Re: [ccp4bb] Difficult Molecular replacement

[Marco Bravo]

https://www.dropbox.com/scl/fo/pmw9nisdmq53yir2jqcmu/h?rlkey=zaj6cnknkjgkowrzf0vrmqdyf=0
  

Here is a link to my Molecular replacement and asymmetric unit contents 
logfiles. 
 
I ran Simple molecular replacement phaser MR through ccp4 cloud with my .mtz 
that was auto-processed at the ALS light source Beamline 831. I truncated my 
Alphaphold model into several domains as suggested and ran separate MR jobs. 
All of them are still running but one MR job for just one of the helicase 
domains finished and that is the molecular replacement job logfile i have 
posted.

I also posted my Data collection output for the crystal in the dropbox file. 

I also attached my XDS.INP and XDSCONV.INP files for more troubleshooting help.

Thank you all for helping me I hope this provides more information to help 
figure out what is going on. I can post more information if needed.



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[Daniele de Sanctis]

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Daniele


-- 

ἀρετή
---
Daniele de Sanctis, PhD
Structural Biology Group
ESRF - The European Synchrotron
Grenoble, France
Tel 33 (0)4 76 88 2869

http://www.esrf.eu/id29



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[ccp4bb] Tenure track position in Protein Science for Pathogen Defense

[Cygler, Miroslaw]

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===
Dr. Miroslaw Cygler, FCAHS
Professor, Dept. Biochem, Microbiol & Immune, U. of Saskatchewan
Canada Research Chair in Molecular Medicine Using Synchrotron Light
Health Sciences Building 3D30.6 (Box 9)
107 Wiggins Road, Saskatoon, SK S7N 5E5 Canada
Email: miroslaw.cyg...@usask.ca
Website: http://research-groups.usask.ca/cyglerlab/
Phone: 306-966-4361, cell: 306-203-2317
===






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CRC2 ad_final.docx
Description: CRC2 ad_final.docx

[ccp4bb] Fwd: postdoc post at world-renowned Astbury Centre, in Leeds, UK

[Ville Uski]

-- Forwarded message -
From: Helen McAllister 
Date: Fri, 16 Feb 2024 at 13:10
Subject: [CCP4] Please could you advertise another post for us?
To: c...@listserv.stfc.ac.uk 


Dear Sirs,



Please could you advertise the following post on your website? (This is by
coincidence the second post in a week, not a duplication.)



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Ranson, working on outer membrane proteins. Please contact either for more
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*https://jobs.leeds.ac.uk/vacancy.aspx?ref=FBSAS1075
*



Very many thanks indeed!



Best wishes



*Helen*



Helen McAllister (Mrs), h.mcallis...@leeds.ac.uk

PA to Professor Sheena Radford, OBE, FRS, FMedSci

School of Cellular and Molecular Biology, Faculty of Biological Sciences

The University of Leeds, Leeds LS2 9JT

Working at home 4 days, and usually on campus on Fridays

--

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Re: [ccp4bb] i2run crank2 problem

[Stuart McNicholas]

I got i2run to work with this data by:

* Importing the data with "Import merged"
* Importing the 2 sequences one at a time with 2 import sequence jobs
* Running a 'Define AU contents' job with both sequences added to the
contents.
* Creating a Crank2 job, then clicking the "Show i2 run command" to produce
script.
* I tweaked the i2run script to put double quotes around strings with
brackets "()<>"

I have attached my script.

