Not sure about protein diffraction images - I would recommend going to any
course where Zbysek Dauter was teaching - he is the master..
And he lectured at severa;l CCP4 Study Weekends. It would be worth looking
up those. I think they are online at the CCP4 website or in Acta Cryst.
On 1 December 2016 at 20:24, Napoleao Fonseca Valadares <n...@ifsc.usp.br>
> Thank you all for the answers.
> Eleanor, you nailed it, thank you! Your observation that the cell in P6
> had a different C axis led me to reprocess the data using a cell of the
> same size of the other space groups. I have commented that processing in P6
> before yielded r_free > 0.50, but now the r_free is 0.3232 without using a
> twin law, and using the twin law h,-h-k,-l which was recommended by Phenix
> Xtriage, yields r_work = 0.1674 and r_free = 0.1960. Thank you for the
> other suggestions and reference, I followed them.
> I reviewed all my analyses and only P6 had the wrong cell size. I don't
> know why.
> - P3 yields r_work = 0.1711 and r_free = 0.1935 with 120 water molecules
> (the asymmetric unit contains 420 residues and the resolution is 2.01 A).
> - P6 yields r_work = 0.1674 and r_free = 0.1960 with 94 water molecules
> (the asymmetric unit contains 210 residues and the resolution is 1.91 A).
> I guess I should stick with P6, since it has higher symmetry, right?
> Regardless of how I process the data, in all space groups, pointless
> always says it is sure that the space group P622. That is probably due to
> the twinning, that causes reflections to lay over each other and gives the
> appearance of higher symmetry.
> A question, does it makes sense to use TLS and a twin law to refine the
> data at this resolution (1.9 A)?
> Carlos, thank you for the references.
> Carlos Frazao, the PDB header generated by Phenix indicates that the twin
> fraction is still 0.490 after refinement in P3 and P6.
> Jacob Keller, I have tried all trigonal and hexagonal space groups, except
> for those I reported (P1, P3 and now P6), all others yield unrefinable
> solutions (R_free > 0.41). However, there are a lot of explanations for it,
> for example, the data might be processed using the wrong cell size, or the
> MR solution might just be wrong.
> Thank you all for the help!
> I'm still looking for references that can teach me how to look at protein
> diffraction images and understand what I am seeing. The basics like
> recognizing bad data and what usually leads to deformity in the spots (for
> example, elliptical or duplicated) would be of great help.
> *De: *"Eleanor Dodson" <eleanor.dod...@york.ac.uk>
> *Para: *CCP4BB@JISCMAIL.AC.UK
> *Enviadas: *Quarta-feira, 30 de Novembro de 2016 14:10:16
> *Assunto: *Re: [ccp4bb] To win or not twinning?
> First - there is a useful document about possible twin laws in the CCP4
> documentation. It references the SHELX description of twinning for small
> It is possible in a trigonal system that you could have two twin operators
> but the twinning stats should lok odd in that case
> For a trigonal space group there are several options, but your technique
> seems to have correctly found SG P3.
> As Jacob Keller says - check POINTLESS to see if P322 etc are options?
> Or ask Zanudu..
> Just to comment - I think you must have the cell dimensions wrong for the
> P63 case? I cant see how the c axis can be longer?
> I like the data processing POINTLESS/AIMLESS/ CTRUNCATE..
> POINTLESS lists all axial reflections so you can make an educated guess at
> screw axes (or let POINTLESS do it for you...)
> POINTLESS analyses each symmetry operator separately. So if the -k, -h, -l
> was slightly less convincing than others, AND the twin tests suggested
> twinning, you might expect that to be a twin operator and not a symmetry
> ctruncate tests each possible twin operator and scores them. (And so does
> XTRIAGE of course..)
> On 30 November 2016 at 14:00, Keller, Jacob <kell...@janelia.hhmi.org>
>> What about spacegroups in PG 32, e.g., p3212?
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *Napoleao Fonseca Valadares
>> *Sent:* Wednesday, November 30, 2016 2:01 AM
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] To win or not twinning?
>> Dear CCP4ers,
>> I'd like to kindly ask your advice. Sorry for the long e-mail.
