Re: [ccp4bb] PDB to AlphaFold via Uniprot

[Robbie Joosten]

Hi Paul,

From the PDB file get the Uniprot primary accession code (see DBREF) and use 
this to get the Alphafold model using 3D-beacons 
(https://www.ebi.ac.uk/pdbe/pdbe-kb/3dbeacons/).
You can also use 3D-beacons to get the related AlphaFill model (just saying...).

Cheers,
Robbie

> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Paul
> Emsley
> Sent: Friday, July 29, 2022 20:31
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] PDB to AlphaFold via Uniprot
> 
> I wonder if others are curious about (or know how to solve) the
> following problem:
> 
> I am looking at a PDB file that I've downloaded from a wwPDB site and I
> would like to see the AlphaFold model(s) overlaid. What the best (or
> easiest) way of doing that? (let's imagine that I can do a bit of
> scripting in python (including urllib) and can use the functions in Coot
> for the superposition).
> 
> Thanks,
> 
> Paul.
> 
> ###
> #
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> 
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[ccp4bb] Postdoc opening in Berkeley

[Jan Kern]

We are seeking two postdoctoral researchers to join our efforts at Lawrence
Berkeley National Lab in Berkeley, CA, to better understand reaction
mechanisms of several important metalloenzymes. We are using XFELs for time
resolved room temperature crystallography in combination with X-ray
spectroscopy and develop new methods for reaction triggering, sample
delivery and data processing. Projects can involve protein biochemistry and
crystallization, metalloenzyme X-ray spectroscopy, methods and
instrumentation development depending on the interests of the candidates.

Some recent publications from our group include:
Ibrahim et al 2020, https://doi.org/10.1073/pnas.2000529117
Hussein et al 2021, https://doi.org/10.1038/s41467-021-26781-z
Butryn et al 2021, https://doi.org/10.1038/s41467-021-24757-7
Rabe et al 2021, https://doi.org/10.1126/sciadv.abh0250

Details for the positions can be found here:
https://lbl.referrals.selectminds.com/jobs/xfel-postdoctoral-fellow-crystallography-spectroscopy-4991

https://lbl.referrals.selectminds.com/jobs/chemist-postdoctoral-fellow-4992

Do not hesitate to contact me directly in case of any questions.

Jan Kern
Staff Scientist - Head of Bioenergetics Department
Lawrence Berkeley National Laboratory
jfk...@lbl.gov



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[ccp4bb] PDB to AlphaFold via Uniprot

[Paul Emsley]

I wonder if others are curious about (or know how to solve) the 
following problem:


I am looking at a PDB file that I've downloaded from a wwPDB site and I 
would like to see the AlphaFold model(s) overlaid. What the best (or 
easiest) way of doing that? (let's imagine that I can do a bit of 
scripting in python (including urllib) and can use the functions in Coot 
for the superposition).


Thanks,

Paul.



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Re: [ccp4bb] Checking X-ray sequence (no more protein).

[David J. Schuller]

I don't know how much effort I would put into that, given how easy nucleic acid 
sequencing has become.

===
 All Things Serve the Beam
 ===
 David J. Schuller
 modern man in a post-modern world
 MacCHESS, Cornell University
 schul...@cornell.edu

From: CCP4 bulletin board  on behalf of Robbie Joosten 

Sent: Friday, July 29, 2022 9:56 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Checking X-ray sequence (no more protein).

Hi Jon,

There are placeholders for ASP/ASN and GLU/GLN ambiguities: ASX and GLX 
respectively. You can just use those. AFAICT there no such thing for VAL/THR 
ambiguities. You could look for the most likely canadidata based on multiple 
sequence alignments. Refinement of both alternatives can give hints in 
B-factors and if you are lucky in difference density. But if hydrogen bonding 
gives no hints, then the residues are also not in a place where the identity 
really matters. You can give your best guess with a CAVEAT record or use the 
name UNK to indicate that you do not know what the residue is. You would loose 
the knowledge that it is either VAL or THR in that case.

Cheers,
Robbie

> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Jon
> Cooper
> Sent: Friday, July 29, 2022 12:14
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Checking X-ray sequence (no more protein).
>
> Hello, I am looking for suggestions of ways to check a 1.7 Angstrom X-ray
> sequence for a protein where it is impractical to do experimental sequencing,
> protein or DNA. The structure refines to publishable R/R-free and the main
> ambiguities seem to be Thr/Val, Asp/Asn and Glu/Gln where alternative H-
> bonding networks are possible. Running alpha-fold seems an interesting
> option? Any suggestions much appreciated.
>
> Cheers, Jon.C.
>
> Sent from ProtonMail mobile
>
>
>
>
> 
>
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1




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Re: [ccp4bb] Checking X-ray sequence (no more protein).

[Robbie Joosten]

Hi Jon,

There are placeholders for ASP/ASN and GLU/GLN ambiguities: ASX and GLX 
respectively. You can just use those. AFAICT there no such thing for VAL/THR 
ambiguities. You could look for the most likely canadidata based on multiple 
sequence alignments. Refinement of both alternatives can give hints in 
B-factors and if you are lucky in difference density. But if hydrogen bonding 
gives no hints, then the residues are also not in a place where the identity 
really matters. You can give your best guess with a CAVEAT record or use the 
name UNK to indicate that you do not know what the residue is. You would loose 
the knowledge that it is either VAL or THR in that case. 

Cheers,
Robbie

> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Jon
> Cooper
> Sent: Friday, July 29, 2022 12:14
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Checking X-ray sequence (no more protein).
> 
> Hello, I am looking for suggestions of ways to check a 1.7 Angstrom X-ray
> sequence for a protein where it is impractical to do experimental sequencing,
> protein or DNA. The structure refines to publishable R/R-free and the main
> ambiguities seem to be Thr/Val, Asp/Asn and Glu/Gln where alternative H-
> bonding networks are possible. Running alpha-fold seems an interesting
> option? Any suggestions much appreciated.
> 
> Cheers, Jon.C.
> 
> Sent from ProtonMail mobile
> 
> 
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1




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Re: [ccp4bb] Checking X-ray sequence (no more protein).

[Jon Cooper]

Thank you, yes, threading style tools to assess the likelihood of having a 
given amino acid in a certain position in the fold would be a good approach. I 
have tried one but wasn't hugely informative, in my hands anyway. All 
suggestions very welcome but big database science is a bit outside my skill 
set. Of course, sequence conservation in the family helps a lot with the 
assignment but there are bigger ambiguities in less conserved surface regions 
e.g. a disordered Lys can refine well as a Ser, Ala or Gly even, but e.g. 
nearby conserved acidic groups might suggest the presence of the basic amino 
acid which could salt-bridge with them, but why then would it be so disordered? 
Tangles we weave...

Sent from ProtonMail mobile

 Original Message 
On 29 Jul 2022, 14:17, Clemens Vonrhein wrote:

> Maybe a crazy idea, but couldn't one use various model/geometry validation 
> tools to figure out some of those ambiguities? As a test one could take a 
> very good 1.7A structure and do some random ASN->ASP, THR->VAL etc mutations 
> followed by refinement (including hydrogens). Wouldn't some validation tool 
> pick up unfavourable conformations, poor rotamers and/or hydrogen clashes and 
> poor H-networks (compared to the initial, correct sequence)? Maybe there is 
> some kind of "fingerprint" in validation results for such incorrect residue 
> assignments that can distinguish correct from incorrect sequences ... Or put 
> another way, if model validation can not pick up such sequence errors: should 
> we be worried about the reliability of our validation criteria? A large scale 
> re-refinement of deposited structures with (1) the current/correct sequence 
> and (2) those ASN/ASP, THR/VAL etc ambiguities artificially introduced, could 
> provide a clever algorithm (AI?) with the data basis to figure out those 
> "fingerprints". Maybe even for the ASN/GLN/HIS side-chain orientations when 
> the sequence is actually correct. Cheers Clemens On Fri, Jul 29, 2022 at 
> 12:08:58PM +, Jon Cooper wrote: > Thank you so much for your replies. I 
> apologise for being unclear. The protein is purified from a plant that hasn't 
> had its genome sequence determined. We know the enzyme family of the protein 
> and therefore the structure was originally solved by MR. The 'X-ray sequence' 
> we have is just determined from looking at the 1.7 Angstrom density, which is 
> good, over several refinement and rebuilding rounds. The resulting sequence 
> has been run through blast and it is up to 58% identical with other family 
> members. To me this seemed low but that degree of identity is typical of 
> other family members. The postgrad who did the work did obtain some peptide 
> sequences and prior to that about 20% of the sequence was determined by the 
> Edman method with the usual Asp/Asn and Glu/Gln ambiguity. However, there 
> isn't any prospect of us doing further experimental work, sorry, but that's 
> the way it is!! > > Best wishes, Jon.C. > > Sent from ProtonMail mobile > > 
>  Original Message  > On 29 Jul 2022, 12:23, Jan Dohnalek 
> wrote: > > > If you know at least something about your protein, organism, 
> type of molecule, ..., you could try mass spectrometry peptide mapping to 
> known sequences, this may give you some answers for the ambiguities you might 
> be seeing, if nothing else .. > > > > Jan > > > > On Fri, Jul 29, 2022 at 
> 12:15 PM Jon Cooper wrote: > > > >> Hello, I am looking for suggestions of 
> ways to check a 1.7 Angstrom X-ray sequence for a protein where it is 
> impractical to do experimental sequencing, protein or DNA. The structure 
> refines to publishable R/R-free and the main ambiguities seem to be Thr/Val, 
> Asp/Asn and Glu/Gln where alternative H-bonding networks are possible. 
> Running alpha-fold seems an interesting option? Any suggestions much 
> appreciated. > >> > >> Cheers, Jon.C. > >> > >> Sent from ProtonMail mobile > 
> >> > >> --- > >> 
> > >> To unsubscribe from the CCP4BB list, click the following link: > >> 
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > > -- > 
> > > > Jan Dohnalek, Ph.D > > Institute of Biotechnology > > > > Academy of 
> Sciences of the Czech Republic > > Biocev > > Prumyslova 595 > > 252 50 
> Vestec near Prague > > Czech Republic > > Tel. +420 325 873 758 > > 
>  > > 
> 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/ -- 
> *-- * Clemens 
> Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * Global Phasing Ltd., 
> 

Re: [ccp4bb] Checking X-ray sequence (no more protein).

[Clemens Vonrhein]

Maybe a crazy idea, but couldn't one use various model/geometry
validation tools to figure out some of those ambiguities? As a test
one could take a very good 1.7A structure and do some random ASN->ASP,
THR->VAL etc mutations followed by refinement (including
hydrogens). Wouldn't some validation tool pick up unfavourable
conformations, poor rotamers and/or hydrogen clashes and poor
H-networks (compared to the initial, correct sequence)?

Maybe there is some kind of "fingerprint" in validation results for
such incorrect residue assignments that can distinguish correct from
incorrect sequences ... Or put another way, if model validation can
not pick up such sequence errors: should we be worried about the
reliability of our validation criteria?

A large scale re-refinement of deposited structures with (1) the
current/correct sequence and (2) those ASN/ASP, THR/VAL etc ambiguities
artificially introduced, could provide a clever algorithm (AI?) with
the data basis to figure out those "fingerprints". Maybe even for the
ASN/GLN/HIS side-chain orientations when the sequence is actually
correct.

Cheers

Clemens


On Fri, Jul 29, 2022 at 12:08:58PM +, Jon Cooper wrote:
> Thank you so much for your replies. I apologise for being unclear. The 
> protein is purified from a plant that hasn't had its genome sequence 
> determined. We know the enzyme family of the protein and therefore the 
> structure was originally solved by MR. The 'X-ray sequence' we have is just 
> determined from looking at the 1.7 Angstrom density, which is good, over 
> several refinement and rebuilding rounds. The resulting sequence has been run 
> through blast and it is up to 58% identical with other family members. To me 
> this seemed low but that degree of identity is typical of other family 
> members. The postgrad who did the work did obtain some peptide sequences and 
> prior to that about 20% of the sequence was determined by the Edman method 
> with the usual Asp/Asn and Glu/Gln ambiguity. However, there isn't any 
> prospect of us doing further experimental work, sorry, but that's the way it 
> is!!
> 
> Best wishes, Jon.C.
> 
> Sent from ProtonMail mobile
> 
>  Original Message 
> On 29 Jul 2022, 12:23, Jan Dohnalek wrote:
> 
> > If you know at least something about your protein, organism, type of 
> > molecule, ..., you could try mass spectrometry peptide mapping to known 
> > sequences, this may give you some answers for the ambiguities you might be 
> > seeing, if nothing else ..
> >
> > Jan
> >
> > On Fri, Jul 29, 2022 at 12:15 PM Jon Cooper 
> > <488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
> >
> >> Hello, I am looking for suggestions of ways to check a 1.7 Angstrom X-ray 
> >> sequence for a protein where it is impractical to do experimental 
> >> sequencing, protein or DNA. The structure refines to publishable R/R-free 
> >> and the main ambiguities seem to be Thr/Val, Asp/Asn and Glu/Gln where 
> >> alternative H-bonding networks are possible. Running alpha-fold seems an 
> >> interesting option? Any suggestions much appreciated.
> >>
> >> Cheers, Jon.C.
> >>
> >> Sent from ProtonMail mobile
> >>
> >> ---
> >>
> >> To unsubscribe from the CCP4BB list, click the following link:
> >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> >
> > --
> >
> > Jan Dohnalek, Ph.D
> > Institute of Biotechnology
> >
> > Academy of Sciences of the Czech Republic
> > Biocev
> > Prumyslova 595
> > 252 50 Vestec near Prague
> > Czech Republic
> > Tel. +420 325 873 758
> 
> 
> 
> 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/

-- 

*--
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
* Global Phasing Ltd., Sheraton House, Castle Park 
* Cambridge CB3 0AX, UK   www.globalphasing.com
*--



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Re: [ccp4bb] Checking X-ray sequence (no more protein).

[Natesh Ramanathan]

Dear Jon,
 We did exactly the same back in 1999's.
1) Natesh R, Bhanumoorthy P, Vithayathil PJ, Sekar K, Ramakumar S,
Viswamitra MA. (1999). Crystal structure at 1.8 Å resolution and proposed
amino acid sequence of a thermostable xylanase from *Thermoascus
aurantiacus*. J. Mol. Biol., 288, 999-1012.


and then updated the 1.8 A crystal structure derived sequence with ultra
high resolution electron density map.  We were also able to model
additional terminal residues in ultra high resolution map (reported in 2003
paper below).

2) Natesh R, Manikandan K, Bhanumoorthy P, Viswamitra MA, Ramakumar S.
(2003). Thermostable xylanase from *Thermoascus aurantiacus* at ultrahigh
resolution (0.89 Å) at 100 K and atomic resolution (1.11 Å) at 293 K
refined anisotropically to small-molecule accuracy. Acta Crystallogr., D
59,105-117. 



I will be able happy to help off the egroup if you need more
information/help.

Best regards,
Natesh

--
--
"Live Simply and do Serious Things .. "
- Dorothy Mary Crowfoot Hodgkin OM, FRS

"In Science truth always wins"
- Max Ferdinand Perutz OM FRS
--
Dr. Ramanathan Natesh
Assistant Professor,
Founding President and President Cryo Electron Microscopy and 3 Dimensional
Image Processing Society of India (CEM3DIPSI)
http://cem3dipsi.iisertvm.ac.in/
School of Biology,
Indian Institute of Science Education and Research Thiruvananthapuram
(IISER-TVM),
Maruthamala P.O., Vithura,
Thiruvananthapuram,  695551, Kerala, India

nat...@iisertvm.ac.in
http://faculty.iisertvm.ac.in/natesh

*Researcher ID*: http://www.researcherid.com/rid/C-4488-2008
*ORCID*: http://orcid.org/-0002-1145-5962
Vidwan-ID : 94134: http://iisertvm.irins.org/profile/94134
*PUBLONS*: https://publons.com/author/1520837/ramanathan-natesh#profile

Office Ph. 0091- 471-2778087

On Fri, 29 Jul 2022 at 15:45, Jon Cooper <
488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello, I am looking for suggestions of ways to check a 1.7 Angstrom X-ray
> sequence for a protein where it is impractical to do experimental
> sequencing, protein or DNA. The structure refines to publishable R/R-free
> and the main ambiguities seem to be Thr/Val, Asp/Asn and Glu/Gln where
> alternative H-bonding networks are possible. Running alpha-fold seems an
> interesting option? Any suggestions much appreciated.
>
> Cheers, Jon.C.
>
> Sent from ProtonMail mobile
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Regarding the correct space group identification

[Andrew Leslie - MRC LMB]

Dear Sayan,

I find this really surprising. According to the ccp4 program 
“othercell” the two cells that you specify (C2 and P222) are NOT compatible, ie 
they cannot both give correct predictions for the diffraction. This is, of 
course, only based on the two images that have been provided, but it does 
suggest that something rather strange is happening. Can you upload a few other 
images, eg rotated 30° and 60° fro image 1 that you uploaded previously?

