 
> diffraction images? If so, and I know its hard to tell with fine-slicing on 
> modern detectors, do they look clean or do they show any splitting of the 
> spots, etc? Also, a bit boring question, but when you say the space group is 
> P32, I guess you mean P3 subscript(2) rather than P321, and I take it you 
> have tried alternative space groups in the MR like P3 subscript(1). Are the 
> systematic absences good, or is it all a bit ambiguous?
> 
> Best wishes, Jon Cooper.
> jon.b.coo...@protonmail.com
> 
> Sent with ProtonMail Secure Email.
> 
> ‐‐‐ Original Message ‐‐‐
>  On Tuesday, 8 September 2020 19:53, Andrew Lovering  
> wrote:
>  
> 
> 
> Dear all,
> 
> 
> 
> 
> 
> I've a project with two historical datasets (i.e. not sure of when/if I can 
> reproduce to solve problem by getting better data) that are twinned.
> 
> 
> 
> 
> 
> The spacegroup is P32, with operator -h, -k, l
> 
> 
> cell = 83.5 83.5 64.2 90 90 120
> 
> 
> 
> 
> 
> protein has three domains d1-d2-d3, likely 50% solvent, 2 copies in asu
> 
> 
> 
> 
> 
> dataset 1, roughly 3.6 angstrom resolution, twin fraction 0.25
> 
> 
> dataset 2, roughly 3.1 angstrom resolution, twin fraction 0.4
> 
> 
> 
> 
> 
> I've managed to solve this (unambiguously) by finding a d3:d3 dimer in 
> detwinned data, then seeing via phenix autobuild that it finds a d2 also in 
> map. At present the ability to interpret the map further stalls there, and it 
> can't be "the limit" because lack of crystal contacts suggest there should be 
> more protein to find.
> 
> 
> 
> 
> 
> I realise I can go two ways, either using the detwinned data or using twinned 
> data and supplying the operator.the former giving ridiculously optimistic 
> R-values with a suspiciously "clean map", the latter giving noisier but maybe 
> more informative maps.
> 
> 
> 
> 
> 
> Two-fold averaging is not going to be great because the d2-d3 arrangement 
> observed in monomer 1 clashes when 

Re: [ccp4bb] Best protocols to advance a low resolution twin

[Rezaul Karim]

Hi Eleanor,Thanks for asking.
Yes, 2nd domain refine alright with Rfree ~25%, inhibitor at the binding site 
and no major issues at all except that I don't see any trace of 1st domain and 
the protein from dissolved crystals are on SDS gel at right MW size = 1st+2nd 
domain.I tried all other possible space group of P3. Only P31 works with ~26% 
Rfree but with 2 monomers of BD2. Strangely, I find this phenomenon only in 
presence of a specific inhibitor. Unliganded protein structure ~2A and with few 
other inhibitors (1.5-2.2A) all have two domains. Just can't wrap my head 
around the mystery.
Thanks,Reza

Md Rezaul Karim
PhD candidate
PhD Program in Integrated Biomedical Sciences
Dept. of Molecular Medicine, Morsani College of Medicine, USF,Tampa
Schonbrunn lab, Moffitt Cancer Center, Tampa
Email: rez...@health.usf.edu, md.ka...@moffitt.org
Phone: (813) 745 4673 ext. 5462
 
 
  On Mon, Sep 14, 2020 at 5:50 AM, Eleanor Dodson 
wrote:   Rezaul: You sayBut I only see the 2nd domain with exact same unit cell 
that I have solved for 2nd domain's  with same space group. 

