Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image
Dear Thomas, Interestingly, I had crystallized the same protein with another ligand before with the same condition except that it had sodium citrate in addition. I was able to collect the data for this and it was a protein-ligand complex, which could be seen in the density. So I was speculating about this data especially because of the closely spaced diffraction spots, and if the crystal has some peculiar defect. Thank you Regards Kavya On 2023-02-03 14:27, Thomas Flower wrote: > Dear Kavya, > > 4 mM is quite a high concentration of TCEP, perhaps they are TCEP crystals. > > You could try a Unit Cell Search of the CCDC to see if you find a match: Unit > Cell Search - WebCSD (cam.ac.uk) [2] > > Best, > > Thomas > > Thomas Flower, PhD > Senior Scientist, Protein Science > > Galapagos > 102 Avenue Gaston Roussel > 93230 Romainville > France > T: +33 7 81 26 95 70 > www.glpg.com [3] > > FROM: CCP4 bulletin board ON BEHALF OF mesters@biochem > SENT: vendredi 3 février 2023 9:53 AM > TO: CCP4BB@JISCMAIL.AC.UK > SUBJECT: Re: [ccp4bb] Regarding the diffraction image > > You don't often get email from mest...@biochem.uni-luebeck.de. Learn why this > is important [4] > > A and B unit cell dimensions are hardly bigger than twice the uni cell of > cubic NaCl and will probably not accommodate a 30 kDa protein. > > Make a SDS gel of washed & dissolved crystals to be sure these are not alt or > inhibitor crystals. You can stain the crystals with IzIt... > > J. > > -- > Dr. math. et dis. nat. Jeroen R. Mesters > https://orcid.org/-0001-8532-6699 [5] > > University of Lübeck > Institute of Biochemistry > https://www.biochem.uni-luebeck.de [6] > phone: +49-451-3101-3105 > Ratzeburger Allee 160 > 23562 Lübeck > Germany > -- > >> Am 03.02.2023 um 09:22 schrieb kavyashreem : >> >> 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 [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 [1] This > e-mail and its attachment(s) (if any) may contain confidential and/or > proprietary information and is intended for its addressee(s) only. Any > unauthorized use of the information contained herein (including, but not > limited to, alteration, reproduction, communication, distribution or any > other form of dissemination) is strictly prohibited. If you are not the > intended addressee, please notify the originator promptly and delete this > e-mail and its attachment(s) (if any) subsequently. Neither Galapagos nor any > of its affiliates shall be liable for direct, special, indirect or > consequential damages arising from alteration of the contents of this message > (by a third party) or as a result of a virus being passed on. > > - > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 Links: -- [1] https://eur05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=05%7C01%7CThomas.flower%40GLPG.COM%7C44212ddb46284392893a08db05c40c61%7C627f3c33bccc48bba033c0a6521f7642%
Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image
Hi Jessica, I am quite sure the protein cannot be fit in this unitcell. I was just curious why the diffraction has closely spaced spots. Thanks On 2023-02-04 00:02, Jessica Bruhn wrote: > Hi Kavya, > > As others have mentioned, the unit cell is too small to contain your protein. > With a volume of ~4820 Ang^3, the unit cell can contain at most ~268 atoms, > excluding hydrogens (divide the volume by 18 to get this number). If the > symmetry is P3, then the asymmetric unit can only contain ~89 atoms (divide > the number of atoms in the unit cell by 3), which is not a lot. It is most > likely something organic from your buffers (the ligand, TCEP, protein > fragment, other buffer components, etc). > > Searching the CCDC (https://www.ccdc.cam.ac.uk/structures/?) or the COD > (https://nanocrystallography.org/search.html) databases may be helpful. The > CCDC also has a unit cell searcher tool (CellCheckCSD) that you can download > and use without a license > (https://www.ccdc.cam.ac.uk/support-and-resources/downloads/). > Collecting higher resolution data (<1 Ang) and trying to solve this with > SHELXT would likely get to the bottom of things if you really want to know. > > Best of luck! > > Kind regards, > Jessica > > On Fri, Feb 3, 2023 at 7:37 AM Artem Evdokimov > wrote: > With 50 mM Zn++ in solution, whatever it is will probably have a Zn salt in > it. So if you wanted to solve it by direct methods or via SAD - that should > do well. Sadly (hur hur) it's probably quite small, whatever it is. > > Artem > > - Cosmic Cats approve of this message > > On Fri, Feb 3, 2023 at 6:20 AM Mark J. van Raaij > wrote: > like others mentioned, looks like something in between a salt and a protein, > perhaps TCEP, the ligand, a peptide cleaved from your protein by trace > protease. > If possible, I would move the detector closer, collect an atomic resolution > dataset and try to solve the structure by direct methods. You never know, it > could be something interesting. > > 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/ > https://namedrop.io/markvanraaij > > 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] Regarding the diffraction image