Best wishes,

Andrew

> On 29 Jul 2022, at 12:17, Sayan Saha  wrote:
> 
> Dear Sir,
> 
> The Rw/Rf for P22121 structure solution is ~29/32%. For C2 structure 
> solution, it is a little higher, 32/35%.
> 
> 
> With best regards,
> Sayan Saha.
> 
> 
> 
> 
> 
> On Fri, Jul 29, 2022 at 3:25 PM Andrew Leslie - MRC LMB 
> mailto:and...@mrc-lmb.cam.ac.uk>> wrote:
> Dear Sayan,
>  
>Using imosflm, based on the two images that you have 
> uploaded, the cell appears to be orthorhombic (approx 80, 85, 111) and there 
> is no evidence for the C2 unit call that you suggested. Using only the second 
> image there is a C2 solution, but the prediction is very poor (high 
> positional error). I am therefore a bit surprised that you found a MR 
> solution in C2. Were the Rfactors that you quoted (29/32%) for the 
> orthorhombic solution or the C2 solution?
> 
> As Herman pointed out, there is definite streaking in some lunes on image 2, 
> but this seems to be restricted to a relatively small part of the diffraction 
> pattern. While this does indicate some kind of disorder, I do not think this 
> is serious enough to prevent a reliable structure determination, but it might 
> account for the slightly high R-factors.
> 
> There is definitely a lot of spot overlap, as the mosaic spread (mosflm 
> definition) is in the region of 1.5°. The oscillation angle would have to be 
> 0.3° or less to avoid this spot overlap (determined from the Strategy option 
> in imosflm). Again, as Kay pointed out, this would lead to higher then 
> expected R-factors. As this is an image plate detector, I can understand why 
> you might not be using an oscillation angle of 0.1°, but you do need to check 
> that the oscillation angle you are using does not give rise to a lot of 
> spatial overlaps and a smaller oscillation angle will generally give improved 
>  quality data, especially if the background level is quite high, as it is in 
> your images.
> 
> Best wishes,
> 
> Andrew
> 
>> On 29 Jul 2022, at 05:06, Sayan Saha > > wrote:
>> 
>> Dear Sir,
>> 
>>  image1.osc 
>> 
>>  image2.osc 
>> 
>> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
>> degree. Please find attached two diffraction images.
>> With best regards,
>> Sayan Saha.
>> 
>> On Thu, Jul 28, 2022 at 9:41 PM Sayan Saha > > wrote:
>> Dear Sir,
>> 
>> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
>> degree. Please find attached two diffraction images.
>> With best regards,
>> Sayan Saha.
>> 
>> 
>> On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs 
>> mailto:kay.diederi...@uni-konstanz.de>> 
>> wrote:
>> Dear Sayan,
>> 
>> On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha > > wrote:
>> 
>> >Dear Sir,
>> >
>> >1. There are no ice-rings. However, diffraction spots seem to be
>> >overlapping. This can be seen during the data processing, as the space
>> >group (C2 or P222) varies even in the consecutive frames.
>> 
>> spot overlap results in inaccurate intensity values. Inaccurate intensities 
>> result in high Rwork/Rfree.
>> 
>> Why do the spots overlap? High mosaicity? Detector distance too small? 
>> Oscillation range too high (0.1° is typically adequate)?
>> 
>> It would be good to see the data, otherwise we can only speculate.
>> 
>> Space group does not change from one frame to the next. If you use XDS, a 
>> good guide to decide between higher and lower-symmetry space groups is to 
>> compare their ISa values.
>> 
>> best,
>> Kay
>> 
>> >
>> >2. Crystal packing of C2 and P22121 seem to be similar (please see the
>> >attached images).
>> >
>> >3. Forgot to mention in my previous email that we have already processed
>> >the data in P1 and MR solution could be found only in P1 (Phaser was used
>> >with an option in all possible space groups of that point group).
>> >
>> >Please let me know if any other information is required.
>> >
>> >With best regards,
>> >Sayan Saha.
>> >
>> >
>> >On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE <
>> >herman.schreu...@sanofi.com > wrote:
>> >
>> >> Dear Sayan,
>> >>
>> >>
>> >>
>> >> If a subunit is correctly oriented, but the translation is incorrect,
>> >> density for a ligand may still show up in the binding 

Re: [ccp4bb] Regarding the correct space group identification

[Clemens Vonrhein]

Small addition on top of the great advice you already received ...

If this is inhouse data with a vertical rotation axis /and/ the images
are shown in true orientation (i.e. your beamstop holder really comes
in from the top): those "streaks" or overlapped spots on image2 seem
to be roughly perpendicular to the rotation axis - so could just be
the effect of a long(er) unit cell axis and your (too wide) 1 degree
rotation.

Maybe this is not so much a case of overlapping (but distinct)
multiple lattices but rather very bad spatial overlap due to
unfortnuate crystal orientation and inadequate image width? In which
case your intensities at 90 degree to image2 will most likely also be
wrong (containing multiple diffraction spots due to the long axis
oriented along the beam, i.e. contained within your 1 degree image
width).

Either orient your crystals differently (longest axis parallel to
rotation axis) or use smaller image widths. Or even better: do both
;-)

Cheers

Clemens

On Fri, Jul 29, 2022 at 10:08:01AM +0200, Andrew Thompson wrote:
> Dear Sayan 
> So much has been said about this already. What struck me, in looking at the 
> second of your images, is that there is almost certain to be spot overlap (to 
> the left / centre of the image) and a chance that the correct unit cell may 
> be larger than the indexing suggests. The lower beam divergence at a 
> synchrotron, combined with fine sliced images, may tell a different story 
> about the indexing. 
> Regards 
> Andy 
> 
> 
> De: "Sayan Saha"  
> À: CCP4BB@JISCMAIL.AC.UK 
> Envoyé: Jeudi 28 Juillet 2022 18:11:54 
> Objet: Re: [ccp4bb] Regarding the correct space group identification 
> 
> Dear Sir, 
> 
> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
> degree. Please find attached two diffraction images. 
> With best regards, 
> Sayan Saha. 
> 
> 
> On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs < [ 
> mailto:kay.diederi...@uni-konstanz.de | kay.diederi...@uni-konstanz.de ] > 
> wrote: 
> 
> 
> Dear Sayan, 
> 
> On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha < [ 
> mailto:ssaha43...@gmail.com | ssaha43...@gmail.com ] > wrote: 
> 
> >Dear Sir, 
> > 
> >1. There are no ice-rings. However, diffraction spots seem to be 
> >overlapping. This can be seen during the data processing, as the space 
> >group (C2 or P222) varies even in the consecutive frames. 
> 
> spot overlap results in inaccurate intensity values. Inaccurate intensities 
> result in high Rwork/Rfree. 
> 
> Why do the spots overlap? High mosaicity? Detector distance too small? 
> Oscillation range too high (0.1° is typically adequate)? 
> 
> It would be good to see the data, otherwise we can only speculate. 
> 
> Space group does not change from one frame to the next. If you use XDS, a 
> good guide to decide between higher and lower-symmetry space groups is to 
> compare their ISa values. 
> 
> best, 
> Kay 
> 
> > 
> >2. Crystal packing of C2 and P22121 seem to be similar (please see the 
> >attached images). 
> > 
> >3. Forgot to mention in my previous email that we have already processed 
> >the data in P1 and MR solution could be found only in P1 (Phaser was used 
> >with an option in all possible space groups of that point group). 
> > 
> >Please let me know if any other information is required. 
> > 
> >With best regards, 
> >Sayan Saha. 
> > 
> > 
> >On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE < 
> > [ mailto:herman.schreu...@sanofi.com | herman.schreu...@sanofi.com ] > 
> > wrote: 
> > 
> >> Dear Sayan, 
> >> 
> >> 
> >> 
> >> If a subunit is correctly oriented, but the translation is incorrect, 
> >> density for a ligand may still show up in the binding site of the protein. 
> >> It might be that one of the 2-fold axes, you think is crystallographic, is 
> >> in fact non crystallographic and a few Angstroms away from the 
> >> crystallographic position. 
> >> 
> >> 
> >> 
> >> What I would do: 
> >> 
> >> 1. Check the images: are there ice-rings or other artifacts that could 
> >> cause scaling problems that would lead to high Rw/Rf values? In that case, 
> >> there is not much you can do. 
> >> 2. Compare the C2 and P22121 solutions: do they have the same overall 
> >> crystal packing (CS+NCS), or are they different? Do they have the same 
> >> Rw/Rf values? Can we learn anything from the differences in overall 
> >> crystal 
> >> packing? 
> >> 3. Process, run MR and refine in P1. Do you get lower R-factors? If 
> >> so, then run Zanuda to find out the real space group. 
> >> 
> >> 
> >> 
> >> Best, 
> >> 
> >> Herman 
> >> 
> >> 
> >> 
> >> *Von:* CCP4 bulletin board < [ mailto:CCP4BB@JISCMAIL.AC.UK | 
> >> CCP4BB@JISCMAIL.AC.UK ] > *Im Auftrag von *Sayan 
> >> Saha 
> >> *Gesendet:* Donnerstag, 28. Juli 2022 08:15 
> >> *An:* [ mailto:CCP4BB@JISCMAIL.AC.UK | CCP4BB@JISCMAIL.AC.UK ] 
> >> *Betreff:* [ccp4bb] Regarding the correct space group identification 
> >> 
> >> 
> >> 
> >> Dear All, 
> >> 
> >> 
> >> 
> >> We have collected home-source 