>> I have crystals of a 12.3 KDa protein that grow in hexagon-like patterns,
>> link for the crystal image:
>> XDS, Phenix and Pointless always suggest that the data sets for these
>> crystals belong to the space group P622. However, Phenix, Phaser and
>> Pointless indicate that twinning is present.
>> "Bad looking" diffraction images, diffracted to 1.6 A (collected 6 months
>> Best data set, diffracted to 2.01 A (collected a month ago):
>> The second data set present better looking images, a better XDS ISa value
>> (around 24) and diffracted to 2.2 A. The "bad looking" data set diffracted
>> to 1.6 A, but I decided to stop working with it (XDS ISa around 10).
>> There is a template with 60% identity, I used XDS to try to process the
>> data in all trigonal/hexagonal space groups from P3 to P6(3)22, and spend a
>> lot of time trying molecular replacement procedures in Phaser and Morda,
>> and refining the candidate solutions. Used Zanuda too, trying to figure out
>> the space group (and read as much as possible in this CCP4 list looking for
>> similar cases).
>> Unit cells and typical MR results:
>> P1: 56.430 77.718 77.673 119.99 89.99 89.97
>> SOLU SET RFZ=9.5 TFZ=* PAK=0 LLG=91 RF++ TFZ=18.2 PAK=0 LLG=311
>> TFZ==18.4 (&
>> TFZ==17.0) LLG+=(311 & 726) LLG=5751 TFZ==64.6 PAK=0 LLG=5751
>> P3: 77.675 77.675 56.409 90.00 90.00 120.00
>> RFZ=5.5 TFZ=8.5 PAK=0 LLG=86 TFZ==9.4 LLG=2575 TFZ==46.5
>> P6: 77.534 77.534 92.986 90.00 90.00 120.00
>> RFZ=11.2 TFZ=16.2 PAK=2 LLG=437 TFZ==25.7 LLG=437 TFZ==25.7
>> P6(3): 77.675 77.675 56.409 90.00 90.00 120.00
>> RFZ=8.8 TFZ=11.5 PAK=0 LLG=66 TFZ==7.6 LLG=66 TFZ==7.6
>> P622: 77.660 77.660 56.400 90.00 90.00 120.00
>> RFZ=4.8 TFZ=5.4 PAK=1 LLG=22 TFZ==4.7 LLG=24 TFZ==4.8
>> In P1 (LLG=5751 TFZ==64.6) there are 12 molecules in the asymmetric unit,
>> and in P3 (LLG=2575 TFZ==46.5) 4 molecules. Packing looks good, in P1 the
>> ASU looks like two superposed hexagonal donuts formed by 6 molecules each.
>> Refining in P1, without adding waters or TLS, yield r_work = 0.2964 and
>> r_free = 0.3428, and it is hard to decrease these values.
>> Refining in P3, I managed to get r_work = 0.2934 and r_free = 0.3399, but
>> looks like it's not getting any better than this.
>> Refining in P6 yields horrible r_free values (>0.50).
>> Trying to refine MR solutions in any other other space groups yield Rfree
>> 0.41 or more.
>> If in the P3 space group I use the twin operator -k,-h,-l (estimated twin
>> fraction of 0.490) suggested by Phenix Xtriage, the values miraculously go
>> down to r_work = 0.1923 r_free = 0.2202, without waters (r_free without
>> using a twin law = 0.3399). Adding 120 water molecules and doing some
>> refining yields 0.1711 r_free = 0.1935 (the asymmetric unit contains 420
>> residues and the resolution is 2.01 A).
>> I've been reading about twinning refinement and how it can drop the
>> Rvalues, but from what I understood if I use it improperly I may compromise
>> the refinement quality.
>> I would like advice on:
>> 1 - References that can teach how to look at protein diffraction images
>> and understand what I am seeing. The basics like recognizing bad data and
>> what usually leads to deformity in the spots (for example, elliptical or
>> duplicated) would be of great help.
>> 2 - Should I look for other space groups? What else could be tried? Is
>> this a case where a twin law should be used in the refinement? If yes, what
>> can I do to confirm the need for a twin law in the refinement?
>> Thank you all in advance.