TCEP is known to form transition metal complexes. With the molecule alone already about 10A across, a ~11x11x46 cell is not unreasonable, and there might be alternatives to P3 (can’t tell from the single image). Would be interesting to collect and, as mentioned, toss into Direct methods assisted by anomalous P (or Zn?) signal...
Have fun, BR From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Mark J. van Raaij Sent: Saturday, February 4, 2023 14:32 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image 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 <mailto: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 <mailto: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 <mailto: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 <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> &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/