I am afraid if that is so that you probably have only crystallised the 2nd 
domain. Does that domain refine all right?Eleanor
On Mon, 14 Sep 2020 at 09:02, Rezaul Karim 
<1e364c8f16de-dmarc-requ...@jiscmail.ac.uk> wrote:

Eagerly following >I have a similar case. A 3A data with P3(1)21 from two 
domains (80+% sequence identity) protein. No major twinning issue. But I only 
see the 2nd domain with exact same unit cell that I have solved for 2nd 
domain's  with same space group. No proteolysis- crystals give protein band on 
SDS at right MW. Data from 20+ crystals from various condition & additive 
screens came out all the same.Resorted to changing the construct to make 
variable N/C-term for future.
Thanks,Reza

Md Rezaul Karim
Postdoctoral Research FellowH. Lee Moffitt Cancer Center and Research 
InstituteTampa, FL
Email: rez...@health.usf.edu, reza.ka...@moffitt.org
Phone: (813) 745 4673 ext. 5462
 
 
  On Sun, Sep 13, 2020 at 7:33 PM, Jon 
Cooper<488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:   Hello, a couple 
of thoughts. 

If your best dataset has a twinning fraction of 40% (i.e. almost 50:50 ;-?) and 
the twin operator corresponds to a 2-fold rotation parallel to the z-axis, is 
it definitely trigonal, rather than hexagonal? The 2-fold NCS operator that you 
found for the d3 domains, is it parallel to an axis, e.g. z? Also, how long did 
the crystals take to grow and do you have the original diffraction images? If 
so, and I know its hard to tell with fine-slicing on modern detectors, do they 
look clean or do they show any splitting of the spots, etc? Also, a bit boring 
question, but when you say the space group is P32, I guess you mean P3 
subscript(2) rather than P321, and I take it you have tried alternative space 
groups in the MR like P3 subscript(1). Are the systematic absences good, or is 
it all a bit ambiguous?

Best wishes, Jon Cooper.
jon.b.coo...@protonmail.com

Sent with ProtonMail Secure Email.

‐‐‐ Original Message ‐‐‐
 On Tuesday, 8 September 2020 19:53, Andrew Lovering  
wrote:
 


Dear all,





I've a project with two historical datasets (i.e. not sure of when/if I can 
reproduce to solve problem by getting better data) that are twinned.





The spacegroup is P32, with operator -h, -k, l


cell = 83.5 83.5 64.2 90 90 120





protein has three domains d1-d2-d3, likely 50% solvent, 2 copies in asu





dataset 1, roughly 3.6 angstrom resolution, twin fraction 0.25


dataset 2, roughly 3.1 angstrom resolution, twin fraction 0.4





I've managed to solve this (unambiguously) by finding a d3:d3 dimer in 
detwinned data, then seeing via phenix autobuild that it finds a d2 also in 
map. At present the ability to interpret the map further stalls there, and it 
can't be "the limit" because lack of crystal contacts suggest there should be 
more protein to find.





I realise I can go two ways, either using the detwinned data or using twinned 
data and supplying the operator.the former giving ridiculously optimistic 
R-values with a suspiciously "clean map", the latter giving noisier but maybe 
more informative maps.





Two-fold averaging is not going to be great because the d2-d3 arrangement 
observed in monomer 1 clashes when placed over monomer 2 (so at present can 
only average the small % of ASU between d3 copies). Critically, looking for a 
second d2 using MR fails but the lack of crystal contacts suggests success 
should be possibleits really unlikely that a third entity is in there (d1 
and d2 are actually 50% similar, so by searching for d2 and failing, I'm 
actually missing three spots it could potentially occupy if the protein is not 
proteolysed)





Sorry for long email but the devil is in the details. My question would be, 
what is best practice in a low res poor twinned dataset where you can only fill 
roughly just over half of what should be in there?





Feel free to tell 

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Re: [ccp4bb] [ccpem] Averaging data for gain correction

[Marin van Heel]

One more remark on the camera correction issue...

