Dear all, I would like to thank all the comments, specially from Kay and Andy.
The space group was correct P212121. More than 100 sites were found with occupancy higher 0.5. The keywords mentioned by Andy and the improvement of the data consistency at low resolution as pointed by kay seemed to be instrumental to get few solutions in SHELXD. Only SIRAS resulted in a very few solutions after thousands runs and many fine-tuning of those parameters in SHELXD. Different kappas (0, 30 and 60) were also essential. Only a single kappa or even two did not yield any solution applying the same strategy. Maps are of great quality and auto-building resulted in crystallographic residuals below 30% without water molecules. Chains are 99% complete. 10 molecules in AU. Thank you Mario De: Holton, James M <jmhol...@slac.stanford.edu> Enviada em: sábado, 20 de julho de 2019 15:55 Para: Mario Tyago Murakami <mario.murak...@lnbr.cnpem.br>; CCP4BB@JISCMAIL.AC.UK Assunto: Re: [ccp4bb] RES: [ccp4bb] Se-SAD phasing It would not surprise me at all if pseudotranslation (aka tNCS) were holding you back. It is like kryptonite to all phasing techniques, MR, MAD, SAD, MIR, you name it. I recently did an analysis of old, never-solved data sets from my beamline. These are MAD/SAD data that don't match anything in the PDB, despite being 10 years old or more. At least 45% of them suffer from pseudotranslation. Another 21% are twinned. In contrast, if you search the PDB for the word "twin" only 0.5% of all entries contain that string. Might explain why methods development on these two fronts has been slow. I don't suppose you'd be up for crowdsourcing? As in making your raw data publicly available? -James Holton MAD Scientist On 7/18/2019 11:34 AM, Mario Tyago Murakami wrote: Curiously, in the native data we observed a strong peak of 28%. However, the same was not seen in the Semet crystals. There is isomorphism between these crystals. Please see below the unit cell parameters. In the previous email I sent attached pointless, index.lp from xds and shelxc logs. Native -> UNIT_CELL_CONSTANTS= 118.082 151.969 166.266 90.000 90.000 90.000 (tNCS 28% F.C. = 0.5 0.135 0.50) Semet -> UNIT_CELL_CONSTANTS= 118.730 151.831 167.070 90.000 90.000 90.000 (tNCS 8% F.C. = 0.5 0.140 0.50) De: Eleanor Dodson <eleanor.dod...@york.ac.uk><mailto:eleanor.dod...@york.ac.uk> Enviada em: quinta-feira, 18 de julho de 2019 12:34 Para: Mario Tyago Murakami <mario.murak...@lnbr.cnpem.br><mailto:mario.murak...@lnbr.cnpem.br> Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Assunto: Re: [ccp4bb] Se-SAD phasing Good advice above.. Someting extra - I would look at information about how the molecules might be arranged. Is there any non-crystallographic translation? ( One with a coordinate 0f say z= 0.5 could mean the spacegroup could be P21212 or P212121 ) Sometimes there is a sub-cell and you can search for fewer sites in that cell.. Eleanor On Thu, 18 Jul 2019 at 16:28, Mario Tyago Murakami <mario.murak...@lnbr.cnpem.br<mailto:mario.murak...@lnbr.cnpem.br>> wrote: Dear Andy, Clemens and Michal The solution for orthrombic SG is very clear. P1 indexing results in a very similar Unit cell parameters without appreciable deviations in angles <0.1degrees. It seems more likely the problem pointed by Andy of a lot of sites. I will try all the suggestions and hope to come back soon with good news. Thanks Mario Em 18 de jul de 2019 11:44, THOMPSON Andrew <andrew.thomp...@synchrotron-soleil.fr<mailto:andrew.thomp...@synchrotron-soleil.fr>> escreveu: Dear Mario The data seem very good but you are looking for an awful lot of sites. Keep trying with SHELX, but there are some keywords that you might use which may help (see extract from SHELX doc). This worked for me for a couple of structures with > 100 sites. You should also try with and without Patterson seeding. You may need many thousand trials…. I would also start with the data cutoff somewhere between 3.5 - 4 A (to get rid of anisotropy) . You could also try manually modifying the Emin value to use just your strongest signal. Don’t expect the solution to come out easily - you may have to try different shelx runs for days before getting a single solution. At the same time, you really do need to be absolutely certain of the space group ……any tests you can do Good luck Andy For large selenomethionine substructures (which behave more like equal atom ab initiostructure solution of small molecules) it may be worth increasing the number of Pattersonpeaks used for the Patterson seeding (e.g. PATT 200; the default is 100) and adding theinstructions WEED 0.3 (random omit maps) and SKIP 0.5 (uranium atom removal). Thelatter two are the defaults when PLOP is present but are switched off by default if PLOP isabsent. When PATS is used, WEED produces a much smaller additional improvement in thehit ratio than when PATS is absent. For small substructures (<10 sites), WEED and SKIP cando more harm than good by eliminating too many correct sites at once. De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] De la part de Mario Tyago Murakami Envoyé : jeudi 18 juillet 2019 16:19 À : CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Objet : [ccp4bb] Se-SAD phasing Dear all, I am trying to solve the phases of the following SeMet data, but so far unsuccessfully. Suggestions are very welcome. Please see below some details about the case. The statistics below is from a merged data from different kappas of the same crystal to increase redundancy. We used the fixed energy 12675 eV since the fluorescence detector was not working at the used beamline to get best energy values for this crystal. Xtriage did not indicate any crystallographic pathology, except moderate anisotropy. The unit cells parameters are 118.72 151.82 167.05 90.000 90.000 90.000 (P212121) containing from 8 to 12 molecules in the asymmetric unit. The protein has ~28.5 kDa and 10 Met residues, excluding those from the N- and C-termini, probably with low occupancy. Thus, something 80 to 120 scatterers are expected. Phenix_anomalous_signal indicates a probability of 99% to solve it and the anomalous signal is theoretically in a very good range. I have tried SHELXD with different resolutions and number of sites. I have also used Hyss. But all attempts failed. Thanks in advance Mario Aviso Legal: Esta mensagem e seus anexos podem conter informações confidenciais e/ou de uso restrito. Observe atentamente seu conteúdo e considere eventual consulta ao remetente antes de copiá-la, divulgá-la ou distribuí-la. Se você recebeu esta mensagem por engano, por favor avise o remetente e apague-a imediatamente. Disclaimer: This email and its attachments may contain confidential and/or privileged information. Observe its content carefully and consider possible querying to the sender before copying, disclosing or distributing it. If you have received this email by mistake, please notify the sender and delete it immediately. ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