Dear Mark, I did think it was salt, so I checked the same batch of protein which went for crystallization by running a gel, it was intact, no cleavage. My doubts arose because of two things 1. I crystallized the same protein with another ligand, in very similar condition (10% PEG3350, 50mM Zn acetate, 0.25M Sod citrate) which diffracted and ligand density was visible in the protein structure 2. When can salt crystals give closely spaced spots which is seen in the image that was attached Thank you Regards Kavya On 2023-02-03 16:50, Mark J. van Raaij wrote: > like others mentioned, looks like something in between a salt and a protein, > perhaps TCEP, the ligand, a peptide cleaved from your protein by trace > protease. > If possible, I would move the detector closer, collect an atomic resolution > dataset and try to solve the structure by direct methods. You never know, it > could be something interesting. > > 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/ > https://namedrop.io/markvanraaij > >> 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 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] Regarding the diffraction image
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 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/
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] Regarding the diffraction image
Dear Ben, It was a single crystal that was mounted. I did use IZIT dye, did not trun blue much. Probably its a TCEp crystal. But I did try a negative control without protein+ligand and rest everything same, the drop was clear! Thank you Regards Kavya On 2023-02-06 22:49, Ben Bax wrote: > Hi Kavya, More than one crystal? > Epitaxial growth (protein crystal growing on salt crystal or vice versa) > Did you try IZIT? > (https://hamptonresearch.com/product-Izit-Crystal-Dye-33.html). > > I have seen salt and protein crystals in the same drop before now - but not > growing together. > Ben > > On 3 Feb 2023, at 22:15, CRAIG A BINGMAN > <21371e2fba31-dmarc-requ...@jiscmail.ac.uk> wrote: > > Oxidation products of TCEP crystallize in the presence of zinc ions. These > crystals fluoresce strongly under UV, which seems like a dirty trick, if your > expectation is that only protein crystals can do that. The brightfield white > light image of the crystals is also consistent with not-protein crystals, > because of the high refractive index contrast between the crystals and the > mother liquor. > > FROM: CCP4 bulletin board on behalf of kavyashreem > > DATE: Friday, February 3, 2023 at 2:32 AM > TO: CCP4BB@JISCMAIL.AC.UK > SUBJECT: [ccp4bb] Regarding the diffraction image > > 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/
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/
[ccp4bb] Crystallizing a tough target
Dear All, Has anyone worked on a protein which is highly soluble even at 80mg/ml? We have one such candidate, which does not precipitate even at 80mg/ml instead forms phase separated globules in crystallization plate, which eventually hardens over a period of 1 to 1.5 months (which is florescent under UV microscope.) We tried screening at different pH, but failed to get any hits. Since we got few conditions in which the phase separated globules solidified, we focused on them and expanded with 120mg/ml protein, still there were not visible precipitates except for the phase separation. This has been a challenging target so far. We have tried with different constructs, which unfortunately are not soluble! Does POMs help in such cases? Or do you have any other suggestion. Thank you Regards Kavya CONFIDENTIAL: This email and any files transmitted with it are confidential and intended solely for the use of the individual or group to whom they are addressed. If you have received this email in error, please notify the sender immediately and delete this e-mail from your system. It is NOT AUTHORISED to copy, forward, or in any way reveal the contents of this message to anyone. 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] [ccp4bb] Crystallizing a tough target
