Re: [ccp4bb] Difficult Molecular replacement

[Marco Bravo]

https://www.dropbox.com/scl/fi/aqxm6s28mwbvufx5vkt4z/j7pointless.log?rlkey=075kia624ehmtzugnu5n8ymy0=0

Here is a link to the pointless file , thank you for your help!



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[ccp4bb] Positions available UK

[Panne, Daniel (Prof.)]

Dear all,

We currently have Postdoc positions available in our group funded by the 
Wellcome Trust. Our project focuses on understanding molecular mechanisms that 
control the regulation of 3D genome organisation and how they contribute to 
different aspects of genome function. The project is an interdisciplinary, 
collaborative and international project involving groups in Imperial College 
London, the Netherlands Cancer Institute (NKI) Amsterdam, the University of 
Oxford, and the University of Leicester, UK. We combine expertise in structural 
biology, biochemistry, cell biology, model organisms and integrative imaging 
across scales. The aim is to link atomic structures to molecular mechanisms and 
organismal function.

We are seeking creative and highly motivated scientists that are keen to work 
on interdisciplinary projects in an international setting. You should have a 
PhD degree in structural biology or equivalent and have a demonstrated record 
of accomplishment in conducting high quality original research. Experience in 
protein biochemistry and single particle cryoEM or xray crystallography, are 
essential. Depending on interests, fellows can develop additional skills in 
cellular imaging using cryoET.

We offer a well-balanced training programme focused on transferable skills for 
scientists. Workshops cover career development, research integrity/good 
scientific practice and open science and data management initiatives. In 
addition, fellows can complete trainings of their choice based on their future 
career goals and interests.

Applicants are required to outline their suitability for this role via the 
online application process and should include a CV, summary of their 
accomplishment and research interests, and publication list.

The initial closing date for applications is 22th March 2024, however 
applications will be reviewed and interviewed on an ongoing basis until this 
vacancy is filled.

Research Associate; Structural biology of genome 
regulation
For further information about this role please contact Daniel Panne 
(daniel.pa...@le.ac.uk).



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Re: [ccp4bb] Difficult Molecular replacement

[Oganesyan, Vaheh]

Thank you!
At the end it appears that Zanuda is not really a "зануда", translated from 
Russian as something being depressing, disagreeable or unsatisfactory.

Vaheh

From: CCP4 bulletin board  on behalf of Randy John Read 

Sent: Wednesday, February 21, 2024 11:55 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Difficult Molecular replacement

Hi,

This is also one of my favourite methods, although I hadn’t realised that you 
could just say “labin F=FC” instead of making fake intensities!

However, at a recent workshop I ran into a case where pointless misled me. This 
was a case with tNCS where one of the translation components was half a unit 
cell edge. As a result, the calculated diffraction data would have alternately 
strong and weak reflections along that axis regardless of whether there was a 
crystallographic 2(1) axis or an NCS translation of 1/2 parallel to that axis. 
Pointless assigned a 2(1) axis where there should have been a pure two-fold. On 
the other hand, Zanuda tried refinement in all the different possibilities and 
only one refined well.

Best wishes,

Randy

> On 21 Feb 2024, at 16:05, Kay Diederichs  
> wrote:
>
> Hi Vaheh,
>
> for this purpose, I use
>
> pointless hklin refmacXY.mtz < labin F=FC
> eof
>
> Thus, pointless determines the space group, including the crystallographic 
> screw axes, from the Fcalc.
>
> Best wishes,
> Kay
>
> On Wed, 21 Feb 2024 15:40:07 +, Oganesyan, Vaheh 
>  wrote:
> ...
>> This might be a silly question, but I do not know the answer: After 
>> refinement in P1 how do I distinguish which axis is crystallographic and 
>> which one in non-crystallographic?
>>
>> Vaheh
>
> 
>
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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] i2 won't accept 90 degree angles in moniclinic

[Phil Evans]

That looks like an unnecessarily severe test buried in the i2 library, which 
maybe should just be a warning. I can probably have a look at it

Phil

Sent from my iPad

> On 21 Feb 2024, at 16:52, Winter, Graeme (DLSLtd,RAL,LSCI) 
> <6a19cead4548-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> 
> CAUTION: This email originated from outside of the LMB:
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> Do not click links or open attachments unless you recognize the sender and 
> know the content is safe.
> If you think this is a phishing email, please forward it to 
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> Tried reporting to i2 dev list but it got bounced - feels like something 
> others may hit so don’t feel _too_ bad sending to the bb with a tiny 
> attachment
> 
> 
> 
> 
> 
> 
> Hi Folks
> 
> Helping someone out with some rather specialist data processing challenges 
> and she hit a problem: I can reproduce this with very boring thaumatin data 
> so I think this is a bug
> 
> (it obliquely relates some to a current CCP4bb thread about MR, in passing)
> 
> Processing a data set in lower than necessary symmetry e.g. tetragonal as 
> monoclinic you _cannot_ import the merged MTZ file into i2 because it is 
> impossible to have 90 degree angles for P21
> 
> Well, it is possible, and also, it is still correct to under merge the data 
> so this is a bug? Is this a reasonable viewpoint to hold?
> 
> Many thanks Graeme
>  
> 
> -- 
> 
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Re: [ccp4bb] Difficult Molecular replacement

[Randy John Read]

Hi,

This is also one of my favourite methods, although I hadn’t realised that you 
could just say “labin F=FC” instead of making fake intensities!

However, at a recent workshop I ran into a case where pointless misled me. This 
was a case with tNCS where one of the translation components was half a unit 
cell edge. As a result, the calculated diffraction data would have alternately 
strong and weak reflections along that axis regardless of whether there was a 
crystallographic 2(1) axis or an NCS translation of 1/2 parallel to that axis. 
Pointless assigned a 2(1) axis where there should have been a pure two-fold. On 
the other hand, Zanuda tried refinement in all the different possibilities and 
only one refined well.