Re: [ccp4bb] Checking X-ray sequence (no more protein).

[Jon Cooper]

Thank you so much for your replies. I apologise for being unclear. The protein 
is purified from a plant that hasn't had its genome sequence determined. We 
know the enzyme family of the protein and therefore the structure was 
originally solved by MR. The 'X-ray sequence' we have is just determined from 
looking at the 1.7 Angstrom density, which is good, over several refinement and 
rebuilding rounds. The resulting sequence has been run through blast and it is 
up to 58% identical with other family members. To me this seemed low but that 
degree of identity is typical of other family members. The postgrad who did the 
work did obtain some peptide sequences and prior to that about 20% of the 
sequence was determined by the Edman method with the usual Asp/Asn and Glu/Gln 
ambiguity. However, there isn't any prospect of us doing further experimental 
work, sorry, but that's the way it is!!

Best wishes, Jon.C.

Sent from ProtonMail mobile

 Original Message 
On 29 Jul 2022, 12:23, Jan Dohnalek wrote:

> If you know at least something about your protein, organism, type of 
> molecule, ..., you could try mass spectrometry peptide mapping to known 
> sequences, this may give you some answers for the ambiguities you might be 
> seeing, if nothing else ..
>
> Jan
>
> On Fri, Jul 29, 2022 at 12:15 PM Jon Cooper 
> <488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> Hello, I am looking for suggestions of ways to check a 1.7 Angstrom X-ray 
>> sequence for a protein where it is impractical to do experimental 
>> sequencing, protein or DNA. The structure refines to publishable R/R-free 
>> and the main ambiguities seem to be Thr/Val, Asp/Asn and Glu/Gln where 
>> alternative H-bonding networks are possible. Running alpha-fold seems an 
>> interesting option? Any suggestions much appreciated.
>>
>> Cheers, Jon.C.
>>
>> Sent from ProtonMail mobile
>>
>> ---
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> --
>
> Jan Dohnalek, Ph.D
> Institute of Biotechnology
>
> Academy of Sciences of the Czech Republic
> Biocev
> Prumyslova 595
> 252 50 Vestec near Prague
> Czech Republic
> Tel. +420 325 873 758



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Re: [ccp4bb] Checking X-ray sequence (no more protein).

[Jan Dohnalek]

If you know at least something about your protein, organism, type of
molecule, ..., you could try mass spectrometry peptide mapping to known
sequences, this may give you some answers for the ambiguities you might be
seeing, if nothing else ..

Jan


On Fri, Jul 29, 2022 at 12:15 PM Jon Cooper <
488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello, I am looking for suggestions of ways to check a 1.7 Angstrom X-ray
> sequence for a protein where it is impractical to do experimental
> sequencing, protein or DNA. The structure refines to publishable R/R-free
> and the main ambiguities seem to be Thr/Val, Asp/Asn and Glu/Gln where
> alternative H-bonding networks are possible. Running alpha-fold seems an
> interesting option? Any suggestions much appreciated.
>
> Cheers, Jon.C.
>
> Sent from ProtonMail mobile
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
Jan Dohnalek, Ph.D
Institute of Biotechnology
Academy of Sciences of the Czech Republic
Biocev
Prumyslova 595
252 50 Vestec near Prague
Czech Republic

Tel. +420 325 873 758



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Re: [ccp4bb] Discrepancy in His library used by CCP4 and Molprobity?

[Jan Dohnalek]

Dear all,
We also have had problems with FAD, FMN and I think NAD when depositing to
the PDB.
The ligand checks in PDB were expecting values different for some bonds and
angles and our values either corresponded to the CCP4 dictionaries (and/or)
to our defined values where there were modifications, always taken from the
CSD average values and checked against similar situations found in the CCP4
dictionaries.

I.e. we left the "raised flags" in the PDB without much attention as we
assumed that for some reasons the geometrical values being used there are
not what CCP4 and we are convinced would be the right ones.

Now, Jan's question triggered mine ..

Anybody else had similar experience with such ligands?

Jan Dohnalek



On Fri, Jul 29, 2022 at 11:54 AM Jan Stransky 
wrote:

> Hi all,
>
> while validating X-ray structure using Molprobity (web service), we got
> systematic outlier flags on CE1-NE2 distance in histidines. The distance
> is 1.36A.
>
> I have tested it also using high resolution lysozyme structure, I have
> laying around. There the distance refines as 1.31A and Molprobity is
> happy, but when Bong Angels are called in Coot (Regularize zone button
> :-)) the bond length goes to 1.36A and Molprobity  is unhappy again.
>
> So I would like to ask, who is right or where else the problem can be.
>
> Best regards,
>
> Jan
>
> P.S. CCP4 is version 8.0.002
>
> 
>
> 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/
>


-- 
Jan Dohnalek, Ph.D
Institute of Biotechnology
Academy of Sciences of the Czech Republic
Biocev
Prumyslova 595
252 50 Vestec near Prague
Czech Republic

Tel. +420 325 873 758



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Re: [ccp4bb] Yes, there are now 214 *million* structures in the AlphaFold Protein Structure Database

[Frank von Delft]

Gerard, Sameer - that is highly impressive indeed


About that Editorial (nice read!), it frames these developments as a 
"crisis" that heralds "the end of structural biology" - or at the very 
least, you're responding to people having said that.  I remain puzzled 
that such stuff receive any editorial oxygen - to me it's like saying 
that that higher flux and automation at synchrotrons threaten 
crystallography.  Or something.  AlphaFold is just another tool - a 
sensationally powerful one, of course, but it doesn't Do Science.


Unless there have been noises from Funders - in which case we do need a 
calls to arms, to scream some sense into them.


Btw, for whoever missed it, AlphaFold models are as accurate as 3.5A 
structures: 
https://twitter.com/LindorffLarsen/status/1527410977213403147. So 
they're amazing, but Real Crystallographers know the experimental 
distance between 3.5 and the 2.2 needed for structure-supported ligand 
design, to name just one.


Frank



On 28/07/2022 16:19, Gerard Kleywegt wrote:

Hi all,

I thought Sameer was burying the lead a tad in his message... :-) So, 
for those of you who -like me- are not on social media:


==> As of today, the AlphaFold Protein Structure Database contains 214 
million models predicted with AlphaFold, covering almost all of 
UniProt. <==


So, if your favourite protein was not available in the database before 
today, it's worth checking in again at 
https://www.alphafold.ebi.ac.uk/ now.


