Re: [ccp4bb] cryo-EM
Hi Rana, in contrast to Brads answer, and in case I understand you well, Cryo EM is not in ts infancy stage (as long as you do not talk about Cryo EM on crystals. For cryo EM you need only several ul of a nM solution. I can always suggest you the 3dem bb. There are of course still limitataions on the size of protein target and resolution, but those are slowly falling. Some reading material http://www.ncbi.nlm.nih.gov/pubmed/23181775 http://www.ncbi.nlm.nih.gov/pubmed/25071206 Cheers Marcus Quoting Brad Bennett bradbennet...@gmail.com: Yes, but the technique is still in its infancy stage. DOI: 10.1016/j.sbi.2014.03.004 Cheers- Brad On Fri, Aug 29, 2014 at 6:34 AM, rana ibd 0285de88044a-dmarc-requ...@jiscmail.ac.uk wrote: Dear CCP4 I wanted to ask has anyone tested 3D protein structure by cryo-electron microscopy? what is the suitable concentration required for this procedure Best Regards Rana -- Marcus Fislage, PhD Howard Hughes Medical Institute (HHMI) Columbia University Department of Biochemistry and Biophysics Lab of Joachim Frank New York, NY Phone: 212.305.9524 Fax: 212.305.9500
[ccp4bb] CC1/2, XDS and resolution cut off
Dear all, We have in our lab a data set collected and are discussing where to cut the resolution for refinement. According to the work of Kai Diederichs and Andy Karplus one should use CC 1/2 of 12.5% (in case it is significant) to determine the highest resolution independent of the I/sigI and R factor rules used earlier. But I would like to know if this also counts for low completeness data? The problem is that we have in the highest resolution shell an I/sigI of 4, a good cc1/2 but only a completeness of 30%. Which I guess means we measured the high resolution data very accurate but not complete. Would you still use the low complete data in the highest resolution shell or should that be still a valid argument to cut your data towards lower resolution? My guess would be to use the data still even if the completeness drops, since the data we measured is good and according to CC1/2 significant. Are we right to do so or would you disagree? Thanks for any input Marcus -- Marcus Fislage Structural Biology Brussels Vrije Universiteit Brussel Department of Structural Biology, VIB Oefenplein, Gebouw E Pleinlaan 2, 1050 Brussel Belgium Tel: +32-2-629 18 51 Email : marcus.fisl...@vib-vub.be Url: http://www.verseeslab.structuralbiology.be
Re: [ccp4bb] CC1/2, XDS and resolution cut off
Hello Edward A. Berry wrote: What about collecting in the corners of a square detector? Due to the crystal diffracting better than expected or the need to sacrifice resolution for spot separation? This is actually our reason that we have problem. The strategy initially suggested lower resolution, but after we shot the crystal and analyzed the data we could see diffraction way into the corners of the detector. Ian Tickle wrote: Instead I use the average (I / sigma(I)) for all reflections, i.e. including unmeasured for which I take (I / sigma(I)) = 0, as the cut-off criterion with a cut-off value of 1. Do you think that this averaged I/sig I could also be transferred to a averaged CC1/2 giving unobserved spots a CC1/2 of 0. -- CC1/2_all = shell_completeness * CC1/2_measured? Marcus On Mo, 2012-08-06 at 09:33 -0400, Edward A. Berry wrote: Ian Tickle wrote: below the noise threshold. This does make the tacit assumption that the unmeasured reflections are distributed randomly in reciprocal space, which is clearly not entirely true, but it's hard to see how one could account for the non-random distribution. Again, in any case What about collecting in the corners of a square detector? Due to the crystal diffracting better than expected or the need to sacrifice resolution for spot separation? eab
[ccp4bb]
Dear Abraham, after you added all TER signal and have all rows with atom number present you can use pdbset to renumber all atoms. For this take care that you call first your TER signal ATOM (otherwise this row will be deleted). Regards Marcus - Original Message - From: protein chemistry To: CCP4BB@JISCMAIL.AC.UK Sent: Sunday, April 01, 2012 8:42 PM Subject: [ccp4bb] Dear All, I have one query regarding the atom number in the coordinates. Before i began with my coordinate submission i need to put TER cap at the end of chains/polymers, but that may lead to change in atom number for the subsequent residues/molecules. i need to know is there any way to do this or i shall leave it and submit the coordinates directly. Thanks in advance Abraham
[ccp4bb] Refmac: sidechain bond breaks
Dear all, I might excuse myself for the silly question but it is the first time I solve an x-ray structure. After modell building in coot and running of refmac with restrained refinement I have the problem that the pdb output file contains distances between e.g. ILE Cb and Cg that are so long that the Cg is displayed as single atom (distance ~1.64 A instead of 1.5 A). This seems to happen to me for aminoacid sidechains where I added an alternative conformation (especially if the 2nd conformation is oriented not far away from the first one) and if I set the occupancy of sidechains like Glu for Cd and further to zero (Here refmac gives a bond break between the last atom with occupancy 0 and the first with occupancy 1). I can adjust it of course in coot back to normal but after the next refmac run the same happens again. The option card in refmac for geometric restraints I kept untouched. Am I missing to set on an option in refmac, or is there something else going wrong? Thanks a lot for the help Marcus