Best wishes,

Randy

> On 21 Feb 2024, at 16:05, Kay Diederichs  
> wrote:
>
> Hi Vaheh,
>
> for this purpose, I use
>
> pointless hklin refmacXY.mtz < labin F=FC
> eof
>
> Thus, pointless determines the space group, including the crystallographic 
> screw axes, from the Fcalc.
>
> Best wishes,
> Kay
>
> On Wed, 21 Feb 2024 15:40:07 +, Oganesyan, Vaheh 
>  wrote:
> ...
>> This might be a silly question, but I do not know the answer: After 
>> refinement in P1 how do I distinguish which axis is crystallographic and 
>> which one in non-crystallographic?
>>
>> Vaheh
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
> This message was issued to members of http://www.jiscmail.ac.uk/CCP4BB, a 
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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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[ccp4bb] i2 won't accept 90 degree angles in moniclinic

[Winter, Graeme (DLSLtd,RAL,LSCI)]

Tried reporting to i2 dev list but it got bounced - feels like something others 
may hit so don’t feel _too_ bad sending to the bb with a tiny attachment


[cid:CA3CE9EB-841D-41A3-A41E-F79714492B21]

Hi Folks

Helping someone out with some rather specialist data processing challenges and 
she hit a problem: I can reproduce this with very boring thaumatin data so I 
think this is a bug

(it obliquely relates some to a current CCP4bb thread about MR, in passing)

Processing a data set in lower than necessary symmetry e.g. tetragonal as 
monoclinic you _cannot_ import the merged MTZ file into i2 because it is 
impossible to have 90 degree angles for P21

Well, it is possible, and also, it is still correct to under merge the data so 
this is a bug? Is this a reasonable viewpoint to hold?

Many thanks Graeme

-- 
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are not the intended addressee or an authorised recipient of the addressee 
please notify us of receipt by returning the e-mail and do not use, copy, 
retain, distribute or disclose the information in or attached to the e-mail.
Any opinions expressed within this e-mail are those of the individual and not 
necessarily of Diamond Light Source Ltd. 
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may sustain as a result of software viruses which may be transmitted in or with 
the message.
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Wales with its registered office at Diamond House, Harwell Science and 
Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom




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

[Eleanor Dodson]

Various (all depressing) possibilities..
You don’t say what the sequence ID to the models us

Your protein is flexible with poor fit to the available models. How well do
those overlap?
The bioinformatics tools in i2 give you pictures showing this. Look at them
and see if there are sensible places to divide models up for the search.

You have crystallised the wrong protein - not common but it does happen..
I use cco4i2 fir most things and there is a MR option there to use SINBAD .
That checks against the pdb whether there is a deposited structure that has
a) your point group and cell, then whether that model fits your data.

Next go back to the data processing. The ccp4i2 report needs to be read
carefully.
Is the data poor?
Inspect the graphs and warning messages..
What is the  Rmerge?
Are there bad batches? There is a graph showing the variation against
batch.. if there are wild variations you may need to exclude some batches..
It will suggest there might be twinning ,
look st the Wilson plot graphs .


Is there non- crystallographic translation? ( not harmful but if the
translation is (x,y,z=1/2) say then the space group selection is
problematic - the fact that all l=2n+1 ( used to suggest the soacegroup is
P2i2i21) might be caused by that translation..

Then how many copies of your protein can fit into the crystal lattice?
There is a task - “import sequence “ then another “define AU contents”. You
input sequence and experimental diffraction data and it tells you if one or
two or many molecules can fit. That is only a guesstimate but if the cell
can hold many copies the MR search will be more challenging.
If there could be several copies a self rotation function ( in ccp4i2 part
of the data processing folder) might tell you there is a dimer ? Trimer?
Etc .. but you say you expect a monomer..

The good news is that you have a solution with r factors below 0.5. Have
you tried rebuilding from that starting point? In some cases Buccaneer/
modelcraft can take that model, discard the worst defined bits and rebuild
the model. Not terribly likely to succeed with 2.8A data and a poor MR
solution but worth a try..

On Tue, 20 Feb 2024 at 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
>
> [You don't often get email from mbrav...@ucr.edu. Learn why this is
> important at https://aka.ms/LearnAboutSenderIdentification ]
>
> 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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Re: [ccp4bb] Difficult Molecular replacement

[Eleanor Dodson]

Cant answer your question
"This might be a silly question, but I do not know the answer: After
refinement in P1 how do I distinguish which axis is crystallographic and
which one in non-crystallographic?"

Space group validation with Zanuda
[image: image.png]
CCP4
https://legacy.ccp4.ac.uk › newsletter48 › articles › zanuda

The program Zanuda presented in this article was developed to automate the
validation of space group assignment in such circumstances. In addition,
the program ...



On Wed, 21 Feb 2024 at 15:40, Oganesyan, Vaheh <
vaheh.oganes...@astrazeneca.com> wrote:

> Hi All,
>
> Interesting discussion as I have a similar case. In my case molecular
> replacement solution can be found easily in P21, P212121, with very similar
> looking electron densities. However, R-factors remain relatively high (mid
> 30s). In P1 completeness suffers (75% completeness), maps look decent, R
> factors are in high 20's. Resolution limit is 1.9A with CC1/2 in high res.
> shell ~0.7.
> This might be a silly question, but I do not know the answer: After
> refinement in P1 how do I distinguish which axis is crystallographic and
> which one in non-crystallographic?
>
> Vaheh
> --
> *From:* CCP4 bulletin board  on behalf of Randy
> John Read 
> *Sent:* Wednesday, February 21, 2024 9:18 AM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* Re: [ccp4bb] Difficult Molecular replacement
>
> Hi,
>
> It’s possible the true space group is P2(1) with the b-axis unique, and
> that subset of true symmetry is found repeatedly with different incorrect
> backgrounds of other copies. But I think the easiest way to resolve this
> unambiguously is to solve in P1, and let that uncover the true symmetry.
>
> Best wishes,
>
> Randy
>
> > On 21 Feb 2024, at 13:56, Pedro Matias  wrote:
> >
> > But curiously, all the 4 best solutions correspond to a SG with a 21
> screw along b.
> > And amazingly none of the TF solutions is rejected due to clashes.
> > On 21/02/2024 12:20, Eleanor Dodson wrote:
> >> Lots of comments, but it would be easier to actually look at your
> integrated data!
> >> Some of the stats look a bit ropey -
> >> 621 reflections labelled as outliers by PHASER?
> >> Very anisotropic
> >> Moments go mad at the highest resolution..
> >>
> >> The good news - extremely strong signal from the rotation function
> means the model is probably a good one.
> >> Bad news - translation function results do not select a definitive
> solution..
> >> Possible reasons
> >> Unit Cell: 72.61 73.73 147.23 90.00 90.00 90.00
> >> Most likely data problems - a axis ~ = b axis so twinning is possible
> >>
> >> I could add more comments if either you could share the unmerged data,
> or at least a pointless logfile..
> >> Cheers Eleanor
> >>
> >>
> >>
> >>
> >>
> >> On Wed, 21 Feb 2024 at 11:06, Randy John Read  wrote:
> >> Hi Marco,
> >>
> >> To add to what Kay has said:
> >>
> >> The intensity moments from Phaser (between 1.5 and 2 for the second
> moments after correcting for anisotropy) are indicative of likely twinning.
> With the cell dimensions, it might be possible to have pseudomerohedral
> twinning in an orthorhombic space group, but given the lack of distinction
> among possible choices of orthorhombic spac group (noted by Kay), it seems
> much more likely that the true symmetry is lower and that you have
> pseudosymmetry combined with perfect twinning.
> >>
> >> Judging from the strong and unambiguous rotation peak, your model is
> clearly very good, so I think it would be easy to ask Phaser to solve this
> by looking for 4 copies in space group P1. You can get P1 data either by
> expanding the orthorhombic data to P1 or by re-merging the data in P1. If
> the merging statistics were good, that would indicate that any twinning
> would be close to perfect, so just expanding the data would be a reasonable
> choice. Alternatively, you have reasonable redundancy so merging in P1
> would be a plausible choice. I would probably go with expanding the data,
> figuring out from the MR solution what the real symmetry is, and then
> merging the data with that symmetry.
> >>
> >> Unless there are some other pathologies, I think the MR in P1 is very
> likely to give a clear answer (or maybe 2 answers related by the twin
> operator). It’s formally possible that you could have a different number of
> copies (e.g. 6 in the unit cell) if the true symmetry were monoclinic, so
> keep an open mind on that question. You could just try finding the largest
> domain from splitting the AlphaFold model (presumably the domain for which
> you sent the log file), work out the symmetry from that solution, and then
> run a job to search for all the domains in the right space group. It’s
> generally a good idea, by the way, to ask Phaser to look for everything you
> expect to find in one job, because it has built-in logic to predict the
> best search order but then update 