Dear all, Thank you all for your valuable experiences, inputs and references!! I shall try them and hope for some good news! Its good to know there are so many examples of crystallization at such high concentrations. A curious question - is it logical to say that if the protein is highly charged (either negative or positive), it is likely to be more soluble and resist crystallization due to electrostatic repulsion? Our protein has highly positively charged surface, although with some small negative patches. Following are some of the suggestions indicated: 1. Use water (will try) 2. Change buffers, pH temperature - (done) 3. Seeding (will try) 4. Methylating lysines (will try!!) 5. Surface entropy reduction (ongoing) 6. Optimize constructs (done) 7. Different ratios (done) 8. Keep concentrating (will try, the problem is yield is low!) 9. High salt concentrations (will try) 10. Organic solvents (though about it) 11. Mutations (ongoing) Thank you Regards Kavya On 2024-02-05 15:57, kavyashreem wrote: > Dear All, > > Has anyone worked on a protein which is highly soluble even at 80mg/ml? > > We have one such candidate, which does not precipitate even at 80mg/ml > instead forms phase separated globules in crystallization plate, which > eventually hardens over a period of 1 to 1.5 months (which is florescent > under UV microscope.) > > We tried screening at different pH, but failed to get any hits. > > Since we got few conditions in which the phase separated globules solidified, > we focused on them and expanded with 120mg/ml protein, still there were not > visible precipitates except for the phase separation. This has been a > challenging target so far. We have tried with different constructs, which > unfortunately are not soluble! > > Does POMs help in such cases? Or do you have any other suggestion. > > Thank you > > Regards > > Kavya > > CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL > AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE > ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER > IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO > COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. > > CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL > AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE > ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER > IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO > COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. > > - > > 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 IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM > **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT > CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE > FROM ADDRESS AND URLs* CONFIDENTIAL: This email and any files transmitted with it are confidential and intended solely for the use of the individual or group to whom they are addressed. If you have received this email in error, please notify the sender immediately and delete this e-mail from your system. It is NOT AUTHORISED to copy, forward, or in any way reveal the contents of this message to anyone. 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] [ccp4bb] Crystallizing a tough target
Dear Guillaume, That is right, if not for cations it would not have been possible. We will check with the surface entropy reduction. Since we are designing inhibitors for it, we cannot afford to do to much modifications. Thank you Regards Kavya On 2024-02-06 03:49, Guillaume Gaullier wrote: > Hello, > > Regarding this question: > >> A curious question - is it logical to say that if the protein is highly >> charged (either negative or positive), it is likely to be more soluble and >> resist crystallization due to electrostatic repulsion? Our protein has >> highly positively charged surface, although with some small negative patches. > > A counter example coming to mind is DNA: it is very strongly charged, yet has > been crystallized many times (you can find multiple examples of DNA-only > crystal structures in the PDB). Actually charges arranged so regularly along > the molecule actually help crystallization, when using an adequate > concentration of divalent cations. > Since fusion to a crystallizable scaffold protein has been suggested, I would > like to add that you could take inspiration from this paper from David > Baker's lab to design the scaffold protein (instead of using MBP or similar > ones): https://doi.org/10.1038/s41563-023-01683-1 > Pretty crazy things in there: spontaneous crystallization upon mixing E. coli > lysates, crystals that survive being autoclaved... If you know somebody who > knows how to use Rosetta for de novo computational design, this might be > worth a try. > An alternative would be to design de novo a high-affinity and crystallizable > binder for your protein. Similarly as the fusion protein approach, de novo > design may or may not be easier than alternatives, depending on protein > design expertise near you. > > I hope this helps, > > Guillaume > > On 5 Feb 2024, at 22:01, Tao-Hsin Chang wrote: > > Dear Kavya, > > I wanted to share with you that we have faced the same issue with a few > projects. In addition to the great suggestions given earlier, I recommend > trying out fusion proteins, such as the surface entropy reduction MBP mutant, > or using protein binding partners like antibodies or natural binders. These > strategies can help alter the protein properties and facilitate new crystal > contacts. Additionally, it may be worth experimenting with reduced salt > concentrations or using a salt-free buffer, akin to using water in place of a > buffer. > > PMID: 32541044 > PMID: 26850170 > > Wishing you the best of luck, > Tao-Hsin Chang > > On Feb 5, 2024, at 10:05 AM, kavyashreem wrote: > > Dear all, > > Thank you all for your valuable experiences, inputs and references!! I shall > try them and hope for some good news! Its good to know there are so many > examples of crystallization at such high concentrations. > > A curious question - is it logical to say that if the protein is highly > charged (either negative or positive), it is likely to be more soluble and > resist crystallization due to electrostatic repulsion? Our protein has highly > positively charged surface, although with some small negative patches. > > Following are some of the suggestions indicated: > > 1. Use water (will try) > > 2. Change buffers, pH temperature - (done) > > 3. Seeding (will try) > > 4. Methylating lysines (will try!!) > > 5. Surface entropy reduction (ongoing) > > 6. Optimize constructs (done) > > 7. Different ratios (done) > > 8. Keep concentrating (will try, the problem is yield is low!) > > 9. High salt concentrations (will try) > > 10. Organic solvents (though about it) > > 11. Mutations (ongoing) > > Thank you > > Regards > > Kavya > > On 2024-02-05 15:57, kavyashreem wrote: > > Dear All, > > Has anyone worked on a protein which is highly soluble even at 80mg/ml? > > We have one such candidate, which does not precipitate even at 80mg/ml > instead forms phase separated globules in crystallization plate, which > eventually hardens over a period of 1 to 1.5 months (which is florescent > under UV microscope.) > > We tried screening at different pH, but failed to get any hits. > > Since we got few conditions in which the phase separated globules solidified, > we focused on them and expanded with 120mg/ml protein, still there were not > visible precipitates except for the phase separation. This has been a > challenging target so far. We have tried with different constructs, which > unfortunately are not soluble! > > Does POMs help in such cases? Or do you have any other suggestion. > > Than
Re: [ccp4bb] [EXT] Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target
Dear Patrick, Thank you for the insight! Regards Kavya On 2024-02-06 00:29, Patrick Shaw Stewart wrote: >> is it logical to say that if the protein is highly charged (either negative >> or positive), it is likely to be more soluble and resist crystallization due >> to electrostatic repulsion? > > Hi Kavyashreem > > The project that I mentioned, where we looked at the crystallization > conditions reported in the PDB, also looked at the areas of amino acids (and > atoms) on the surfaces of proteins and tried to correlate the various groups > with crystallization conditions. > > Positive, negative, charged, polar or hydrophobic groups had very little > effect on the concentration of precipitant (we looked at PEG and ammonium > sulfate) and seemed not to affect the choice of precipitant (looking at all > precipitants). > > The only thing you could say was that the area of all charged and polar > groups as a proportion of the total surface area was roughly constant. If > the charged/polar area was high, the protein was crystallized at slightly > higher protein concentrations, and slightly higher concentrations of > precipitant were used on average. > > But it should be possible to make predictions. DeepMind is probably working > on this right now! > > Best wishes, > > Patrick > > On Mon, Feb 5, 2024 at 3:06 PM kavyashreem wrote: > > Dear all, > > Thank you all for your valuable experiences, inputs and references!! I shall > try them and hope for some good news! Its good to know there are so many > examples of crystallization at such high concentrations. > > A curious question - is it logical to say that if the protein is highly > charged (either negative or positive), it is likely to be more soluble and > resist crystallization due to electrostatic repulsion? Our protein has highly > positively charged surface, although with some small negative patches. > > Following are some of the suggestions indicated: > > 1. Use water (will try) > > 2. Change buffers, pH temperature - (done) > > 3. Seeding (will try) > > 4. Methylating lysines (will try!!) > > 5. Surface entropy reduction (ongoing) > > 6. Optimize constructs (done) > > 7. Different ratios (done) > > 8. Keep concentrating (will try, the problem is yield is low!) > > 9. High salt concentrations (will try) > > 10. Organic solvents (though about it) > > 11. Mutations (ongoing) > > Thank you > > Regards > > Kavya > > On 2024-02-05 15:57, kavyashreem wrote: > > Dear All, > > Has anyone worked on a protein which is highly soluble even at 80mg/ml? > > We have one such candidate, which does not precipitate even at 80mg/ml > instead forms phase separated globules in crystallization plate, which > eventually hardens over a period of 1 to 1.5 months (which is florescent > under UV microscope.) > > We tried screening at