See also:

- EMBL-EBI press release: 
https://www.ebi.ac.uk/about/news/technology-and-innovation/alphafold-200-million/


- Nature news: https://www.nature.com/articles/d41586-022-02083-2

- IUCr J guest editorial about the potential impact of all this on 
structural biologists (shameless plug): 
https://journals.iucr.org/m/issues/2022/04/00/me6185/index.html



Best wishes,

--Gerard





On Thu, 28 Jul 2022, Sameer Velankar wrote:


Dear All,

You may have seen our announcement today about expanding the 
AlphaFold Protein Structure Database to 214M predicted models. To 
enable this expansion, we’ve updated the Predicted Aligned Error 
(PAE) JSON format to make it compact (about 4x smaller):


The PAE JSON numbers are now rounded to the closest integer, giving 
~75% compressed size reduction. The integer resolution is sufficient 
for analytical purposes.
The indices are not stored anymore since we store the full 2D PAE 
matrix rather than a sparse one, giving ~4% compressed size reduction.
The “distances” field has been renamed to “predicted_aligned_error” 
and is now stored as a 2D array of shape (num_res, num_res) rather 
than a 1D array. We renamed the field on purpose so that existing 
code breaks rather than potentially silently returning wrong values.


For a protein of length num_res, the PAE JSON file has now the 
following format:


[{
 "predicted_aligned_error": [[0, 1, 4, 7, 9, ...], ...],  # Shape: 
(num_res, num_res).

 "max_predicted_aligned_error": 31.75  # Scalar.
}]

The fields in the JSON file are:
predicted_aligned_error: The PAE value of the residue pair, rounded 
to the closest integer. For PAE value on position (i, j), i is the 
residue on which the structure is aligned for the predicted error, j 
is the residue on which the error is predicted.
max_predicted_aligned_error: A number that denotes the largest 
possible unrounded value of PAE that could occur in the PAE array. 
The smallest possible value of PAE is 0.


The updated PAE format is only available from the AlphaFold Protein 
Structure Database. The PAE format from the AlphaFold Colab notebook 
is not updated.


If you require support with this change, please email 
alphaf...@deepmind.com  and they may 
be able to assist.


Best Wishes,

Sameer Velankar


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Best wishes,

--Gerard

**
   Gerard J. Kleywegt

  http://xray.bmc.uu.se/gerard   mailto:ger...@xray.bmc.uu.se
**
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
**
   Little known gastromathematical curiosity: let "z" be the
   radius and "a" the thickness of a pizza. Then the volume
    of that pizza is equal to pi*z*z*a !
**



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Re: [ccp4bb] Regarding the correct space group identification

[Sayan Saha]

Dear Sir,

The Rw/Rf for P22121 structure solution is ~29/32%. For C2 structure
solution, it is a little higher, 32/35%.


With best regards,
Sayan Saha.





On Fri, Jul 29, 2022 at 3:25 PM Andrew Leslie - MRC LMB <
and...@mrc-lmb.cam.ac.uk> wrote:

> Dear Sayan,
>
>Using imosflm, based on the two images that you have
> uploaded, the cell appears to be orthorhombic (approx 80, 85, 111) and
> there is no evidence for the C2 unit call that you suggested. Using only
> the second image there is a C2 solution, but the prediction is very poor
> (high positional error). I am therefore a bit surprised that you found a MR
> solution in C2. Were the Rfactors that you quoted (29/32%) for the
> orthorhombic solution or the C2 solution?
>
> As Herman pointed out, there is definite streaking in some lunes on image
> 2, but this seems to be restricted to a relatively small part of the
> diffraction pattern. While this does indicate some kind of disorder, I do
> not think this is serious enough to prevent a reliable structure
> determination, but it might account for the slightly high R-factors.
>
> There is definitely a lot of spot overlap, as the mosaic spread (mosflm
> definition) is in the region of 1.5°. The oscillation angle would have to
> be 0.3° or less to avoid this spot overlap (determined from the Strategy
> option in imosflm). Again, as Kay pointed out, this would lead to higher
> then expected R-factors. As this is an image plate detector, I can
> understand why you might not be using an oscillation angle of 0.1°, but you
> do need to check that the oscillation angle you are using does not give
> rise to a lot of spatial overlaps and a smaller oscillation angle will
> generally give improved  quality data, especially if the background level
> is quite high, as it is in your images.
>
> Best wishes,
>
> Andrew
>
> On 29 Jul 2022, at 05:06, Sayan Saha  wrote:
>
> Dear Sir,
>
>  image1.osc
> 
>  image2.osc
> 
> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0
> degree. Please find attached two diffraction images.
> With best regards,
> Sayan Saha.
>
> On Thu, Jul 28, 2022 at 9:41 PM Sayan Saha  wrote:
>
>> Dear Sir,
>>
>> The detector-to-crystal distance was 190 mm. The Oscillation range was
>> 1.0 degree. Please find attached two diffraction images.
>> With best regards,
>> Sayan Saha.
>>
>>
>> On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs <
>> kay.diederi...@uni-konstanz.de> wrote:
>>
>>> Dear Sayan,
>>>
>>> On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha 
>>> wrote:
>>>
>>> >Dear Sir,
>>> >
>>> >1. There are no ice-rings. However, diffraction spots seem to be
>>> >overlapping. This can be seen during the data processing, as the space
>>> >group (C2 or P222) varies even in the consecutive frames.
>>>
>>> spot overlap results in inaccurate intensity values. Inaccurate
>>> intensities result in high Rwork/Rfree.
>>>
>>> Why do the spots overlap? High mosaicity? Detector distance too small?
>>> Oscillation range too high (0.1° is typically adequate)?
>>>
>>> It would be good to see the data, otherwise we can only speculate.
>>>
>>> Space group does not change from one frame to the next. If you use XDS,
>>> a good guide to decide between higher and lower-symmetry space groups is to
>>> compare their ISa values.
>>>
>>> best,
>>> Kay
>>>
>>> >
>>> >2. Crystal packing of C2 and P22121 seem to be similar (please see the
>>> >attached images).
>>> >
>>> >3. Forgot to mention in my previous email that we have already processed
>>> >the data in P1 and MR solution could be found only in P1 (Phaser was
>>> used
>>> >with an option in all possible space groups of that point group).
>>> >
>>> >Please let me know if any other information is required.
>>> >
>>> >With best regards,
>>> >Sayan Saha.
>>> >
>>> >
>>> >On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE <
>>> >herman.schreu...@sanofi.com> wrote:
>>> >
>>> >> Dear Sayan,
>>> >>
>>> >>
>>> >>
>>> >> If a subunit is correctly oriented, but the translation is incorrect,
>>> >> density for a ligand may still show up in the binding site of the
>>> protein.
>>> >> It might be that one of the 2-fold axes, you think is
>>> crystallographic, is
>>> >> in fact non crystallographic and a few Angstroms away from the
>>> >> crystallographic position.
>>> >>
>>> >>
>>> >>
>>> >> What I would do:
>>> >>
>>> >>1. Check the images: are there ice-rings or other artifacts that
>>> could
>>> >>cause scaling problems that would lead to high Rw/Rf values? In
>>> that case,
>>> >>there is not much you can do.
>>> >>2. Compare the C2 and P22121 solutions: do they have the same
>>> overall
>>> >>crystal packing (CS+NCS), or are they different? Do they have the
>>> same
>>> >>Rw/Rf values? Can we learn anything from the differences in
>>> overall 

[ccp4bb] Sorry typo:Re: [ccp4bb] Discrepancy in His library used by CCP4 and Molprobity?

[Fei Long]

It should be

The new value from aceDRG and the latest version of CCP4 monomer lib will
be about 1.32.

Fei

Dear Jan,

It is a bug in aceDRG. It has been fixed in aceDRG and the latest version
of CCP4 monomer lib. It will be in the next CCP4 updating.

Thanks,

Fei




Hi all,

while validating X-ray structure using Molprobity (web service), we got
systematic outlier flags on CE1-NE2 distance in histidines. The distance
is 1.36A.

I have tested it also using high resolution lysozyme structure, I have
laying around. There the distance refines as 1.31A and Molprobity is
happy, but when Bong Angels are called in Coot (Regularize zone button
:-)) the bond length goes to 1.36A and Molprobity  is unhappy again.

So I would like to ask, who is right or where else the problem can be.

Best regards,

Jan

P.S. CCP4 is version 8.0.002



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-- 
Dr Fei Long
Structural Studies Division
MRC Laboratory of Molecular Biology
Francis Crick Avenue,
Cambridge Biomedical Campus,
Cambridge
CB2 0QH UK
Email:fl...@mrc-lmb.cam.ac.uk
Tel:+44 1223 402200



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Re: [ccp4bb] Discrepancy in His library used by CCP4 and Molprobity?