On Tue, 20 Feb 2024 at 12:05, Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hmm - I can't help with the scripting question, but it solved easily from
> the I2 interface?
>
> This is run on a Mac.
>
> CRANK2 called SHELX - found SE with weak signal..
> Some difference between hand
> It traced ALA - R factor 41%
>
> Then BUCCANEER or MODELFIR quickly built and sequenced the rest..
> Eleanor
>
>
> On Thu, 15 Feb 2024 at 09:07, zx2...@connect.hku.hk 
> wrote:
>
>> Dear Colleagues,
>>
>> I hope this message finds you well. I am reaching out to inquire if
>> anyone has experience with *running Crank2 using the CCP4i2 i2run
>> command*. Unfortunately, I've encountered some difficulties and would
>> greatly appreciate any guidance or advice.
>>
>> For context, I've attached the script and files utilized in my attempts.
>> Specifically, I'm interested in understanding how to address the errors
>> I've encountered and would like to know the minimum input requirements for
>> successfully running this script.
>>
>> Below are the errors I've received:
>>
>>
>> Error in wrapper crank2 0.02: -ERROR- crank2:0 Error in wrapper crank2
>> 0.02::
>> *Anomalous scattering coeficients could not be determined.  Please input
>> them or the wavelength.*
>>
>> Traceback (most recent call last):
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/ccp4i2crank.py",
>> line 41, in CallCrankFromCCP4i2
>> raise
>> RuntimeError: No active exception to reraise
>>
>> During handling of the above exception, another exception occurred:
>>
>> Traceback (most recent call last):
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/script/crank2_script.py",
>> line 466, in process
>> crank2 = ccp4i2crank.CallCrankFromCCP4i2(self, inpfile=inpfile,
>> defaults=defaults, rvapi_style=rvapi_style)
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/ccp4i2crank.py",
>> line 43, in CallCrankFromCCP4i2
>> raise error
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/ccp4i2crank.py",
>> line 29, in CallCrankFromCCP4i2
>> crank =
>> parser.ParseAndRun(ccp4i2crank,defaults,rvapi_style=rvapi_style)
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/parse.py",
>> line 58, in ParseAndRun
>> crank_prep.Run()
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/process.py",
>> line 1017, in Run
>>
>> self.RunPreprocess(rundir=rundir,clear_out=clearout,save=restore,*args,**kwargs)
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/manager.py",
>> line 107, in RunPreprocess
>> self.GetAnomScatCoefs()
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/manager.py",
>> line 327, in GetAnomScatCoefs
>> common.Error('Anomalous scattering coeficients could not be
>> determined.  Please input them or the wavelength.')
>>   File
>> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/common.py",
>> line 34, in Error
>> raise CrankError(message)
>> common.CrankError: Anomalous scattering coeficients could not be
>> determined.  Please input them or the wavelength.
>>
>> I would be grateful for any insights or suggestions you might have
>> regarding this issue. Thank you very much for your time and assistance.
>>
>> Best regards,
>> Xin
>>
>> --
>>
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i2_run_crank.sh
Description: Bourne shell script

Re: [ccp4bb] Difficult Molecular replacement

[Artem Evdokimov]

Hi Marco,

To follow up: if your protein is something like WRN or BLM helicase - you
mention recA like fold after all - (I worked on these in the past hehe)
then the split into domains is almost mandatory - these proteins are very
plastic and doing MR on them is equivalent to doing antibody MR where you
absolutely have to treat domains as separately mobile rigid bodies.

I would love to help more but it would require more sharing that you might
be justifiably unwilling or unable to do :)

Best of luck,

Artem



On Mon, Feb 19, 2024 at 6:51 PM Artem Evdokimov 
wrote:

> Hello Marco,
>
> First: compare your unit cell parameters with RCSB (there is an option for
> that in the search, very helpful). Give it say 5% error margin.
>
> Worst case scenario - cry a little, having found that your protein is
> something else...
>
> Next, check in depth for space group oddities, twinning, or general data
> misbehavior.
>
> Next, split your helicase into two lobes (if this is the right kind of
> helicase) and do MR with both halves, like it was a complex of two proteins
> (requires a bit of skill with MR - but nothing terrifyingly hard tbh). If
> there are more domains - split into more (RQC, HRDC, etc.)
>
> Hope this helps.
>
> Artem
>
>
>
>
> On Mon, Feb 19, 2024, 5:54 PM Marco Bravo  wrote:
>
>> Hello all,
>> I recently collected data on some plate crystals for a previously
>> uncharacterized protein at the ALS light source. The XDS auto-data
>> processing log output indicates that my resolution is 2.8 angstroms. The
>> protein is a helicase with homologs already in the protein data bank making
>> it a suitable target for molecular replacement which I thought initially.
>> However after trying molecular replacements with all known homologs in the
>> protein data bank the R values remain high after MR >0.5. After an initial
>> round of Rigid body or restrained refinement. The R values still remain
>> very high at around >.5. I have tried MR with Rosetta and alphaphold models
>> but the problem of high R values persists. The best solution I get is from
>> the CCP4 cloud automatic molecular replacement and model building pipeline
>> which gives me a free R value of 0.46.. However the solution is only for
>> residues ~100-326 out of a 543 amino acid long protein. And even then the
>> model still has a lot of missing residues and truncated sidechains and
>> overall fits the map quite poorly. Does anyone have any suggestions about
>> how I can solve my structure if at all possible at this point? I ran the
>> crystals and pre-crystalized samples on a gel and it appears that the
>> protein remains stable during crystallization as the molecular weight did
>> not change or any degradation does not appear.
>>
>> 
>>
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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Difficult Molecular replacement

[Harry Powell]

Hi Marco

Further to Pedro’s comment, one way to avoid missing data in cusps is to use a 
multi-sweep strategy for data collection (as championed by the good folk at 
Global Phasing over many years). Many of the problems encountered in structure 
solution can be traced back to a sub-optimal data collection.

You don’t say which beamline at ALS you used, but if it’s got a kappa or 
similar fitted the beamline staff should be able to help.

best wishes

Harry

> On 20 Feb 2024, at 09:20, Pedro Matias  wrote:
> 
> Hi Marco,
> 
> Further to Zhen's comment, that can also happen if you have a cusp containing 
> one of the reciprocal lattice axes. The SG may be P212121 but one of the 21 
> is never located due to the missing data.