different pH, but failed to get any hits. > > Since we got few conditions in which the phase separated globules solidified, > we focused on them and expanded with 120mg/ml protein, still there were not > visible precipitates except for the phase separation. This has been a > challenging target so far. We have tried with different constructs, which > unfortunately are not soluble! > > Does POMs help in such cases? Or do you have any other suggestion. > > Thank you > > Regards > > Kavya > > CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL > AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE > ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER > IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO > COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. > > CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL > AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE > ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER > IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO > COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. > > - > > 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 IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM > **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT > CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE > FROM ADDRES
Re: [ccp4bb] [EXT] Re: [ccp4bb] Crystallizing a tough target
Dear Patrick, Thank you for the suggestions and for the detailed the statistics!! Regards Kavya On 2024-02-06 23:06, Patrick Shaw Stewart wrote: > Hi Kavya > > 1. Make a seed stock from the globules or anything else that you think might > be crystalline, and recreen. In other words, you should add your seed stock > to _random screens_ (not optimization experiments). There could be many > conditions that are in the metastable zone of the phase diagram in your > normal screens - this method can give you crystals in those conditions. > > If this works, you'll be in a better position anyway because you'll have more > control - by diluting the seed stock, you can control the number of crystals > per drop. > > References: > >> D'Arcy, A., Villard, F. and Marsh, M., 2007. An automated microseed >> matrix-screening method for protein crystallization. _Acta Crystallographica >> Section D: Biological Crystallography_, _63_(4), pp.550-554. >> >> Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock, >> P.F., 2011. Random microseeding: a theoretical and practical exploration of >> seed stability and seeding techniques for successful protein >> crystallization. _Crystal Growth & Design_, _11_(8), pp.3432-3441. > > This is how we normally make the seed: > >> https://www.douglas.co.uk/f_ftp1/rMMS_Procedure.pdf > > 2. Many years ago, we did some data mining of the crystallization conditions > in a remark in the PDB. The concentrations that people reported are below. > There were eight reports where over 100 mg/mL was used. It only goes up to > 2004. > >> https://www.douglas.co.uk/PDB_data.htm [1] > > Good luck, > > Patrick > > On Mon, Feb 5, 2024 at 10:27 AM kavyashreem > wrote: > >> Dear All, >> >> Has anyone worked on a protein which is highly soluble even at 80mg/ml? >> >> We have one such candidate, which does not precipitate even at 80mg/ml >> instead forms phase separated globules in crystallization plate, which >> eventually hardens over a period of 1 to 1.5 months (which is florescent >> under UV microscope.) >> >> We tried screening at different pH, but failed to get any hits. >> >> Since we got few conditions in which the phase separated globules >> solidified, we focused on them and expanded with 120mg/ml protein, still >> there were not visible precipitates except for the phase separation. This >> has been a challenging target so far. We have tried with different >> constructs, which unfortunately are not soluble! >> >> Does POMs help in such cases? Or do you have any other suggestion. >> >> Thank you >> >> Regards >> >> Kavya >> >> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL >> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE >> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE >> SENDER IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT >> AUTHORISED TO COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS >> MESSAGE TO ANYONE. >> >> - >> >> 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 IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM > **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT > CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE > FROM ADDRESS AND URLs* > > CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL > AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE > ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER > IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO > COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. Links: -- [1] https://www.douglas.co.uk/PDB_data.htm CONFIDENTIAL: This email and any files tr
