We recently had a projects where removing the HIS-tag changed an unsolvable crystal form into one where Molecular Replacement worked immediately (and with an NMR ensemble as the search model to boot). In this case the HIS-tag was about 5% of the total domain mass and the tags were probably tangled up in the centre of the unit cell.
Along with all the other suggestions, I would also put a word in for crystallisation at a lower/different temperature. We've had several projects where this has improved diffraction from worse than 4 Ang. to better than 2 Ang. or where different forms have grown. You can try all the other suggestions in the cold room as well as well as your normal condition. I've been trying to get all our users to screen new crystal forms (both new projects and new conditions in existing projects) in-situ, before trying cryo-cooled data collection. This gives a baseline before opening the drops for cryo-protection and cryo-cooling and may also give some guidelines for dehydration etc. Good luck. Andy Purkiss On Thu, 2020-12-17 at 21:30 +0000, Rafael Marques wrote: > Hello all, how are you doing? > > I have been working with two different proteins for 3 years. I was > able to purify them by IMAC and SEC, no problems at all, and I have > got some crystals of both. However, the best data I have got until > now is around 4A. The diffraction experiment was carried out several > times at DLS. > > The crystals for each protein are different. One is really small > whereas the other one is a regular size (Images below). Some people > have told me to try microseeding and I did this, but with no success > at all. > > So I am asking here if there is someone experienced in this kind of > witchcraft that could give me a hand and also answer me these two > things: > > Have you ever had good size crystals that didn’t diffract beyond 4A > and after trying seeding you have got very good diffraction data (in > these case with no change concerning the crystal size)? > > What is the best protocol that you have tried to increase the size of > your crystals and make them diffract better? > > Many thanks in advance. > > Best > > > > > Rafael Marques da Silva > Mestrando em Física Biomolecular > Universidade de São Paulo > > Bacharel em Ciências Biológicas > Universidade Federal de São Carlos > > phone: +55 16 99766-0021 > > "A sorte acompanha uma mente bem treinada" > ________________________________________________ > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 -- Andrew Purkiss Structural Biology STP The Francis Crick Institute ######################################################################## 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/
