No love for RNA!? Ouch ! I was in the similar problem ten years ago when I was solving riboswitch RNAs as a graduate student. B12 riboswitch,stuck at 5-6A for derivatives, but had pretty reasonable native data.
It was a combination of experimental phasing using clusters at low resolution and then extending the data with smaller derivatives (iridium/cobalt hexammine), multiple crystal averaging, SHARP, and major assistance from people who have been in a similar resolution. You can find the details in my thesis here https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/6969z093h Or you can reach out to me directly. Francis > On May 6, 2022, at 8:34 AM, Eleanor Dodson > <0000176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote: > > How I hate RNA! > However, structures have been solved.. > > As others say: > Look for twinning > Look for translation non-cryst symmetry. > It seems likely when you have a doubling of the a axis for the Zn derivative. > > I use CCP4I2 and the data processing report will do both these tests. > Then you need to decide whether the SG is P6i or P6i22 > > IF there is twinning then the apparent P6i22 symmetry could be due to this.. > > Where are your sites? If they are related by crystal symmetry then it is hard > to distinguish the hand. > And low solvent content hampers DM > Good luck Eleanor > > > > > On Fri, 6 May 2022 at 12:51, Petr Kolenko <petr.kole...@fjfi.cvut.cz > <mailto:petr.kole...@fjfi.cvut.cz>> wrote: > Dear Charlie Nichols, > when optimizing your SHELX runs, SHELIXIR may help you in some cases > (https://kmlinux.fjfi.cvut.cz/~kolenpe1/shelixir > <https://kmlinux.fjfi.cvut.cz/~kolenpe1/shelixir>). > A couple of days ago, I created quick and dirty tutorial to SHELIXIR command > line: https://www.youtube.com/watch?v=dqi5_yLhWOc > <https://www.youtube.com/watch?v=dqi5_yLhWOc> and also the same quality > tutorial to the GUI: https://youtu.be/CZIziPv28hA > <https://youtu.be/CZIziPv28hA> > There may be some parameters to further optimize in which SHELIXIR may help > you (trying more space groups, optimizing of high or low resolution cut-off, > solvent content). However, SHELX C/D/E is generally known to have problems at > lower resolution (say 3.5 AA and lower). To say a bit more about your > problem, would you share the files off the list? > Btw, the great thing about SHELX is that you do not even need to know the > content of the AU in some cases! > Best regards, > Petr > ________________________________________ > From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK > <mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Nichols, Charlie > <charles.nich...@crl.com <mailto:charles.nich...@crl.com>> > Sent: Friday, May 6, 2022 1:17:20 PM > To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> > Subject: [ccp4bb] Phasing a difficult RNA heteroduplex structure > > Dear all, > > I am trying to solve the structure of an RNA heteroduplex + ligand with > approximate MW of 6800. > > * Structure likely to have a core helical region and a couple of bases of > single stranded material at both ends on both strands > > I have datasets from visually similar crystals with different, but related > unit cells: > > Form1: > 32.70 32.70 54.55 90.00 90.00 120.00 – best native ~2.9A > - different pipelines reported P62 2 2, P62 and P32 from auto-indexing > - P6222 impossible to fit 1 complete heteroduplex > - P62, most consistent indexing choice, 37% solvent, low Matthews probability > but possible to fit 1 heteroduplex > > However, Xia/Dials report tNCS > > > * If the ASU only has room for 1 copy the heteroduplex there can’t be > tNCS, does this therefore mean we must have twinning? > * There is a reasonable similarity NMR structure in the PDB, this is ~45A > long > * I am therefore guessing that the duplexes are most probably making > end-end contacts to form long fibres that are ~aligned along the Z-axis and > that the crystal either contains fibres bound both ways up, or that the > duplex can bind either way up to create the fibres, the twinning then > superposes the two orientations to create two identical repeats mimicking > tNCS – does this seem a reasonable interpretation? > > > I have a dataset with Zn/Mg exchange and good anomalous signal to ~3.8A, > ShelX finds good sites but DM / phase-extension with the 2.9A native data > creates a mess and there is little difference between the two hands – can you > give any advice on how I might try to proceed with experimental phasing in > this case? > > Form2: > 62.40 62.40 54.82 90.00 90.00 120.00 – best native ~2.5A > - Auto-indexes as P62 / P64, higher resolution data than Form1 > - Again, Xia/Dials report tNCS > - Cell has a doubling of a/b dimensions but c is the same > - Molecular replacement fails completely > - Cobalt-hexammine soak gave strong anomalous signal but only to ~6A, again > ShelX finds good sites, but DM / phase-extension gives a mess > - From the pathology of Form1, I am concerned that we have exactly the same > issue with overlaid flipped orientations along the Z-axis > > Any advice on how to proceed would be greatly appreciated. > > Thanks, take care, > > Dr Charlie Nichols > Charles River Laboratories > > ________________________________ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB > <http://www.jiscmail.ac.uk/CCP4BB>, a mailing list hosted by > www.jiscmail.ac.uk <http://www.jiscmail.ac.uk/>, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > <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&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=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/