Re: [ccp4bb] Difficult Molecular replacement

[Kay Diederichs]

Hi Vaheh,

for this purpose, I use

pointless hklin refmacXY.mtz < wrote:
...
>This might be a silly question, but I do not know the answer: After refinement 
>in P1 how do I distinguish which axis is crystallographic and which one in 
>non-crystallographic?
>
>Vaheh



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Re: [ccp4bb] Difficult Molecular replacement

[Oganesyan, Vaheh]

Hi All,

Interesting discussion as I have a similar case. In my case molecular 
replacement solution can be found easily in P21, P212121, with very similar 
looking electron densities. However, R-factors remain relatively high (mid 
30s). In P1 completeness suffers (75% completeness), maps look decent, R 
factors are in high 20's. Resolution limit is 1.9A with CC1/2 in high res. 
shell ~0.7.
This might be a silly question, but I do not know the answer: After refinement 
in P1 how do I distinguish which axis is crystallographic and which one in 
non-crystallographic?

Vaheh

From: CCP4 bulletin board  on behalf of Randy John Read 

Sent: Wednesday, February 21, 2024 9:18 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Difficult Molecular replacement

Hi,

It’s possible the true space group is P2(1) with the b-axis unique, and that 
subset of true symmetry is found repeatedly with different incorrect 
backgrounds of other copies. But I think the easiest way to resolve this 
unambiguously is to solve in P1, and let that uncover the true symmetry.

Best wishes,

Randy

> On 21 Feb 2024, at 13:56, Pedro Matias  wrote:
>
> But curiously, all the 4 best solutions correspond to a SG with a 21 screw 
> along b.
> And amazingly none of the TF solutions is rejected due to clashes.
> On 21/02/2024 12:20, Eleanor Dodson wrote:
>> Lots of comments, but it would be easier to actually look at your integrated 
>> data!
>> Some of the stats look a bit ropey -
>> 621 reflections labelled as outliers by PHASER?
>> Very anisotropic
>> Moments go mad at the highest resolution..
>>
>> The good news - extremely strong signal from the rotation function means the 
>> model is probably a good one.
>> Bad news - translation function results do not select a definitive solution..
>> Possible reasons
>> Unit Cell: 72.61 73.73 147.23 90.00 90.00 90.00
>> Most likely data problems - a axis ~ = b axis so twinning is possible
>>
>> I could add more comments if either you could share the unmerged data, or at 
>> least a pointless logfile..
>> Cheers Eleanor
>>
>>
>>
>>
>>
>> On Wed, 21 Feb 2024 at 11:06, Randy John Read  wrote:
>> Hi Marco,
>>
>> To add to what Kay has said:
>>
>> The intensity moments from Phaser (between 1.5 and 2 for the second moments 
>> after correcting for anisotropy) are indicative of likely twinning. With the 
>> cell dimensions, it might be possible to have pseudomerohedral twinning in 
>> an orthorhombic space group, but given the lack of distinction among 
>> possible choices of orthorhombic spac group (noted by Kay), it seems much 
>> more likely that the true symmetry is lower and that you have pseudosymmetry 
>> combined with perfect twinning.
>>
>> Judging from the strong and unambiguous rotation peak, your model is clearly 
>> very good, so I think it would be easy to ask Phaser to solve this by 
>> looking for 4 copies in space group P1. You can get P1 data either by 
>> expanding the orthorhombic data to P1 or by re-merging the data in P1. If 
>> the merging statistics were good, that would indicate that any twinning 
>> would be close to perfect, so just expanding the data would be a reasonable 
>> choice. Alternatively, you have reasonable redundancy so merging in P1 would 
>> be a plausible choice. I would probably go with expanding the data, figuring 
>> out from the MR solution what the real symmetry is, and then merging the 
>> data with that symmetry.
>>
>> Unless there are some other pathologies, I think the MR in P1 is very likely 
>> to give a clear answer (or maybe 2 answers related by the twin operator). 
>> It’s formally possible that you could have a different number of copies 
>> (e.g. 6 in the unit cell) if the true symmetry were monoclinic, so keep an 
>> open mind on that question. You could just try finding the largest domain 
>> from splitting the AlphaFold model (presumably the domain for which you sent 
>> the log file), work out the symmetry from that solution, and then run a job 
>> to search for all the domains in the right space group. It’s generally a 
>> good idea, by the way, to ask Phaser to look for everything you expect to 
>> find in one job, because it has built-in logic to predict the best search 
>> order but then update it on the basis of preliminary results.
>>
>> There are different ways to sort out the true symmetry from the MR solution, 
>> but within CCP4 the Zanuda procedure is a very effective choice.
>>
>> Get in touch if you have any difficulties following this procedure, and it 
>> would be great to let the BB know the outcome!
>>
>> Best wishes,
>>
>> Randy Read
>>
>> > On 21 Feb 2024, at 08:42, Kay Diederichs  
>> > wrote:
>> >
>> > Hi Marco:
>> >
>> > short comments (I sent you also a private mail):
>> > - the stats in CORRECT.LP look ok, but I'd like to know what ISa is, and 
>> > what the number of outliers is ("misfits"). Seeing the delta-CC1/2 stats 
>> > as a function of frame number is also useful for judging e.g. 