The camera correction does actually matter quite a bit, especially where it
concerns the movie alignments in the early phases of processing, as Tanaka
mentioned too.  What I want to point out here is that the effect of the
camera correction needs to be judged directly at the camera level by the
FRC, or better still, by the FRI (https://arxiv.org/abs/2009.03223) and not
at the end of a long processing chain where the influence of tons of
unrelated intermediate processing decisions have been made. (Yes the
quality of the shoes of a football player has an influence on who wins the
game, but blaming the outcome of a game on the quality of shoes of the
football player..., well... ). Since the camera correction significantly
influences the movie alignment procedures, and assuming all processing is
done adhering to the appropriate sampling and processing rules, the main
influence on the 3D output of the game will be in the number of particles
effectively available for 3D reconstruction. That number will affect the
FSC (Harauz & van Heel 1986), yes, but less than it will affect the
R-weighted Fourier Shell Information FSI (https://arxiv.org/abs/2009.03223)!
Bottom line: take care of your metrics and stick to the rules and you will
win the game!

Two more pennies on the issue,
Marin

On Mon, Sep 14, 2020 at 3:50 PM Thomas Cleveland 
wrote:

> Thanks everyone for the tool suggestions and comments. I particularly
> enjoyed the correction of Mars rover photos as an example.
>
> Summary:
>
>1. sum_all_tiffs in cisTEM
>2. relion_estimate_gain
>3. camera-correction command in IMAGIC (
>https://www.nature.com/articles/srep10317)
>
> Tom
>
> On Mon, Sep 14, 2020 at 2:11 PM Thomas Cleveland <
> thomas.clevel...@gmail.com> wrote:
>
>> Hi all,
>>
>> I've heard of folks averaging an entire set of unaligned movie files in
>> order to make a new gain reference after data collection. Can anyone
>> comment on how well this works? Is there any command that does it in a
>> really simple way? (I realize it would not be too hard to just loop through
>> and average all the movies, but thought I would ask, in case this is
>> already implemented in a tool somewhere).
>>
>> Best,
>> Tom
>>
>
> --
>
> To unsubscribe from the CCPEM list, click the following link:
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>



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Re: [ccp4bb] How to save two superposed protein structures in PDB format

[Philip D. Jeffrey]

Hello Abhik

In coot use: Calculate>Merge Molecules to append one structure to another.  
Coot will change the chain labels for you, which might work out OK in this case.

Alternatively, I've done a lot of this in my time:
Make a copy of the reference PDB file
Open it in a simple text edit (emacs, vi etc)
Go to the end, remove the PDB 'END' statement
(Many programs stop reading a PDB file when they see an END)
Append the second, superimposed file.
For most chain labels you can also change them in a text editor but 'C' 'O' and 
'N' will cause you trouble.  You can do it easily in Coot.
This would take no more than 15 seconds in emacs.

>From the command line:
grep -v 'END' reference.pdb > reference2.pdb
egrep '^ATOM|^HETATM' superimposed.pdb >> reference2.pdb
(text editor to change chain labels, if you care, but there are ways to do that 
from the command line too)

Cheers
Phil Jeffrey
Princeton


From: CCP4 bulletin board  on behalf of Abhik 
Mukhopadhyay 
Sent: Monday, September 14, 2020 4:55 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] How to save two superposed protein structures in PDB format

Hi,
How can I save two superposed protein structures in  PDB format? Is there any 
way I can do this in coot or pymol? There is only one chain in those two PDB 
structures.

Thanks in advance,
Abhik





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[ccp4bb] How to save two superposed protein structures in PDB format

[Abhik Mukhopadhyay]

Hi,
How can I save two superposed protein structures in  PDB format? Is there
any way I can do this in coot or pymol? There is only one chain in those
two PDB structures.

Thanks in advance,
Abhik



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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Best protocols to advance a low resolution twin

[Huw Jenkins]

Hi Randy,

> On 14 Sep 2020, at 18:52, Randy Read  wrote:
> 
> Thanks for pointing that out.  I guess I hadn’t presumed to brand myself as 
> an expert!  I’ll have to find out now what else I’ve been missing...