[Fei Long]

Dear Jan,

It is a bug in aceDRG. It has been fixed in aceDRG and the latest version
of CCP4 monomer lib. It will be in the next CCP4 updating.

Thanks,

Fei




Hi all,

while validating X-ray structure using Molprobity (web service), we got
systematic outlier flags on CE1-NE2 distance in histidines. The distance
is 1.36A.

I have tested it also using high resolution lysozyme structure, I have
laying around. There the distance refines as 1.31A and Molprobity is
happy, but when Bong Angels are called in Coot (Regularize zone button
:-)) the bond length goes to 1.36A and Molprobity  is unhappy again.

So I would like to ask, who is right or where else the problem can be.

Best regards,

Jan

P.S. CCP4 is version 8.0.002



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-- 
Dr Fei Long
Structural Studies Division
MRC Laboratory of Molecular Biology
Francis Crick Avenue,
Cambridge Biomedical Campus,
Cambridge
CB2 0QH UK
Email:fl...@mrc-lmb.cam.ac.uk
Tel:+44 1223 402200



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[ccp4bb] Checking X-ray sequence (no more protein).

[Jon Cooper]

Hello, I am looking for suggestions of ways to check a 1.7 Angstrom X-ray 
sequence for a protein where it is impractical to do experimental sequencing, 
protein or DNA. The structure refines to publishable R/R-free and the main 
ambiguities seem to be Thr/Val, Asp/Asn and Glu/Gln where alternative H-bonding 
networks are possible. Running alpha-fold seems an interesting option? Any 
suggestions much appreciated.

Cheers, Jon.C.

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Re: [ccp4bb] Regarding the correct space group identification

[Harry Powell]

Hi

The mosaicity (as Herman and Andrew have pointed out) is very high - so I’d 
contemplate collecting at least some images at ambient temperature to see if 
the cryocooling has increased it substantially. If images from RT data 
collection shows that the mosaicity has blown up during cooling (I hope not 
freezing…) the crystal, I would take some time to optimise the cryocooling 
conditions.

I’d also think about moving the detector further away (maybe 300mm?) so you 
aren’t “wasting” so much of it - the diffraction only goes ~2/3 of the way to 
the edge.

Just some thoughts.

Harry

> On 29 Jul 2022, at 05:06, Sayan Saha  wrote:
> 
> Dear Sir,
> 
>  image1.osc
>  image2.osc
> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
> degree. Please find attached two diffraction images.
> With best regards,
> Sayan Saha.
> 
> On Thu, Jul 28, 2022 at 9:41 PM Sayan Saha  wrote:
> Dear Sir,
> 
> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
> degree. Please find attached two diffraction images.
> With best regards,
> Sayan Saha.
> 
> 
> On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs 
>  wrote:
> Dear Sayan,
> 
> On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha  wrote:
> 
> >Dear Sir,
> >
> >1. There are no ice-rings. However, diffraction spots seem to be
> >overlapping. This can be seen during the data processing, as the space
> >group (C2 or P222) varies even in the consecutive frames.
> 
> spot overlap results in inaccurate intensity values. Inaccurate intensities 
> result in high Rwork/Rfree.
> 
> Why do the spots overlap? High mosaicity? Detector distance too small? 
> Oscillation range too high (0.1° is typically adequate)?
> 
> It would be good to see the data, otherwise we can only speculate.
> 
> Space group does not change from one frame to the next. If you use XDS, a 
> good guide to decide between higher and lower-symmetry space groups is to 
> compare their ISa values.
> 
> best,
> Kay
> 
> >
> >2. Crystal packing of C2 and P22121 seem to be similar (please see the
> >attached images).
> >
> >3. Forgot to mention in my previous email that we have already processed
> >the data in P1 and MR solution could be found only in P1 (Phaser was used
> >with an option in all possible space groups of that point group).
> >
> >Please let me know if any other information is required.
> >
> >With best regards,
> >Sayan Saha.
> >
> >
> >On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE <
> >herman.schreu...@sanofi.com> wrote:
> >
> >> Dear Sayan,
> >>
> >>
> >>
> >> If a subunit is correctly oriented, but the translation is incorrect,
> >> density for a ligand may still show up in the binding site of the protein.
> >> It might be that one of the 2-fold axes, you think is crystallographic, is
> >> in fact non crystallographic and a few Angstroms away from the
> >> crystallographic position.
> >>
> >>
> >>
> >> What I would do:
> >>
> >>1. Check the images: are there ice-rings or other artifacts that could
> >>cause scaling problems that would lead to high Rw/Rf values? In that 
> >> case,
> >>there is not much you can do.
> >>2. Compare the C2 and P22121 solutions: do they have the same overall
> >>crystal packing (CS+NCS), or are they different? Do they have the same
> >>Rw/Rf values? Can we learn anything from the differences in overall 
> >> crystal
> >>packing?
> >>3. Process, run MR and refine in P1. Do you get lower R-factors? If
> >>so, then run Zanuda to find out the real space group.
> >>
> >>
> >>
> >> Best,
> >>
> >> Herman
> >>
> >>
> >>
> >> *Von:* CCP4 bulletin board  *Im Auftrag von *Sayan
> >> Saha
> >> *Gesendet:* Donnerstag, 28. Juli 2022 08:15
> >> *An:* CCP4BB@JISCMAIL.AC.UK
> >> *Betreff:* [ccp4bb] Regarding the correct space group identification
> >>
> >>
> >>
> >> Dear All,
> >>
> >>
> >>
> >> We have collected home-source X-ray intensity data for a protein at 2.6
> >> Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and
> >> beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be obtained
> >> in both the space groups. However, the solution can be refined with an
> >> Rw/Rf of 29/32% only. The protein is bound to a ligand (co-crystallization)
> >> for which a clear density can be observed.
> >>
> >>
> >>
> >> Any help and suggestion in this regard would be very helpful.
> >>
> >>
> >>
> >> With best regards,
> >>
> >> Sayan Saha.
> >>
> >>
> >>
> >>
> >> --
> >>
> >> To unsubscribe from the CCP4BB list, click the following link:
> >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> >>
> >
> >
> >
> >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 & 

Re: [ccp4bb] Regarding the correct space group identification

[Andrew Leslie - MRC LMB]

Dear Sayan,
 
   Using imosflm, based on the two images that you have 
uploaded, the cell appears to be orthorhombic (approx 80, 85, 111) and there is 
no evidence for the C2 unit call that you suggested. Using only the second 
image there is a C2 solution, but the prediction is very poor (high positional 
error). I am therefore a bit surprised that you found a MR solution in C2. Were 
the Rfactors that you quoted (29/32%) for the orthorhombic solution or the C2 
solution?

As Herman pointed out, there is definite streaking in some lunes on image 2, 
but this seems to be restricted to a relatively small part of the diffraction 
pattern. While this does indicate some kind of disorder, I do not think this is 
serious enough to prevent a reliable structure determination, but it might 
account for the slightly high R-factors.

There is definitely a lot of spot overlap, as the mosaic spread (mosflm 
definition) is in the region of 1.5°. The oscillation angle would have to be 
0.3° or less to avoid this spot overlap (determined from the Strategy option in 
imosflm). Again, as Kay pointed out, this would lead to higher then expected 
R-factors. As this is an image plate detector, I can understand why you might 
not be using an oscillation angle of 0.1°, but you do need to check that the 
oscillation angle you are using does not give rise to a lot of spatial overlaps 
and a smaller oscillation angle will generally give improved  quality data, 
especially if the background level is quite high, as it is in your images.