[ccp4bb] IMCBio International PhD program 2024

[Albert Weixlbaumer]

Hi everyone,
 
I would like to draw your attention to the International PhD program of the 
Integrative Molecular and Cellular Biology (IMCBio) graduate school at the 
University of Strasbourg, France:
https://imcbio-phdprogram.unistra.fr

When you click on the link, scroll down to the section on Integrated Structural 
Biology to read about some of the exciting projects available for candidates 
keen to work on challenging questions.
My lab is also interested in recruiting candidates that want to use 
biochemistry and structural biology to work on supramolecular complexes (e.g. 
to study the cooperation between RNA polymerase and other cellular machineries 
involved in gene expression).

The IMCBio graduate school builds on the strong research developed in five 
different institutes. This includes the IGBMC, which is a European Instruct 
center and thus houses state-of-the-art facilities for structural biology (NMR, 
X-ray crystallography, SAXS, as well as latest instrumentation for Cryo-EM 
including two Titan KRIOS with various direct detectors and a Glacios for 
screening).

Structural biology at the IGBMC has a long tradition and focus on functional 
complexes of large macromolecular machines involved in gene expression 
(transcription, translation, and splicing machineries), Chromatin organization, 
epigenetics and DNA topology (epigenetic regulators, topoisomerases, retroviral 
integrases, histone chaperones and deacetylases, methyltransferases), viral 
oncoproteins, motor proteins, and nuclear receptors.
Strasbourg is a cosmopolitan city in the heart of Europe. The IGBMC is a 
vibrant and highly international institute (over 45 nationalities, working 
language is English) with about 250 PhD students and postdocs.
 
Initial registration for the PhD program needs to be done until March 10th, 
2024. The deadline to complete the application is March 17th, 2024
 
Please forward this to any interested candidate.
 
Thanks,
Albert



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[ccp4bb] Reminder: CBMS Lecture Series - Olga Rechkoblit - February 21st, 13:30 (EST)

[Stojanoff, Vivian]

You are cordially invited to join  the Center for Biomolecular Structure 
Lecture Series ………..





Olga  Rechkoblit

Mount Sinai School of Medicine



WEDNESDAY, February 21, 13:30 (EST)



"Activation of bacterial immune system by cyclic nucleotides"



Register in advance for this meeting:


https://bnl.zoomgov.com/meeting/register/vJIsdOGprDojEjg7-CJFcbqdzjJEXsKmbYk



   Time conversion Link: 
https://www.worldtimebuddy.com/



Abstract: The bacterial CBASS system is similar to the cGAS-STING system in 
humans, containing an enzyme that synthesizes a cyclic nucleotide upon viral 
infection and an effector that senses the second messenger for the anti-viral 
response. Cap5, containing a SAVED domain coupled to an HNH DNA endonuclease 
domain, is the most abundant CBASS effector, yet the mechanism by which it 
becomes activated for cell killing remains unknown. We present here 
high-resolution structures of full-length Cap5 from Pseudomonas syringae (Ps) 
with second messengers. The key to PsCap5 activation is a dimer-to-tetramer 
transition, whereby the binding of second messenger to dimer triggers an 
open-to-closed transformation of the SAVED domains, furnishing a surface for 
assembly of the tetramer. This movement propagates to the HNH domains, 
juxtaposing and converting two HNH domains into states for DNA destruction. 
These results show how Cap5 effects bacterial cell suicide and we provide 
proof-in-principle data that the CBASS can be extrinsically activated to limit 
bacterial infections.



==



Vivian Stojanoff, PhD

Education, Training, Outreach

User Program

p 1(631) 344 8375

e lifescie...@bnl.gov

w https://www.bnl.gov/ps/lifesciences/



Address:

Center for Biomolecular Structure

National Synchrotron Light Source II

Building 745

Brookhaven National Laboratory

Upton NY 11973





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[ccp4bb] EMBO course on “Structural characterisation of macromolecular complexes”, 1-8 June 2024, Grenoble, France

[Wojtek Galej]

Dear Colleagues,
I would like to draw your attention to the upcoming EMBO Course on 
“Structural characterisation of macromolecular complexes”, which will 
take place on the 1st-8th of June 2024 at the EPN Campus in Grenoble.


This course has been organised regularly since 2002 and provides 
participants with a comprehensive overview of state-of-the-art 
structural biology methods, focusing on sample preparation, 
characterisation, and data integration strategies. We have a fantastic 
lineup of speakers covering a broad range of topics.


The course is aimed at advanced PhD students and postdocs, currently 
working on challenging projects in structural biology that would benefit 
from hybrid approaches.


The application deadline is the 10th of March 2024.

For more details, please visit the course website:
https://www.embl.org/about/info/course-and-conference-office/events/mmo24-01/

With best wishes,
Wojtek

--
Wojciech Galej, PhD
Group Leader
EMBL Grenoble
71 Avenue des Martyrs
38042 Grenoble Cedex 09
France
=
https://www.embl.org/groups/galej/
https://twitter.com/WojtekGalej
https://orcid.org/-0001-8859-5229
=



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Re: [ccp4bb] Difficult Molecular replacement

[Randy John Read]

Hi,

It’s possible the true space group is P2(1) with the b-axis unique, and that 
subset of true symmetry is found repeatedly with different incorrect 
backgrounds of other copies. But I think the easiest way to resolve this 
unambiguously is to solve in P1, and let that uncover the true symmetry.