Surely you should identify yourself as developer and have access to all the 
options! 

I've never been that happy with the way that I implemented 'expert level' 
selection in the CCP4i2 Phaser interface and the code that shows and hides the 
options is pretty ugly. Perhaps an i1 style series of expanding subsections 
'user parameters', 'expert parameters' etc would be more in keeping with other 
CCP4i2 task interfaces?

Best regards,


Huw


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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Best protocols to advance a low resolution twin

[Randy Read]

Hi Huw,

Thanks for pointing that out.  I guess I hadn’t presumed to brand myself as an 
expert!  I’ll have to find out now what else I’ve been missing...

Randy

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building   Fax: +44 1223 336827
Hills Road   E-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.  
www-structmed.cimr.cam.ac.uk

> On 14 Sep 2020, at 12:36, Huw Jenkins 
> <288da93ae744-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hi Randy,
> 
>> On 14 Sep 2020, at 12:09, Randy Read  wrote:
>> 
>>   Unfortunately, it looks like this is one of the few options that isn’t yet 
>> available from the ccp4i2 interface! 
> 
> Isn't it this one? 
> 
> 
> 
> 
> Note this only appears when 'expert' options are shown:
> 
> 
> 
> 
> Best regards,
> 
> 
> Huw
> 
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Re: [ccp4bb] Refmac use - water addition

[Christian Roth]

Hi Luca,
as Eleanor mentioned, in the i2 interface there is in the option menu an
add water button. That will automatically run the coot find waters script
similar to the old interface.  Paul did use some very sensible values to
look for blobs, which might be waters. These ones are used (basically the
same defaults if you press the interactive find waters button in Coot
alone).  Afterwards there is another quick 5 cycle refmac run to refine
position and B-values of the added waters.
The R value is the R work. If the value does not reach the given values
during the refinement process, the  additional water picking step is
skipped. The R value is based on your experience and one has to take
resolution, status of the model etc. into account. It is often not helpful
to add waters at an early stage of model building and refinement when R
values are hight or the model is very incomplete.
Does that make sense?

Best
Christian

On Sat, Sep 12, 2020 at 8:38 PM Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> You don’t say quite how you are doing this. There is an option in the i2
> pipeline to add waters using coot when the r factor falls below some
> assigned value.
> This is done using COOT.
> One can debate whether it is a useful option or whether the user would be
> better o open COOT and supervise the water search..
> Eleanor
>
> On Sat, 12 Sep 2020 at 16:56, Jon Cooper <
> 488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> Hello, I'm not totally up-to-the minute with this but I didn't know that
>> refmac itself added waters so maybe it's another program in an i2 pipeline,
>> or something. However, I know another excellent refinement program that
>> does ;-) and an excellent graphics program that does, too ;-0
>>
>> In the past, we have added well-defined water molecule when the R-factor
>> goes into the mid-to-low thirties, or lower.
>>
>> Anything above 2 sigma in a delta-F map making sensible H-bonds is likely
>> to be real but you can lower that cutoff a bit (~1.5) if your resolution is
>> not brilliant.
>>
>> Glad to be corrected on this.
>>
>> Best wishes, Jon Cooper.
>>
>> jon.b.coo...@protonmail.com
>>
>>
>>
>>
>>
>>  Original Message 
>> On 12 Sep 2020, 11:06, Luca Mazzei < luca.mazz...@unibo.it> wrote:
>>
>>
>> Dear CCP4 people,
>>
>>
>> I am approaching in these days to the new CCP4i2 interface and, in
>> particular, to REFMAC. Can anyone quickly explain what the factor R, that
>> must be chosen in order to automatically add water molecules, is? How can I
>> relate this value with the previous DELFWT and FWT map values?
>>
>>
>> Thanks,
>>
>>
>> Luca
>>
>>
>> Luca Mazzei - PhD
>>
>>
>> Department of Pharmacy and Biotechnology
>>
>>
>> University of Bologna
>> 
>>
>>
>> Viale Giuseppe Fanin, 40 - 40127
>> 
>>
>>
>> Bologna - Italy
>> 
>>
>>
>> 
>>
>>
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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Best protocols to advance a low resolution twin