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Re: [ccp4bb] i2run crank2 problem

[Eleanor Dodson]

Hmm - I can't help with the scripting question, but it solved easily from
the I2 interface?

This is run on a Mac.

CRANK2 called SHELX - found SE with weak signal..
Some difference between hand
It traced ALA - R factor 41%

Then BUCCANEER or MODELFIR quickly built and sequenced the rest..
Eleanor


On Thu, 15 Feb 2024 at 09:07, zx2...@connect.hku.hk 
wrote:

> Dear Colleagues,
>
> I hope this message finds you well. I am reaching out to inquire if anyone
> has experience with *running Crank2 using the CCP4i2 i2run command*.
> Unfortunately, I've encountered some difficulties and would greatly
> appreciate any guidance or advice.
>
> For context, I've attached the script and files utilized in my attempts.
> Specifically, I'm interested in understanding how to address the errors
> I've encountered and would like to know the minimum input requirements for
> successfully running this script.
>
> Below are the errors I've received:
>
>
> Error in wrapper crank2 0.02: -ERROR- crank2:0 Error in wrapper crank2
> 0.02::
> *Anomalous scattering coeficients could not be determined.  Please input
> them or the wavelength.*
>
> Traceback (most recent call last):
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/ccp4i2crank.py",
> line 41, in CallCrankFromCCP4i2
> raise
> RuntimeError: No active exception to reraise
>
> During handling of the above exception, another exception occurred:
>
> Traceback (most recent call last):
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/script/crank2_script.py",
> line 466, in process
> crank2 = ccp4i2crank.CallCrankFromCCP4i2(self, inpfile=inpfile,
> defaults=defaults, rvapi_style=rvapi_style)
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/ccp4i2crank.py",
> line 43, in CallCrankFromCCP4i2
> raise error
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/ccp4i2crank.py",
> line 29, in CallCrankFromCCP4i2
> crank =
> parser.ParseAndRun(ccp4i2crank,defaults,rvapi_style=rvapi_style)
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/parse.py",
> line 58, in ParseAndRun
> crank_prep.Run()
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/process.py",
> line 1017, in Run
>
> self.RunPreprocess(rundir=rundir,clear_out=clearout,save=restore,*args,**kwargs)
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/manager.py",
> line 107, in RunPreprocess
> self.GetAnomScatCoefs()
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/manager.py",
> line 327, in GetAnomScatCoefs
> common.Error('Anomalous scattering coeficients could not be
> determined.  Please input them or the wavelength.')
>   File
> "/home/Xin/programs/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/pipelines/crank2/crank2/common.py",
> line 34, in Error
> raise CrankError(message)
> common.CrankError: Anomalous scattering coeficients could not be
> determined.  Please input them or the wavelength.
>
> I would be grateful for any insights or suggestions you might have
> regarding this issue. Thank you very much for your time and assistance.
>
> Best regards,
> Xin
>
> --
>
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Re: [ccp4bb] Difficult Molecular replacement

[Randy John Read]

Hi Marco,

I was just about to write with a different suggestion when Kay’s reply came in. 
What Kay suggests sounds like the best thing to do first!

However, if that isn’t the issue, then I’m wondering if it could be a problem 
with incorrectly diagnosed translational non-crystallographic symmetry (tNCS). 
The ambiguity about which axes are pure 2-folds or 2-fold screws is the kind of 
thing that occurs when there is an NCS translation with a component of 1/2 
along at least one of the axes.

I couldn’t find anything about how many copies you have in the asymmetric unit 
of the crystal, but is it an even number? Do any programs (including Phaser) 
find large native Patterson peaks away from the origin? Are the intensity 
distributions and/or moments unusual?

You could send me a copy of the log file from a Phaser run if you don’t know 
where to find this information.

Good luck!