Best wishes,

Randy

> On 21 Feb 2024, at 13:56, Pedro Matias  wrote:
> 
> But curiously, all the 4 best solutions correspond to a SG with a 21 screw 
> along b.
> And amazingly none of the TF solutions is rejected due to clashes.
> On 21/02/2024 12:20, Eleanor Dodson wrote:
>> Lots of comments, but it would be easier to actually look at your integrated 
>> data!  
>> Some of the stats look a bit ropey - 
>> 621 reflections labelled as outliers by PHASER?
>> Very anisotropic 
>> Moments go mad at the highest resolution..
>> 
>> The good news - extremely strong signal from the rotation function means the 
>> model is probably a good one.
>> Bad news - translation function results do not select a definitive solution..
>> Possible reasons 
>> Unit Cell:   72.61   73.73  147.23   90.00   90.00   90.00
>> Most likely data problems - a axis ~ = b axis so twinning is possible 
>> 
>> I could add more comments if either you could share the unmerged data, or at 
>> least a pointless logfile..
>> Cheers Eleanor
>> 
>> 
>> 
>> 
>> 
>> On Wed, 21 Feb 2024 at 11:06, Randy John Read  wrote:
>> Hi Marco,
>> 
>> To add to what Kay has said:
>> 
>> The intensity moments from Phaser (between 1.5 and 2 for the second moments 
>> after correcting for anisotropy) are indicative of likely twinning. With the 
>> cell dimensions, it might be possible to have pseudomerohedral twinning in 
>> an orthorhombic space group, but given the lack of distinction among 
>> possible choices of orthorhombic spac group (noted by Kay), it seems much 
>> more likely that the true symmetry is lower and that you have pseudosymmetry 
>> combined with perfect twinning.
>> 
>> Judging from the strong and unambiguous rotation peak, your model is clearly 
>> very good, so I think it would be easy to ask Phaser to solve this by 
>> looking for 4 copies in space group P1. You can get P1 data either by 
>> expanding the orthorhombic data to P1 or by re-merging the data in P1. If 
>> the merging statistics were good, that would indicate that any twinning 
>> would be close to perfect, so just expanding the data would be a reasonable 
>> choice. Alternatively, you have reasonable redundancy so merging in P1 would 
>> be a plausible choice. I would probably go with expanding the data, figuring 
>> out from the MR solution what the real symmetry is, and then merging the 
>> data with that symmetry.
>> 
>> Unless there are some other pathologies, I think the MR in P1 is very likely 
>> to give a clear answer (or maybe 2 answers related by the twin operator). 
>> It’s formally possible that you could have a different number of copies 
>> (e.g. 6 in the unit cell) if the true symmetry were monoclinic, so keep an 
>> open mind on that question. You could just try finding the largest domain 
>> from splitting the AlphaFold model (presumably the domain for which you sent 
>> the log file), work out the symmetry from that solution, and then run a job 
>> to search for all the domains in the right space group. It’s generally a 
>> good idea, by the way, to ask Phaser to look for everything you expect to 
>> find in one job, because it has built-in logic to predict the best search 
>> order but then update it on the basis of preliminary results.
>> 
>> There are different ways to sort out the true symmetry from the MR solution, 
>> but within CCP4 the Zanuda procedure is a very effective choice.
>> 
>> Get in touch if you have any difficulties following this procedure, and it 
>> would be great to let the BB know the outcome!
>> 
>> Best wishes,
>> 
>> Randy Read
>> 
>> > On 21 Feb 2024, at 08:42, Kay Diederichs  
>> > wrote:
>> >
>> > Hi Marco:
>> >
>> > short comments (I sent you also a private mail):
>> > - the stats in CORRECT.LP look ok, but I'd like to know what ISa is, and 
>> > what the number of outliers is ("misfits"). Seeing the delta-CC1/2 stats 
>> > as a function of frame number is also useful for judging e.g. the 
>> > radiation damage - this is available from XDSGUI in the "statistics" tab. 
>> > Another remark: SPOT_RANGE=1 11 in COLSPOT will sample reciprocal space 
>> > poorly - I always use the first half of the DATA_RANGE as SPOT_RANGE, at a 
>> > negligible cost in CPU time.
>> > - the Phaser logfile shows that the rotation function (table at line 1344) 
>> > has only a single solution, but the translation function (table at line 
>> > 7028)  does not even allow to determine the space group - there is very 
>> > little contrast difference between potential "solutions".
>> >
>> > Best wishes,
>> > Kay
>> >
>> > On Tue, 20 Feb 2024 21:45:12 +, Marco Bravo  wrote:
>> >
>> >> 

Re: [ccp4bb] Difficult Molecular replacement

[Pedro Matias]

But curiously, all the 4 best solutions correspond to a SG with a 21 
screw along b.


And amazingly none of the TF solutions is rejected due to clashes.

On 21/02/2024 12:20, Eleanor Dodson wrote:
Lots of comments, but it would be easier to actually look at your 
integrated data!

Some of the stats look a bit ropey -
621 reflections labelled as outliers by PHASER?
Very anisotropic
Moments go mad at the highest resolution..

The good news - extremely strong signal from the rotation function 
means the model is probably a good one.
Bad news - translation function results do not select a definitive 
solution..

Possible reasons
Unit Cell:   72.61   73.73  147.23   90.00   90.00   90.00
Most likely data problems - a axis ~ = b axis so twinning is possible

I could add more comments if either you could share the unmerged data, 
or at least a pointless logfile..

Cheers Eleanor





On Wed, 21 Feb 2024 at 11:06, Randy John Read  wrote:

Hi Marco,

To add to what Kay has said:

The intensity moments from Phaser (between 1.5 and 2 for the
second moments after correcting for anisotropy) are indicative of
likely twinning. With the cell dimensions, it might be possible to
have pseudomerohedral twinning in an orthorhombic space group, but
given the lack of distinction among possible choices of
orthorhombic spac group (noted by Kay), it seems much more likely
that the true symmetry is lower and that you have pseudosymmetry
combined with perfect twinning.

Judging from the strong and unambiguous rotation peak, your model
is clearly very good, so I think it would be easy to ask Phaser to
solve this by looking for 4 copies in space group P1. You can get
P1 data either by expanding the orthorhombic data to P1 or by
re-merging the data in P1. If the merging statistics were good,
that would indicate that any twinning would be close to perfect,
so just expanding the data would be a reasonable choice.
Alternatively, you have reasonable redundancy so merging in P1
would be a plausible choice. I would probably go with expanding
the data, figuring out from the MR solution what the real symmetry
is, and then merging the data with that symmetry.

Unless there are some other pathologies, I think the MR in P1 is
very likely to give a clear answer (or maybe 2 answers related by
the twin operator). It’s formally possible that you could have a
different number of copies (e.g. 6 in the unit cell) if the true
symmetry were monoclinic, so keep an open mind on that question.
You could just try finding the largest domain from splitting the
AlphaFold model (presumably the domain for which you sent the log
file), work out the symmetry from that solution, and then run a
job to search for all the domains in the right space group. It’s
generally a good idea, by the way, to ask Phaser to look for
everything you expect to find in one job, because it has built-in
logic to predict the best search order but then update it on the
basis of preliminary results.

