> > As to why previously in a very similar condition you did get your desired > protein plus (other) ligand
Kavya, did you use microseeding? That's the way to get consistent results. Since you're changing the ligand I suggest you go back and run a few random screens (with crushed crystals of your protein with the other ligand) - so-called microseed matrix-screening. https://doi.org/10.1107/S0907444907007652 Good luck Patrick On Sat, Feb 4, 2023 at 10:32 PM Mark J. van Raaij <mjvanra...@cnb.csic.es> wrote: > PS it’s probably not a salt crystal…TCEP is not a salt, your ligand I > presume is also not a salt, a small cleaved peptide neither. As to why > previously in a very similar condition you did get your desired protein > plus (other) ligand crystal, it just means the molecule (TCEP') > crystallises in a similar condition to your protein - I don’t think you can > conclude much more than that (unless there is some other difference like > the TCEP being older this time and more oxidised, for example). > > Mark J van Raaij > Dpto de Estructura de Macromoleculas, lab 20B > Centro Nacional de Biotecnologia - CSIC > calle Darwin 3 > E-28049 Madrid, Spain > tel. +34 91 585 4616 (internal 432092) > Section Editor Acta Crystallographica F > https://journals.iucr.org/f/ > > > On 4 Feb 2023, at 15:48, kavyashreem <kavyashr...@instem.res.in> wrote: > > Dear all, > > Sorry for the confusion created, I did not mean that a protein would have > fit in the small unit cell. My question was - > > 1. Why are there closely spaced spots arising in salt crystal? > > 2. If TCEP could crystallize in the condition, I have got a protein (same > as this)+ligand (different ligand) complex in very close condition. (ligand > size is within 500Da). > > Thank you > > Kavya > > > On 2023-02-03 14:35, a.perra...@nki.nl wrote: > > Hi Kavya, > > Try https://csb.wfu.edu/tools/vmcalc/vm.html > > This tells you that a 30kD protein simply does not fit the cell. > > I am pretty sure you crystallised the ligand, or TCEP actually. > > Also, if you look at the diffractions pattern, its clear the crystal > diffracts beyond 1.0A, diffraction spots are really very very very strong > at 2.0A. > > > > On 3 Feb 2023, at 09:22, kavyashreem <kavyashr...@instem.res.in> wrote: > > Dear all, > > We crystallized a protein (30kDa) + ligand (by cocrystallization), in the > condition 10%PEG3350, 50mM Zinc acetate. > > Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH > 8. > > Crystal: Crystal: > crystal under UV m > > <b06fc576.png> <e091c7fd.png> <8ef9453e.png> > > When we collected the data at an in-house facility, it looked something > like this: > > <b903961d.png> > > The minimum resolution spot is around 9Ang and maximum ~2.2Ang. > > I have not come across a protein diffraction like this, nor of a salt. > When I ran the gel for the incubated protein (protein+ligand), there was no > degradation. > > Although, I was sure there is some problem with this image I tried > processing, which could not be, But indexing showed a unit cell of 11Ang, > 11Ang, 46Ang in P3. which was quite expected for two of the axes but not > the third. > > Can anyone please shed some light on this diffraction image? > > How can it happen? > > > Thank you > > Regards > > Kavya > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > 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 > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > 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 > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 ######################################################################## 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/