Best wishes,

Andrew

> On 29 Jul 2022, at 05:06, Sayan Saha  wrote:
> 
> Dear Sir,
> 
>  image1.osc 
> 
>  image2.osc 
> 
> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
> degree. Please find attached two diffraction images.
> With best regards,
> Sayan Saha.
> 
> On Thu, Jul 28, 2022 at 9:41 PM Sayan Saha  > wrote:
> Dear Sir,
> 
> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
> degree. Please find attached two diffraction images.
> With best regards,
> Sayan Saha.
> 
> 
> On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs 
> mailto:kay.diederi...@uni-konstanz.de>> 
> wrote:
> Dear Sayan,
> 
> On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha  > wrote:
> 
> >Dear Sir,
> >
> >1. There are no ice-rings. However, diffraction spots seem to be
> >overlapping. This can be seen during the data processing, as the space
> >group (C2 or P222) varies even in the consecutive frames.
> 
> spot overlap results in inaccurate intensity values. Inaccurate intensities 
> result in high Rwork/Rfree.
> 
> Why do the spots overlap? High mosaicity? Detector distance too small? 
> Oscillation range too high (0.1° is typically adequate)?
> 
> It would be good to see the data, otherwise we can only speculate.
> 
> Space group does not change from one frame to the next. If you use XDS, a 
> good guide to decide between higher and lower-symmetry space groups is to 
> compare their ISa values.
> 
> best,
> Kay
> 
> >
> >2. Crystal packing of C2 and P22121 seem to be similar (please see the
> >attached images).
> >
> >3. Forgot to mention in my previous email that we have already processed
> >the data in P1 and MR solution could be found only in P1 (Phaser was used
> >with an option in all possible space groups of that point group).
> >
> >Please let me know if any other information is required.
> >
> >With best regards,
> >Sayan Saha.
> >
> >
> >On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE <
> >herman.schreu...@sanofi.com > wrote:
> >
> >> Dear Sayan,
> >>
> >>
> >>
> >> If a subunit is correctly oriented, but the translation is incorrect,
> >> density for a ligand may still show up in the binding site of the protein.
> >> It might be that one of the 2-fold axes, you think is crystallographic, is
> >> in fact non crystallographic and a few Angstroms away from the
> >> crystallographic position.
> >>
> >>
> >>
> >> What I would do:
> >>
> >>1. Check the images: are there ice-rings or other artifacts that could
> >>cause scaling problems that would lead to high Rw/Rf values? In that 
> >> case,
> >>there is not much you can do.
> >>2. Compare the C2 and P22121 solutions: do they have the same overall
> >>crystal packing (CS+NCS), or are they different? Do they have the same
> >>Rw/Rf values? Can we learn anything from the differences in overall 
> >> crystal
> >>packing?
> >>3. Process, run MR and refine in P1. Do you get lower R-factors? If
> >>so, then run Zanuda to find out the real space group.
> >>
> >>
> >>
> >> Best,
> >>
> >> Herman
> >>
> >>
> >>
> >> *Von:* CCP4 bulletin board  >> > *Im Auftrag von *Sayan
> >> Saha

[ccp4bb] Discrepancy in His library used by CCP4 and Molprobity?

[Jan Stransky]

Hi all,

while validating X-ray structure using Molprobity (web service), we got 
systematic outlier flags on CE1-NE2 distance in histidines. The distance 
is 1.36A.


I have tested it also using high resolution lysozyme structure, I have 
laying around. There the distance refines as 1.31A and Molprobity is 
happy, but when Bong Angels are called in Coot (Regularize zone button 
:-)) the bond length goes to 1.36A and Molprobity  is unhappy again.


So I would like to ask, who is right or where else the problem can be.

Best regards,

Jan

P.S. CCP4 is version 8.0.002



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Re: [ccp4bb] Regarding the correct space group identification

[THOMPSON Andrew]

Dear Sayan 
So much has been said about this already. What struck me, in looking at the 
second of your images, is that there is almost certain to be spot overlap (to 
the left / centre of the image) and a chance that the correct unit cell may be 
larger than the indexing suggests. The lower beam divergence at a synchrotron, 
combined with fine sliced images, may tell a different story about the 
indexing. 
Regards 
Andy 


De: "Sayan Saha"  
À: CCP4BB@JISCMAIL.AC.UK 
Envoyé: Jeudi 28 Juillet 2022 18:11:54 
Objet: Re: [ccp4bb] Regarding the correct space group identification 

Dear Sir, 

The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
degree. Please find attached two diffraction images. 
With best regards, 
Sayan Saha. 


On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs < [ 
mailto:kay.diederi...@uni-konstanz.de | kay.diederi...@uni-konstanz.de ] > 
wrote: 


Dear Sayan, 

On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha < [ mailto:ssaha43...@gmail.com 
| ssaha43...@gmail.com ] > wrote: 

>Dear Sir, 
> 
>1. There are no ice-rings. However, diffraction spots seem to be 
>overlapping. This can be seen during the data processing, as the space 
>group (C2 or P222) varies even in the consecutive frames. 

spot overlap results in inaccurate intensity values. Inaccurate intensities 
result in high Rwork/Rfree. 

Why do the spots overlap? High mosaicity? Detector distance too small? 
Oscillation range too high (0.1° is typically adequate)? 

It would be good to see the data, otherwise we can only speculate. 

Space group does not change from one frame to the next. If you use XDS, a good 
guide to decide between higher and lower-symmetry space groups is to compare 
their ISa values. 

best, 
Kay 

> 
>2. Crystal packing of C2 and P22121 seem to be similar (please see the 
>attached images). 
> 
>3. Forgot to mention in my previous email that we have already processed 
>the data in P1 and MR solution could be found only in P1 (Phaser was used 
>with an option in all possible space groups of that point group). 
> 
>Please let me know if any other information is required. 
> 
>With best regards, 
>Sayan Saha. 
> 
> 
>On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE < 
> [ mailto:herman.schreu...@sanofi.com | herman.schreu...@sanofi.com ] > wrote: 
> 
>> Dear Sayan, 
>> 
>> 
>> 
>> If a subunit is correctly oriented, but the translation is incorrect, 
>> density for a ligand may still show up in the binding site of the protein. 
>> It might be that one of the 2-fold axes, you think is crystallographic, is 
>> in fact non crystallographic and a few Angstroms away from the 
>> crystallographic position. 
>> 
>> 
>> 
>> What I would do: 
>> 
>> 1. Check the images: are there ice-rings or other artifacts that could 
>> cause scaling problems that would lead to high Rw/Rf values? In that case, 
>> there is not much you can do. 
>> 2. Compare the C2 and P22121 solutions: do they have the same overall 
>> crystal packing (CS+NCS), or are they different? Do they have the same 
>> Rw/Rf values? Can we learn anything from the differences in overall crystal 
>> packing? 
>> 3. Process, run MR and refine in P1. Do you get lower R-factors? If 
>> so, then run Zanuda to find out the real space group. 
>> 
>> 
>> 
>> Best, 
>> 
>> Herman 
>> 
>> 
>> 
>> *Von:* CCP4 bulletin board < [ mailto:CCP4BB@JISCMAIL.AC.UK | 
>> CCP4BB@JISCMAIL.AC.UK ] > *Im Auftrag von *Sayan 
>> Saha 
>> *Gesendet:* Donnerstag, 28. Juli 2022 08:15 
>> *An:* [ mailto:CCP4BB@JISCMAIL.AC.UK | CCP4BB@JISCMAIL.AC.UK ] 
>> *Betreff:* [ccp4bb] Regarding the correct space group identification 
>> 
>> 
>> 
>> Dear All, 
>> 
>> 
>> 
>> We have collected home-source X-ray intensity data for a protein at 2.6 
>> Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and 
>> beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be obtained 
>> in both the space groups. However, the solution can be refined with an 
>> Rw/Rf of 29/32% only. The protein is bound to a ligand (co-crystallization) 
>> for which a clear density can be observed. 
>> 
>> 
>> 
>> Any help and suggestion in this regard would be very helpful. 
>> 
>> 
>> 
>> With best regards, 
>> 
>> Sayan Saha. 
>> 
>> 
>> 
>> 
>> -- 
>> 
>> To unsubscribe from the CCP4BB list, click the following link: 
>> [ https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 | 
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 ] 
>> 
> 
> 
> 
>To unsubscribe from the CCP4BB list, click the following link: 
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> 
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Re: [ccp4bb] Regarding the correct space group identification

[Phil Evans]

It might be worth reminding people that 1 degree rotation range/image is 
(almost) never appropriate, and is likely to lead to spot overlap. Typical 
values with modern detectors are 0.05-0.1 deg, and I am surprised to see 1 deg 
images
Phil