There are different ways to sort out the true symmetry from the MR
solution, but within CCP4 the Zanuda procedure is a very effective
choice.

Get in touch if you have any difficulties following this
procedure, and it would be great to let the BB know the outcome!

Best wishes,

Randy Read

> On 21 Feb 2024, at 08:42, Kay Diederichs
 wrote:
>
> Hi Marco:
>
> short comments (I sent you also a private mail):
> - the stats in CORRECT.LP look ok, but I'd like to know what ISa
is, and what the number of outliers is ("misfits"). Seeing the
delta-CC1/2 stats as a function of frame number is also useful for
judging e.g. the radiation damage - this is available from XDSGUI
in the "statistics" tab. Another remark: SPOT_RANGE=1 11 in
COLSPOT will sample reciprocal space poorly - I always use the
first half of the DATA_RANGE as SPOT_RANGE, at a negligible cost
in CPU time.
> - the Phaser logfile shows that the rotation function (table at
line 1344) has only a single solution, but the translation
function (table at line 7028)  does not even allow to determine
the space group - there is very little contrast difference between
potential "solutions".
>
> Best wishes,
> Kay
>
> On Tue, 20 Feb 2024 21:45:12 +, Marco Bravo
 wrote:
>
>>

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 

Re: [ccp4bb] Difficult Molecular replacement

[Randy John Read]

Hi Eleanor,

> On 21 Feb 2024, at 12:20, Eleanor Dodson  wrote:
> 
> Lots of comments, but it would be easier to actually look at your integrated 
> data! 
> Some of the stats look a bit ropey - 
> 621 reflections labelled as outliers by PHASER?
> Very anisotropic 
> Moments go mad at the highest resolution..

These 3 observations are actually all consequences of the strong anisotropy. 
Phaser flags reflections with very little information content as outliers, 
because they’re being ignored (as they would have almost no effect on the 
likelihood calculation), and these come from the data in the weak directions. 
The moments go mad, but in a way that is predictable from the sigmas on the 
intensities, as seen by the fact that the predicted and observed moments track 
each other as a function of resolution.

Best wishes,

Randy

> 
> The good news - extremely strong signal from the rotation function means the 
> model is probably a good one.
> Bad news - translation function results do not select a definitive solution..
> Possible reasons 
> Unit Cell:   72.61   73.73  147.23   90.00   90.00   90.00
> Most likely data problems - a axis ~ = b axis so twinning is possible 
> 
> I could add more comments if either you could share the unmerged data, or at 
> least a pointless logfile..
> Cheers Eleanor
> 
> 
> 
> 
> 
> On Wed, 21 Feb 2024 at 11:06, Randy John Read  wrote:
> Hi Marco,
> 
> To add to what Kay has said:
> 
> The intensity moments from Phaser (between 1.5 and 2 for the second moments 
> after correcting for anisotropy) are indicative of likely twinning. With the 
> cell dimensions, it might be possible to have pseudomerohedral twinning in an 
> orthorhombic space group, but given the lack of distinction among possible 
> choices of orthorhombic spac group (noted by Kay), it seems much more likely 
> that the true symmetry is lower and that you have pseudosymmetry combined 
> with perfect twinning.
> 
> Judging from the strong and unambiguous rotation peak, your model is clearly 
> very good, so I think it would be easy to ask Phaser to solve this by looking 
> for 4 copies in space group P1. You can get P1 data either by expanding the 
> orthorhombic data to P1 or by re-merging the data in P1. If the merging 
> statistics were good, that would indicate that any twinning would be close to 
> perfect, so just expanding the data would be a reasonable choice. 
> Alternatively, you have reasonable redundancy so merging in P1 would be a 
> plausible choice. I would probably go with expanding the data, figuring out 
> from the MR solution what the real symmetry is, and then merging the data 
> with that symmetry.
> 
> Unless there are some other pathologies, I think the MR in P1 is very likely 
> to give a clear answer (or maybe 2 answers related by the twin operator). 
> It’s formally possible that you could have a different number of copies (e.g. 
> 6 in the unit cell) if the true symmetry were monoclinic, so keep an open 
> mind on that question. You could just try finding the largest domain from 
> splitting the AlphaFold model (presumably the domain for which you sent the 
> log file), work out the symmetry from that solution, and then run a job to 
> search for all the domains in the right space group. It’s generally a good 
> idea, by the way, to ask Phaser to look for everything you expect to find in 
> one job, because it has built-in logic to predict the best search order but 
> then update it on the basis of preliminary results.
> 
> There are different ways to sort out the true symmetry from the MR solution, 
> but within CCP4 the Zanuda procedure is a very effective choice.
> 
> Get in touch if you have any difficulties following this procedure, and it 
> would be great to let the BB know the outcome!
> 
> Best wishes,
> 
> Randy Read
> 
> > On 21 Feb 2024, at 08:42, Kay Diederichs  
> > wrote:
> >
> > Hi Marco:
> >
> > short comments (I sent you also a private mail):
> > - the stats in CORRECT.LP look ok, but I'd like to know what ISa is, and 
> > what the number of outliers is ("misfits"). Seeing the delta-CC1/2 stats as 
> > a function of frame number is also useful for judging e.g. the radiation 
> > damage - this is available from XDSGUI in the "statistics" tab. Another 
> > remark: SPOT_RANGE=1 11 in COLSPOT will sample reciprocal space poorly - I 
> > always use the first half of the DATA_RANGE as SPOT_RANGE, at a negligible 
> > cost in CPU time.
> > - the Phaser logfile shows that the rotation function (table at line 1344) 
> > has only a single solution, but the translation function (table at line 
> > 7028)  does not even allow to determine the space group - there is very 
> > little contrast difference between potential "solutions".
> >
> > Best wishes,
> > Kay
> >
> > On Tue, 20 Feb 2024 21:45:12 +, Marco Bravo  wrote:
> >
> >> https://www.dropbox.com/scl/fo/pmw9nisdmq53yir2jqcmu/h?rlkey=zaj6cnknkjgkowrzf0vrmqdyf=0
> >>
> >> Here is a link to my Molecular replacement and 

[ccp4bb] Recruitment of the future Director of the Institut de Biologie Structurale (IBS) in Grenoble

[Antoine Royant]

*Recruitment **of the future Director of the Institut de Biologie 
Structurale (IBS) in Grenoble*


The Institutde BiologieStructurale(IBS) in Grenoble, France,is currently 
seeking a Director to lead its upcoming national multi-annualplan 
(HCERES wave A, 2027-2031). We are looking for an individualwith a 
distinguished international reputationin the field of structural 
biologywho possesses outstanding leadership and management skills. The 
ideal candidate will be able to leveragethe Institute's state-of-the-art 
facilitiesto develop their own research activity.