[Randy Read]

Just to follow up on this — if you’ve already tried searching for the 
additional domain with the already placed ones fixed, and that didn’t work, 
it’s possible that the missing domain is less well-ordered than the ones you’ve 
already placed.  So one thing you could try is increasing the relative B-factor 
of the new domain you’re searching for, with the SEARCH BFACTOR command in a 
keyword script, or activating the “Search with bfactor” option in the Expert 
parameters section of ccp4i.  Unfortunately, it looks like this is one of the 
few options that isn’t yet available from the ccp4i2 interface!  I’d probably 
try a number like 20 A^2 as a guess.

If this doesn’t work, then it’s probably worth cutting out the density and 
treating it as a real-space MR problem, using the phased translation target at 
the translation step of the search.

Best wishes,

Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building   Fax: +44 1223 336827
Hills Road   E-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.  
www-structmed.cimr.cam.ac.uk

> On 14 Sep 2020, at 11:27, Schreuder, Herman /DE  
> wrote:
> 
> Dear Andy,
>  
> I few thoughts from my side, but no solution I am afraid:
>   • Your twinning operator -h, -k, l is the standard alternative indexing 
> for P3x space group, which makes a lot of sense.
>   • P32 is a low symmetry space group, which makes MR easier, but this is 
> offset by the NCS.
>   • In my hands, MR is surprisingly insensitive to twinning, so I would 
> search for the molecules using the twinned data. Also, for MR one does not 
> need extremely high resolution data. 
>   • However, twinning and low resolution data seriously hamper “de novo” 
> model building. So if you have good MR models for your d1 and d2 domains, you 
> may have a good chance of solving the structure. If you would have to build 
> them “de novo” in a MR electron density map, you may be doomed.
>   • What I would do, is to look at the phaser map using a very large map 
> radius: say 35-40 Å or more and look in the solvent region if there are 
> places with higher density that may suggest the presence of the missing 
> domains. If something shows up, you can focus on those regions in your MR, or 
> even try to manually fit the Ca chain of your MR model.
>   • You certainly have already done it, but Phaser has to option to 
> search for additional domains, given the domains you already found.
>  
> Best,
> Herman
>  
>  
> Von: CCP4 bulletin board  Im Auftrag von Andrew 
> Lovering
> Gesendet: Montag, 14. September 2020 11:17
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] Re: [ccp4bb] Best protocols to advance a low resolution 
> twin
>  
> EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk
> 
>  
> 
> To follow on from this thread:
>  
> To answer Jon, I did try to see if P6 subgroups were a possibility but can 
> rule this out for a few reasons (MR doesn’t give solutions, merging stats not 
> suggestive of P6, the other dataset with the twin fraction that is 
> significantly further from 50:50); and the d3:d3 NCS is not parallel to any 
> crystallographic axis
>  
> The spacegroup is indeed P3 sub 2, not P321, and the solution again only 
> possible in P3 sub 2 not P3 sub 1, so spacegroup confidence is high
>  
> I did get one reply from Petrus Zwart that twin refinement / map improvement 
> is a subject being worked on
>  
> What I might try is a Phaser MR where the “missing domains” are searched for 
> using cut out density of the one placed domain, rather than model (which 
> could possibly be a better choice at this low resolution? Thoughts 
> appreciated)
>  
> Best wishes & thanks everyone
> Andy
>  
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> 
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[ccp4bb] Videos from CCP4 Study Weekend 2020

[Karen McIntyre - UKRI STFC]

The videos from the CCP4 Study Weekend 2020 held in January are now available 
to view on the CCP4 YouTube channel.