Sent from my iPad

> On 29 Jul 2022, at 05:06, Sayan Saha  wrote:
> 
> 
> Dear Sir,
> 
>  image1.osc
>  image2.osc
> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
> degree. Please find attached two diffraction images.
> With best regards,
> Sayan Saha.
> 
>> On Thu, Jul 28, 2022 at 9:41 PM Sayan Saha  wrote:
>> Dear Sir,
>> 
>> The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
>> degree. Please find attached two diffraction images.
>> With best regards,
>> Sayan Saha.
>> 
>> 
>>> On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs 
>>>  wrote:
>>> Dear Sayan,
>>> 
>>> On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha  wrote:
>>> 
>>> >Dear Sir,
>>> >
>>> >1. There are no ice-rings. However, diffraction spots seem to be
>>> >overlapping. This can be seen during the data processing, as the space
>>> >group (C2 or P222) varies even in the consecutive frames.
>>> 
>>> spot overlap results in inaccurate intensity values. Inaccurate intensities 
>>> result in high Rwork/Rfree.
>>> 
>>> Why do the spots overlap? High mosaicity? Detector distance too small? 
>>> Oscillation range too high (0.1° is typically adequate)?
>>> 
>>> It would be good to see the data, otherwise we can only speculate.
>>> 
>>> Space group does not change from one frame to the next. If you use XDS, a 
>>> good guide to decide between higher and lower-symmetry space groups is to 
>>> compare their ISa values.
>>> 
>>> best,
>>> Kay
>>> 
>>> >
>>> >2. Crystal packing of C2 and P22121 seem to be similar (please see the
>>> >attached images).
>>> >
>>> >3. Forgot to mention in my previous email that we have already processed
>>> >the data in P1 and MR solution could be found only in P1 (Phaser was used
>>> >with an option in all possible space groups of that point group).
>>> >
>>> >Please let me know if any other information is required.
>>> >
>>> >With best regards,
>>> >Sayan Saha.
>>> >
>>> >
>>> >On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE <
>>> >herman.schreu...@sanofi.com> wrote:
>>> >
>>> >> Dear Sayan,
>>> >>
>>> >>
>>> >>
>>> >> If a subunit is correctly oriented, but the translation is incorrect,
>>> >> density for a ligand may still show up in the binding site of the 
>>> >> protein.
>>> >> It might be that one of the 2-fold axes, you think is crystallographic, 
>>> >> is
>>> >> in fact non crystallographic and a few Angstroms away from the
>>> >> crystallographic position.
>>> >>
>>> >>
>>> >>
>>> >> What I would do:
>>> >>
>>> >>1. Check the images: are there ice-rings or other artifacts that could
>>> >>cause scaling problems that would lead to high Rw/Rf values? In that 
>>> >> case,
>>> >>there is not much you can do.
>>> >>2. Compare the C2 and P22121 solutions: do they have the same overall
>>> >>crystal packing (CS+NCS), or are they different? Do they have the same
>>> >>Rw/Rf values? Can we learn anything from the differences in overall 
>>> >> crystal
>>> >>packing?
>>> >>3. Process, run MR and refine in P1. Do you get lower R-factors? If
>>> >>so, then run Zanuda to find out the real space group.
>>> >>
>>> >>
>>> >>
>>> >> Best,
>>> >>
>>> >> Herman
>>> >>
>>> >>
>>> >>
>>> >> *Von:* CCP4 bulletin board  *Im Auftrag von *Sayan
>>> >> Saha
>>> >> *Gesendet:* Donnerstag, 28. Juli 2022 08:15
>>> >> *An:* CCP4BB@JISCMAIL.AC.UK
>>> >> *Betreff:* [ccp4bb] Regarding the correct space group identification
>>> >>
>>> >>
>>> >>
>>> >> Dear All,
>>> >>
>>> >>
>>> >>
>>> >> We have collected home-source X-ray intensity data for a protein at 2.6
>>> >> Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and
>>> >> beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be 
>>> >> obtained
>>> >> in both the space groups. However, the solution can be refined with an
>>> >> Rw/Rf of 29/32% only. The protein is bound to a ligand 
>>> >> (co-crystallization)
>>> >> for which a clear density can be observed.
>>> >>
>>> >>
>>> >>
>>> >> Any help and suggestion in this regard would be very helpful.
>>> >>
>>> >>
>>> >>
>>> >> With best regards,
>>> >>
>>> >> Sayan Saha.
>>> >>
>>> >>
>>> >>
>>> >>
>>> >> --
>>> >>
>>> >> To unsubscribe from the CCP4BB list, click the following link:
>>> >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>> >>
>>> >
>>> >
>>> >
>>> >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 
>>> 

[ccp4bb] AW: [ccp4bb] Regarding the correct space group identification

[Schreuder, Herman /DE]

Dear Sayan,

There are no multiple diffraction patterns in the images you sent us. However, 
the mosaicity appears to be quite high and in this case I would really 
recommend to process the data with XDS (if you have not already done that).

In the left side of image 2, there appear to be alternating loons with strong 
single spots, and loons with rows of streaky spots which are very close 
together, which reminds me of a crystal with lattice translocation disorder I 
once struggled with. However, it might also be bona fide diffraction, with the 
streaky spots caused by a combination of your long cell axis with high 
mosaicity.

You can check this by overlaying the predicted spots on your diffraction 
pattern. All data processing programs have an option to do this.

Finally, I would superimpose your MR solutions of C2 and P222 and look if they 
are exactly the same. If they are different, e.g. if one of the subunits in one 
of the space groups is translated with respect to the other, your crystal may 
contain a mixture both, causing to problems you are facing.

Best,
Herman



Von: CCP4 bulletin board  Im Auftrag von Sayan Saha
Gesendet: Donnerstag, 28. Juli 2022 18:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Regarding the correct space group identification

Dear Sir,

The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
degree. Please find attached two diffraction images.
With best regards,
Sayan Saha.


On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs 
mailto:kay.diederi...@uni-konstanz.de>> wrote:
Dear Sayan,

On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha 
mailto:ssaha43...@gmail.com>> wrote:

>Dear Sir,
>
>1. There are no ice-rings. However, diffraction spots seem to be
>overlapping. This can be seen during the data processing, as the space
>group (C2 or P222) varies even in the consecutive frames.

spot overlap results in inaccurate intensity values. Inaccurate intensities 
result in high Rwork/Rfree.

Why do the spots overlap? High mosaicity? Detector distance too small? 
Oscillation range too high (0.1° is typically adequate)?

It would be good to see the data, otherwise we can only speculate.

Space group does not change from one frame to the next. If you use XDS, a good 
guide to decide between higher and lower-symmetry space groups is to compare 
their ISa values.

best,
Kay

>
>2. Crystal packing of C2 and P22121 seem to be similar (please see the
>attached images).
>
>3. Forgot to mention in my previous email that we have already processed
>the data in P1 and MR solution could be found only in P1 (Phaser was used
>with an option in all possible space groups of that point group).
>
>Please let me know if any other information is required.
>
>With best regards,
>Sayan Saha.
>
>
>On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE <
>herman.schreu...@sanofi.com> wrote:
>
>> Dear Sayan,
>>
>>
>>
>> If a subunit is correctly oriented, but the translation is incorrect,
>> density for a ligand may still show up in the binding site of the protein.
>> It might be that one of the 2-fold axes, you think is crystallographic, is
>> in fact non crystallographic and a few Angstroms away from the
>> crystallographic position.
>>
>>
>>
>> What I would do:
>>
>>1. Check the images: are there ice-rings or other artifacts that could
>>cause scaling problems that would lead to high Rw/Rf values? In that case,
>>there is not much you can do.
>>2. Compare the C2 and P22121 solutions: do they have the same overall
>>crystal packing (CS+NCS), or are they different? Do they have the same
>>Rw/Rf values? Can we learn anything from the differences in overall 
>> crystal
>>packing?
>>3. Process, run MR and refine in P1. Do you get lower R-factors? If
>>so, then run Zanuda to find out the real space group.
>>
>>
>>
>> Best,
>>
>> Herman
>>
>>
>>
>> *Von:* CCP4 bulletin board 
>> mailto:CCP4BB@JISCMAIL.AC.UK>> *Im Auftrag von *Sayan
>> Saha
>> *Gesendet:* Donnerstag, 28. Juli 2022 08:15
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [ccp4bb] Regarding the correct space group identification
>>
>>
>>
>> Dear All,
>>
>>
>>
>> We have collected home-source X-ray intensity data for a protein at 2.6
>> Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and
>> beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be obtained
>> in both the space groups. However, the solution can be refined with an
>> Rw/Rf of 29/32% only. The protein is bound to a ligand (co-crystallization)
>> for which a clear density can be observed.
>>
>>
>>
>> Any help and suggestion in this regard would be very helpful.
>>
>>
>>
>> With best regards,
>>
>> Sayan Saha.
>>
>>
>>
>>
>> --
>>
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