Situated on the European Photon and Neutron (EPN)campus in 
Grenobleadjacent to three international scientific institutes(ESRF, 
ILLand EMBL), the IBS is a joint unit of the CEA, CNRS, and 
UniversitéGrenoble Alpes. As a renowned playerin integrated structural 
biology, the institute fulfilsmultiple roles, functioning asa research 
centre, technical platform, user facility, and scientific training site. 
Our commitment lies in pushing the boundaries ofstructural biology 
research, a pivotal field for decipheringfundamental biological processes.


The incoming Director will be tasked with crafting and executing 
acomprehensive forward-thinking strategyaimed at strengthening the IBS's 
global standing and fostering its future growth. We seek an individual 
with a strongbackground in research and personnel management, adept at 
decision-making, and possessing a nuanced understanding of the French 
academic research landscape, its organization, and operational dynamics. 
Proficiency in both French and English is essential.


*Timetab**le**:*

*First **selection**phase**:*

Applicantsshouldsenda 1-2-page letterof intentand a short curriculum 
vitae no laterthan26/03/2024 to ibs.comite.proposition...@ibs.fr


Applications receivedat thisstage willbetreatedconfidentiallyby the IBS 
Director searchcommittee.


*Second **selection**phase**: *

Candidates shortlistedinthe first phase willbeinvitedto senda 
proposaldetailingtheirleadership projectfor the IBS (5 pages maximum), a 
description of theirresearchactivities(3 pages maximum), and a listof 
publications to: ibs.comite.proposition...@ibs.fr no laterthan31/05/2024.


*Contact* : ibs.comite.proposition...@ibs.fr

--

*Appel à candidature : Directeur/Directrice de l’Institut de Biologie 
Structurale (IBS) à Grenoble

*

L’Institut de Biologie Structurale (IBS) de Grenoble recherche un 
directeur ou une directrice pour le prochain plan national pluriannuel 
(HCERES vague A, 2027-2031), ayant d’excellents antécédents en 
management et une renommée internationale dans le domaine de la biologie 
structurale et qui pourra développer en parallèle une activité de 
recherche propre en bénéficiant des installations mises à disposition.


Situé sur le campus EPN de Grenoble (ESRF/ILL/EMBL/IBS), l’IBS, unité 
mixte de recherche (CEA-CNRS-Université Grenoble Alpes), est un acteur 
d’envergure nationale et internationale dans le domaine de la biologie 
structurale intégrée. A la fois centre de recherche, plateau technique, 
site d'accueil et de formation scientifique, l'IBS a pour vocation le 
développement de recherches en biologie structurale, un champ de 
recherche capital pour la compréhension des mécanismes biologiques 
fondamentaux.


Le candidat ou la candidate saura mener une stratégie globale et 
innovante pour conforter la stature internationale de l’IBS et porter 
les futurs développements de l’Institut. Il ou elle aura de solides 
compétences en gestion de la recherche et de personnels, dans la prise 
de décision et une bonne connaissance du monde de la recherche 
française, de son organisation et de son fonctionnement. Il ou elle 
devra maîtriser les langues française et anglaise.


*Calendrier :*

*1ère phase de sélection :*

Les candidats doivent envoyer une lettre d’intention de 1-2 pages et un 
court curriculum vitae au plus tard le 26/03/2024, à 
ibs.comite.proposition...@ibs.fr


Les candidatures reçues à ce stade seront traitées de façon 
confidentielle par le comité de proposition de DU de l’IBS.


*2de phase de sélection : *

Les candidats retenus à l’issue de la 1ère phase enverront une 
proposition de projet de direction pour l’IBS (5 pages maximum), une 
description de leurs activités de recherche (3 pages maximum), et une 
liste de publications à : ibs.comite.proposition...@ibs.frau plus tard 
le 31/05/2024.


*Les auditions *des candidats retenus à l’issue de la 2de phase se 
dérouleront à partir de mi-juin 2024.


*Contact* : ibs.comite.proposition...@ibs.fr



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Re: [ccp4bb] Difficult Molecular replacement

[Eleanor Dodson]

Lots of comments, but it would be easier to actually look at your
integrated data!
Some of the stats look a bit ropey -
621 reflections labelled as outliers by PHASER?
Very anisotropic
Moments go mad at the highest resolution..

The good news - extremely strong signal from the rotation function means
the model is probably a good one.
Bad news - translation function results do not select a definitive
solution..
Possible reasons
Unit Cell:   72.61   73.73  147.23   90.00   90.00   90.00
Most likely data problems - a axis ~ = b axis so twinning is possible

I could add more comments if either you could share the unmerged data, or
at least a pointless logfile..
Cheers Eleanor





On Wed, 21 Feb 2024 at 11:06, Randy John Read  wrote:

> Hi Marco,
>
> To add to what Kay has said:
>
> The intensity moments from Phaser (between 1.5 and 2 for the second
> moments after correcting for anisotropy) are indicative of likely twinning.
> With the cell dimensions, it might be possible to have pseudomerohedral
> twinning in an orthorhombic space group, but given the lack of distinction
> among possible choices of orthorhombic spac group (noted by Kay), it seems
> much more likely that the true symmetry is lower and that you have
> pseudosymmetry combined with perfect twinning.
>
> Judging from the strong and unambiguous rotation peak, your model is
> clearly very good, so I think it would be easy to ask Phaser to solve this
> by looking for 4 copies in space group P1. You can get P1 data either by
> expanding the orthorhombic data to P1 or by re-merging the data in P1. If
> the merging statistics were good, that would indicate that any twinning
> would be close to perfect, so just expanding the data would be a reasonable
> choice. Alternatively, you have reasonable redundancy so merging in P1
> would be a plausible choice. I would probably go with expanding the data,
> figuring out from the MR solution what the real symmetry is, and then
> merging the data with that symmetry.
>
> Unless there are some other pathologies, I think the MR in P1 is very
> likely to give a clear answer (or maybe 2 answers related by the twin
> operator). It’s formally possible that you could have a different number of
> copies (e.g. 6 in the unit cell) if the true symmetry were monoclinic, so
> keep an open mind on that question. You could just try finding the largest
> domain from splitting the AlphaFold model (presumably the domain for which
> you sent the log file), work out the symmetry from that solution, and then
> run a job to search for all the domains in the right space group. It’s
> generally a good idea, by the way, to ask Phaser to look for everything you
> expect to find in one job, because it has built-in logic to predict the
> best search order but then update it on the basis of preliminary results.
>
> There are different ways to sort out the true symmetry from the MR
> solution, but within CCP4 the Zanuda procedure is a very effective choice.
>
> Get in touch if you have any difficulties following this procedure, and it
> would be great to let the BB know the outcome!
>
> Best wishes,
>
> Randy Read
>
> > On 21 Feb 2024, at 08:42, Kay Diederichs 
> wrote:
> >
> > Hi Marco:
> >
> > short comments (I sent you also a private mail):
> > - the stats in CORRECT.LP look ok, but I'd like to know what ISa is, and
> what the number of outliers is ("misfits"). Seeing the delta-CC1/2 stats as
> a function of frame number is also useful for judging e.g. the radiation
> damage - this is available from XDSGUI in the "statistics" tab. Another
> remark: SPOT_RANGE=1 11 in COLSPOT will sample reciprocal space poorly - I
> always use the first half of the DATA_RANGE as SPOT_RANGE, at a negligible
> cost in CPU time.
> > - the Phaser logfile shows that the rotation function (table at line
> 1344) has only a single solution, but the translation function (table at
> line 7028)  does not even allow to determine the space group - there is
> very little contrast difference between potential "solutions".
> >
> > Best wishes,
> > Kay
> >
> > On Tue, 20 Feb 2024 21:45:12 +, Marco Bravo 
> wrote:
> >
> >>
> 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 

Re: [ccp4bb] Difficult Molecular replacement

[Randy John Read]

Hi Marco,

To add to what Kay has said:

The intensity moments from Phaser (between 1.5 and 2 for the second moments 
after correcting for anisotropy) are indicative of likely twinning. With the 
cell dimensions, it might be possible to have pseudomerohedral twinning in an 
orthorhombic space group, but given the lack of distinction among possible 
choices of orthorhombic spac group (noted by Kay), it seems much more likely 
that the true symmetry is lower and that you have pseudosymmetry combined with 
perfect twinning.

Judging from the strong and unambiguous rotation peak, your model is clearly 
very good, so I think it would be easy to ask Phaser to solve this by looking 
for 4 copies in space group P1. You can get P1 data either by expanding the 
orthorhombic data to P1 or by re-merging the data in P1. If the merging 
statistics were good, that would indicate that any twinning would be close to 
perfect, so just expanding the data would be a reasonable choice. 
Alternatively, you have reasonable redundancy so merging in P1 would be a 
plausible choice. I would probably go with expanding the data, figuring out 
from the MR solution what the real symmetry is, and then merging the data with 
that symmetry.

Unless there are some other pathologies, I think the MR in P1 is very likely to 
give a clear answer (or maybe 2 answers related by the twin operator). It’s 
formally possible that you could have a different number of copies (e.g. 6 in 
the unit cell) if the true symmetry were monoclinic, so keep an open mind on 
that question. You could just try finding the largest domain from splitting the 
AlphaFold model (presumably the domain for which you sent the log file), work 
out the symmetry from that solution, and then run a job to search for all the 
domains in the right space group. It’s generally a good idea, by the way, to 
ask Phaser to look for everything you expect to find in one job, because it has 
built-in logic to predict the best search order but then update it on the basis 
of preliminary results.

There are different ways to sort out the true symmetry from the MR solution, 
but within CCP4 the Zanuda procedure is a very effective choice.

Get in touch if you have any difficulties following this procedure, and it 
would be great to let the BB know the outcome!

Best wishes,

Randy Read

> On 21 Feb 2024, at 08:42, Kay Diederichs  
> wrote:
>
> Hi Marco:
>
> short comments (I sent you also a private mail):
> - the stats in CORRECT.LP look ok, but I'd like to know what ISa is, and what 
> the number of outliers is ("misfits"). Seeing the delta-CC1/2 stats as a 
> function of frame number is also useful for judging e.g. the radiation damage 
> - this is available from XDSGUI in the "statistics" tab. Another remark: 
> SPOT_RANGE=1 11 in COLSPOT will sample reciprocal space poorly - I always use 
> the first half of the DATA_RANGE as SPOT_RANGE, at a negligible cost in CPU 
> time.
> - the Phaser logfile shows that the rotation function (table at line 1344) 
> has only a single solution, but the translation function (table at line 7028) 
>  does not even allow to determine the space group - there is very little 
> contrast difference between potential "solutions".
>
> Best wishes,
> Kay
>
> On Tue, 20 Feb 2024 21:45:12 +, Marco Bravo  wrote:
>
>> 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.
>>
>> 
>>
>> 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 http://www.jiscmail.ac.uk/CCP4BB, a 
>> mailing list hosted by http://www.jiscmail.ac.uk/, terms & conditions are 
>> available at https://www.jiscmail.ac.uk/policyandsecurity/
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> This message was issued to members of http://www.jiscmail.ac.uk/CCP4BB, a 
> mailing list hosted by 

Re: [ccp4bb] Difficult Molecular replacement

[Kay Diederichs]

Hi Marco:

short comments (I sent you also a private mail):
- the stats in CORRECT.LP look ok, but I'd like to know what ISa is, and what 
the number of outliers is ("misfits"). Seeing the delta-CC1/2 stats as a 
function of frame number is also useful for judging e.g. the radiation damage - 
this is available from XDSGUI in the "statistics" tab. Another remark: 
SPOT_RANGE=1 11 in COLSPOT will sample reciprocal space poorly - I always use 
the first half of the DATA_RANGE as SPOT_RANGE, at a negligible cost in CPU 
time. 
- the Phaser logfile shows that the rotation function (table at line 1344) has 
only a single solution, but the translation function (table at line 7028)  does 
not even allow to determine the space group - there is very little contrast 
difference between potential "solutions".

Best wishes,
Kay

On Tue, 20 Feb 2024 21:45:12 +, Marco Bravo  wrote:

>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.
>
>
>
>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 
>https://www.jiscmail.ac.uk/policyandsecurity/



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