Randy Read

> On 20 Feb 2024, at 09:11, Kay Diederichs  
> wrote:
>
> Hello Marco,
>
> this sounds to me like there is space group confusion in the sense that if 
> you use the output model from Phaser, it may say space group P22121 (with 
> a a MTZ file specifying space group P21212 (with a
> If this is indeed your problem, then the way to resolve this is
> a) in XDS.INP, specify UNIT_CELL_CONSTANTS= b c a 90 90 90 !(where a SPACE_GROUP_NUMBER=18,  JOB=CORRECT . Run XDS.
> b) remove UNIT_CELL_CONSTANTS and SPACEGROUP_NUMBER lines from XDSCONV.INP, 
> and run XDSCONV. Or use pointless/aimless
> c) run Phaser again with the resulting MTZ file (check that it has spacegroup 
> 18 i.e. P21212 and cell parameters with c shortest). Check that the resulting 
> PDB file has P21212 and b c a cell parameters in the header.
> d) run refinement
>
> Hope this helps,
> Kay
>
> On Tue, 20 Feb 2024 01:15:49 +, Marco Bravo  wrote:
>
>> Hello Todd, I get the best solution for p22121 space group after MR with an 
>> LLG score of 640 from phaser. and the Rfree is .4748. However after MR 
>> refinement does not lower the Rfree and it appears to make the Rfree worse. 
>> The XDS software indicates that my best solution is P21 21 2. Often Phaser 
>> MR places the solution in P 21 21 2. The helicase is a superfamily 2 
>> helicase and is only monomeric. Its a 543aa long protein with a MW of 62Kda. 
>> It should have two RecA like domains at the core but the protein I have 
>> crystallized has a previously uncharacterized n-terminal domain responsible 
>> for tight single stranded DNA binding.  I have tried different space groups 
>> manually but that resulted in clashing. I will be frank I do need to work on 
>> my crystallography background as crystal lattices, space group, and 
>> self-rotaion functions are limited. Thank you so far for your help , I will 
>> try further trimming down my search model into separate domain and trying 
>> that in the meantime.
>>
>> 
>>
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-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building
Hills Road   E-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.  
www-structmed.cimr.cam.ac.uk




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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Difficult Molecular replacement

[Pedro Matias]

Hi Marco,

Further to Zhen's comment, that can also happen if you have a cusp 
containing one of the reciprocal lattice axes. The SG may be P212121 but 
one of the 21 is never located due to the missing data.


You will always sort of get a solution from MR but you should look at 
the Z-score of the translation function for the solution you get - if it 
is below 8, it's very likely you have the wrong solution, and the most 
likely culprit is the wrong phasing model. As Tom suggested, you should 
validate the crystallized molecule - you might have purified the wrong 
one (it happened to me a few times) even though the MW might be in the 
right ballpark.


Best regards and good luck,

Pedro

On 20/02/2024 01:30, Gong, Zhen wrote:


Hello Marco,

I also feel that it might be due to the wrong space group because all 
the homologous models and alphafold model did not improve the R 
values. Make sure that you turn on “All possible in same pointgroup” 
when you run Phenix.Phaser-MR. I work with P212121 crystals a lot. 
Sometimes, if the diffraction quality was not good, the data will be 
indexed as P222, or P21212 etc, and sometimes even C2221. Try all 
possible in same pointgroup and see what happens. Good luck!


Zhen

*From: *CCP4 bulletin board  on behalf of Marco 
Bravo 

*Date: *Monday, February 19, 2024 at 20:17
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: *[EXTERNAL] Re: [ccp4bb] Difficult Molecular replacement

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Hello Todd, I get the best solution for p22121 space group after MR 
with an LLG score of 640 from phaser. and the Rfree is .4748. However 
after MR refinement does not lower the Rfree and it appears to make 
the Rfree worse. The XDS software indicates that my best solution is 
P21 21 2. Often Phaser MR places the solution in P 21 21 2. The 
helicase is a superfamily 2 helicase and is only monomeric. Its a 
543aa long protein with a MW of 62Kda. It should have two RecA like 
domains at the core but the protein I have crystallized has a 
previously uncharacterized n-terminal domain responsible for tight 
single stranded DNA binding.  I have tried different space groups 
manually but that resulted in clashing. I will be frank I do need to 
work on my crystallography background as crystal lattices, space 
group, and self-rotaion functions are limited. Thank you so far for 
your help , I will try further trimming down my search model into 
separate domain and trying that in the meantime.




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--
Industry and Medicine Applied Crystallography
Macromolecular Crystallography Unit
___
Phones : (351-21) 

Re: [ccp4bb] Difficult Molecular replacement

[Kay Diederichs]

Hello Marco,

this sounds to me like there is space group confusion in the sense that if you 
use the output model from Phaser, it may say space group P22121 (with a wrote:

>Hello Todd, I get the best solution for p22121 space group after MR with an 
>LLG score of 640 from phaser. and the Rfree is .4748. However after MR 
>refinement does not lower the Rfree and it appears to make the Rfree worse. 
>The XDS software indicates that my best solution is P21 21 2. Often Phaser MR 
>places the solution in P 21 21 2. The helicase is a superfamily 2 helicase and 
>is only monomeric. Its a 543aa long protein with a MW of 62Kda. It should have 
>two RecA like domains at the core but the protein I have crystallized has a 
>previously uncharacterized n-terminal domain responsible for tight single 
>stranded DNA binding.  I have tried different space groups manually but that 
>resulted in clashing. I will be frank I do need to work on my crystallography 
>background as crystal lattices, space group, and self-rotaion functions are 
>limited. Thank you so far for your help , I will try further trimming down my 
>search model into separate domain and trying that in the meantime.
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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 
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