Regards

Karen McIntyre
CCP4 Project Administrator
Science and Technology Facilities Council
RCaH 1.22
Rutherford Appleton Laboratory
Harwell Campus
Didcot
OX11 0FA

karen.mcint...@stfc.ac.uk
01235 44 5790


[cid:image001.png@01D68A89.DCD6DDF0]

**Please note that I only work part-time until 1.30pm**


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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Best protocols to advance a low resolution twin

[Schreuder, Herman /DE]

Dear Andy,

I few thoughts from my side, but no solution I am afraid:

  *   Your twinning operator -h, -k, l is the standard alternative indexing for 
P3x space group, which makes a lot of sense.
  *   P32 is a low symmetry space group, which makes MR easier, but this is 
offset by the NCS.
  *   In my hands, MR is surprisingly insensitive to twinning, so I would 
search for the molecules using the twinned data. Also, for MR one does not need 
extremely high resolution data.
  *   However, twinning and low resolution data seriously hamper "de novo" 
model building. So if you have good MR models for your d1 and d2 domains, you 
may have a good chance of solving the structure. If you would have to build 
them "de novo" in a MR electron density map, you may be doomed.
  *   What I would do, is to look at the phaser map using a very large map 
radius: say 35-40 Å or more and look in the solvent region if there are places 
with higher density that may suggest the presence of the missing domains. If 
something shows up, you can focus on those regions in your MR, or even try to 
manually fit the Ca chain of your MR model.
  *   You certainly have already done it, but Phaser has to option to search 
for additional domains, given the domains you already found.

Best,
Herman


Von: CCP4 bulletin board  Im Auftrag von Andrew Lovering
Gesendet: Montag, 14. September 2020 11:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Best protocols to advance a low resolution twin


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

To follow on from this thread:

To answer Jon, I did try to see if P6 subgroups were a possibility but can rule 
this out for a few reasons (MR doesn't give solutions, merging stats not 
suggestive of P6, the other dataset with the twin fraction that is 
significantly further from 50:50); and the d3:d3 NCS is not parallel to any 
crystallographic axis

The spacegroup is indeed P3 sub 2, not P321, and the solution again only 
possible in P3 sub 2 not P3 sub 1, so spacegroup confidence is high

I did get one reply from Petrus Zwart that twin refinement / map improvement is 
a subject being worked on

What I might try is a Phaser MR where the "missing domains" are searched for 
using cut out density of the one placed domain, rather than model (which could 
possibly be a better choice at this low resolution? Thoughts appreciated)

Best wishes & thanks everyone
Andy



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Re: [ccp4bb] Best protocols to advance a low resolution twin

[Eleanor Dodson]

Rezaul: You say
*But I only see the 2nd domain with exact same unit cell that I have solved
for 2nd domain's  with same space group. *

I am afraid if that is so that you probably have only crystallised the 2nd
domain. Does that domain refine all right?
Eleanor

On Mon, 14 Sep 2020 at 09:02, Rezaul Karim <
1e364c8f16de-dmarc-requ...@jiscmail.ac.uk> wrote:

> Eagerly following >
> I have a similar case. A 3A data with P3(1)21 from two domains (80+%
> sequence identity) protein. No major twinning issue. But I only see the 2nd
> domain with exact same unit cell that I have solved for 2nd domain's  with
> same space group.
> No proteolysis- crystals give protein band on SDS at right MW. Data from
> 20+ crystals from various condition & additive screens came out all the
> same.
> Resorted to changing the construct to make variable N/C-term for future.
>
> Thanks,
> Reza
>
> Md Rezaul Karim
> Postdoctoral Research Fellow
> H. Lee Moffitt Cancer Center and Research Institute
> Tampa, FL
> Email: rez...@health.usf.edu, reza.ka...@moffitt.org
> Phone: (813) 745 4673 ext. 5462
>
> On Sun, Sep 13, 2020 at 7:33 PM, Jon Cooper
> <488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
> Hello, a couple of thoughts.
>
> If your best dataset has a twinning fraction of 40% (i.e. almost 50:50
> ;-?) and the twin operator corresponds to a 2-fold rotation parallel to the
> z-axis, is it definitely trigonal, rather than hexagonal? The 2-fold NCS
> operator that you found for the d3 domains, is it parallel to an axis, e.g.
> z? Also, how long did the crystals take to grow and do you have the
> original diffraction images? If so, and I know its hard to tell with
> fine-slicing on modern detectors, do they look clean or do they show any
> splitting of the spots, etc? Also, a bit boring question, but when you say
> the space group is P32, I guess you mean P3 subscript(2) rather than P321,
> and I take it you have tried alternative space groups in the MR like P3
> subscript(1). Are the systematic absences good, or is it all a bit
> ambiguous?
>
> Best wishes, Jon Cooper.
> jon.b.coo...@protonmail.com
>
> Sent with ProtonMail  Secure Email.
>
> ‐‐‐ Original Message ‐‐‐
> On Tuesday, 8 September 2020 19:53, Andrew Lovering 
> wrote:
>
> Dear all,
>
>
> I've a project with two historical datasets (i.e. not sure of when/if I
> can reproduce to solve problem by getting better data) that are twinned.
>
>
> The spacegroup is P32, with operator -h, -k, l
>
> cell = 83.5 83.5 64.2 90 90 120
>
>
> protein has three domains d1-d2-d3, likely 50% solvent, 2 copies in asu
>
>
> dataset 1, roughly 3.6 angstrom resolution, twin fraction 0.25
>
> dataset 2, roughly 3.1 angstrom resolution, twin fraction 0.4
>
>
> I've managed to solve this (unambiguously) by finding a d3:d3 dimer in
> detwinned data, then seeing via phenix autobuild that it finds a d2 also in
> map. At present the ability to interpret the map further stalls there, and
> it can't be "the limit" because lack of crystal contacts suggest there
> should be more protein to find.
>
>
> I realise I can go two ways, either using the detwinned data or using
> twinned data and supplying the operator.the former giving ridiculously
> optimistic R-values with a suspiciously "clean map", the latter giving
> noisier but maybe more informative maps.
>
>
> Two-fold averaging is not going to be great because the d2-d3 arrangement
> observed in monomer 1 clashes when placed over monomer 2 (so at present can
> only average the small % of ASU between d3 copies). Critically, looking for
> a second d2 using MR fails but the lack of crystal contacts suggests
> success should be possibleits really unlikely that a third entity is in
> there (d1 and d2 are actually 50% similar, so by searching for d2 and
> failing, I'm actually missing three spots it could potentially occupy if
> the protein is not proteolysed)
>
>
> Sorry for long email but the devil is in the details. My question would
> be, what is best practice in a low res poor twinned dataset where you can
> only fill roughly just over half of what should be in there?
>
>
> Feel free to tell me its doomed!
>
>
> Best wishes & thanks in advance
>
> Andy
>
> --
>
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This message was issued to members of 

Re: [ccp4bb] Best protocols to advance a low resolution twin

[Andrew Lovering]

To follow on from this thread:

To answer Jon, I did try to see if P6 subgroups were a possibility but can rule 
this out for a few reasons (MR doesn't give solutions, merging stats not 
suggestive of P6, the other dataset with the twin fraction that is 
significantly further from 50:50); and the d3:d3 NCS is not parallel to any 
crystallographic axis

The spacegroup is indeed P3 sub 2, not P321, and the solution again only 
possible in P3 sub 2 not P3 sub 1, so spacegroup confidence is high

I did get one reply from Petrus Zwart that twin refinement / map improvement is 
a subject being worked on

