Re: [ccp4bb] [EXT] Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image
Dear Patrick, I did use microseeding but for a different ligand. Will try with this as well. Thank you REgards Kavya On 2023-02-07 16:20, Patrick Shaw Stewart wrote: >> 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 > 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 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 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 > > <8ef9453e.png> > > When we collected the data at an in-house facility, it looked something like > this: > > > > 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.ukDouglas 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) 148-864-9090US 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/
Re: [ccp4bb] [EXT] RE: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image
Dear Bernhard Rupp, Sure, will look in this direction. Thank you Regards Kavya On 2023-02-06 00:41, Bernhard Rupp wrote: > 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 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 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 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 > > <8ef9453e.png> > > When we collected the data at an in-house facility, it looked something like > this: > > > > 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 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/
Re: [ccp4bb] [EXT] Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image
Dear Mark, Thanks for the clarification. Regards Kavya On 2023-02-05 04:02, Mark J. van Raaij 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 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 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 > > <8ef9453e.png> > > When we collected the data at an in-house facility, it looked something like > this: > > > > 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 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/