What I might try is a Phaser MR where the "missing domains" are searched for 
using cut out density of the one placed domain, rather than model (which could 
possibly be a better choice at this low resolution? Thoughts appreciated)

Best wishes & thanks everyone
Andy



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Re: [ccp4bb] Best protocols to advance a low resolution twin

[Rezaul Karim]

Eagerly following >I have a similar case. A 3A data with P3(1)21 from two 
domains (80+% sequence identity) protein. No major twinning issue. But I only 
see the 2nd domain with exact same unit cell that I have solved for 2nd 
domain's  with same space group. No proteolysis- crystals give protein band on 
SDS at right MW. Data from 20+ crystals from various condition & additive 
screens came out all the same.Resorted to changing the construct to make 
variable N/C-term for future.
Thanks,Reza

Md Rezaul Karim
Postdoctoral Research FellowH. Lee Moffitt Cancer Center and Research 
InstituteTampa, FL
Email: rez...@health.usf.edu, reza.ka...@moffitt.org
Phone: (813) 745 4673 ext. 5462
 
 
  On Sun, Sep 13, 2020 at 7:33 PM, Jon 
Cooper<488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:   Hello, a couple 
of thoughts. 

If your best dataset has a twinning fraction of 40% (i.e. almost 50:50 ;-?) and 
the twin operator corresponds to a 2-fold rotation parallel to the z-axis, is 
it definitely trigonal, rather than hexagonal? The 2-fold NCS operator that you 
found for the d3 domains, is it parallel to an axis, e.g. z? Also, how long did 
the crystals take to grow and do you have the original diffraction images? If 
so, and I know its hard to tell with fine-slicing on modern detectors, do they 
look clean or do they show any splitting of the spots, etc? Also, a bit boring 
question, but when you say the space group is P32, I guess you mean P3 
subscript(2) rather than P321, and I take it you have tried alternative space 
groups in the MR like P3 subscript(1). Are the systematic absences good, or is 
it all a bit ambiguous?

Best wishes, Jon Cooper.
jon.b.coo...@protonmail.com

Sent with ProtonMail Secure Email.

‐‐‐ Original Message ‐‐‐
 On Tuesday, 8 September 2020 19:53, Andrew Lovering  
wrote:
 


Dear all,





I've a project with two historical datasets (i.e. not sure of when/if I can 
reproduce to solve problem by getting better data) that are twinned.





The spacegroup is P32, with operator -h, -k, l


cell = 83.5 83.5 64.2 90 90 120





protein has three domains d1-d2-d3, likely 50% solvent, 2 copies in asu





dataset 1, roughly 3.6 angstrom resolution, twin fraction 0.25


dataset 2, roughly 3.1 angstrom resolution, twin fraction 0.4





I've managed to solve this (unambiguously) by finding a d3:d3 dimer in 
detwinned data, then seeing via phenix autobuild that it finds a d2 also in 
map. At present the ability to interpret the map further stalls there, and it 
can't be "the limit" because lack of crystal contacts suggest there should be 
more protein to find.





I realise I can go two ways, either using the detwinned data or using twinned 
data and supplying the operator.the former giving ridiculously optimistic 
R-values with a suspiciously "clean map", the latter giving noisier but maybe 
more informative maps.





Two-fold averaging is not going to be great because the d2-d3 arrangement 
observed in monomer 1 clashes when placed over monomer 2 (so at present can 
only average the small % of ASU between d3 copies). Critically, looking for a 
second d2 using MR fails but the lack of crystal contacts suggests success 
should be possibleits really unlikely that a third entity is in there (d1 
and d2 are actually 50% similar, so by searching for d2 and failing, I'm 
actually missing three spots it could potentially occupy if the protein is not 
proteolysed)





Sorry for long email but the devil is in the details. My question would be, 
what is best practice in a low res poor twinned dataset where you can only fill 
roughly just over half of what should be in there?





Feel free to tell me its doomed!





Best wishes & thanks in advance


Andy





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