[ccp4bb] Protein-Ligand Crystallization
Hi all, I am working to crystallize a protein-ligand complex. I did a preliminary melting curve analysis for the protein in the absence and presence of 2 ligands (dissolved in protein buffer). I kept the other controls as buffer an a known standard to confirm instrument performance. All expts done in triplicates. Now the results are like : Tm of protein alone is 56 deg, Tm in the presence of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1, 56.0 and 53.5 respectively !! Tm in the presence of Lig 2 conc. of 0.05mM, 0.1mM, 1.0mM, 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6 respectively !! Although the effective delta Tm for both is different at higher concentration, but both are kind of making protein less stable. So i was wondering, will it be difficult to co-crystallize them !! Any suggestions in this regard are highly appreciated !! Thanks Monica
Re: [ccp4bb] Bulk solvent correction in Phaser MR LF
Hi, Bernhard, ok, sure, you are right ! Nevertheless, I would not be so desperate and categorical: you are right , at my knowledge also, there is currently NO known algorithm to take into account the flat mask bulk solvent contribution at the rotation step (maybe I am wrong?), however this does not mean it is impossible. The question is should it be done at all ? When do you the rotation search, you need to use mostly the highest resolution data, and it may be rather useful to remove the low-resolution data (see the tests described in Acta Cryst D51, 888-895, 1995; however, it looks like in practice nobody uses variable resolution for different MR steps). When you work at low resolution with envelopes, you do not care about bulk solvent correction since all this results indeed in a scale coefficient. There is a tiny (but not unusual) situation when one wants to do MR at the worse resolution of 9-12 A (worse in the sense that the maps show neither (already) molecular envelope nor (yet) clear structure. Suppose we could solve the MR problem - what we do then since the maps are so poor ? It is worthy in this case artificially lower the resolution ? I do not know... However, I agree, you question is fully valid and is interesting. All the best , Sacha De : hofkristall...@gmail.commailto:hofkristall...@gmail.com [hofkristall...@gmail.com] Envoyé : mercredi 4 février 2015 14:09 À : Alexandre OURJOUMTSEV; CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Objet : RE: [ccp4bb] Bulk solvent correction in Phaser MR LF Hi Sacha, I was imprecise. With unplaced I meant 'neither rotated nor translated'. Once you become post-rotationally SF based, you can in fact compute a F(env) whole inclusion should improve the TF score. What is not evident to me is how to use a mask and compute the Fs if the orientation (rotation) is yet to be determined? Thx, BR
Re: [ccp4bb] Resolve Domain Sequence Ambiguities
Dear Jacob, Are you sure your MR solution is correct? What is your resolution? Best, D -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: 05 February 2015 15:21 To: ccp4bb Subject: [ccp4bb] Resolve Domain Sequence Ambiguities Hi All, I have 14 identical domains placed in my ASU with reasonable MR scores, but the problem is that there should really be 7+7 or 8+8 of domains A and B (which are structurally similar). Can anyone think of a great and easy way of resolving the ambiguity? I was thinking potentially: -change one domain to polyAla -SA omit map of that one -rebuild/refine -iterate through all domains, noting scores Seems it might be pretty low reliability and a fair amount of work though. Otherwise, could try to go back to a small SAD signal, use partial model phases to find HAs, then phase without the model, rebuild from there. Any thoughts or similar experiences? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org *** -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] Resolve Domain Sequence Ambiguities
Sorry--should have included a bit more info: A huge caveat: data is tetartohedrally twinned, which makes everything an issue. But, I have managed to get Rfree down to ~32% in refmac with intensity-based twin refinement. I know--R's with de-twinning are illusory, but I still think it's solved, since I also can see anomalous signal in partial-model LLG maps from phaser at places predicted by the structure (and I don't think that anomalous info could be encoded in the MR phases (can it?)). So, that was a nice confirmatory moment... Resolution is good, but cut off by detector limit on home system (set at max resolution w/o 2theta) N 1/d^2Dmid CCanomNanom RCRanom CC1/2 NCC1/2 Rsplit CCfit CCanomfit $$ $$ 1 0.0061 12.82 -0.018 938 0.982 0.998 9890.028 1.000 0.001 2 0.0182 7.40 0.026 1831 1.026 0.998 18800.027 1.000 0.006 3 0.0304 5.73 0.062 2334 1.064 0.995 23850.032 1.000 0.010 4 0.0426 4.85 0.048 2806 1.049 0.995 28810.033 1.000 0.014 5 0.0547 4.27 0.036 3164 1.036 0.995 32430.034 1.000 0.019 6 0.0669 3.87 0.025 3524 1.026 0.994 36060.035 1.000 0.023 7 0.0791 3.56 0.011 3758 1.011 0.993 38730.038 1.000 0.027 8 0.0912 3.31 0.021 4111 1.021 0.993 42060.041 0.999 0.032 9 0.1034 3.11 -0.003 4340 0.997 0.991 44410.044 0.999 0.036 10 0.1155 2.94 -0.040 4602 0.961 0.989 47240.049 0.999 0.040 11 0.1277 2.80 0.050 4864 1.051 0.991 49680.048 0.999 0.045 12 0.1399 2.67 0.051 4996 1.052 0.988 51210.052 0.998 0.049 13 0.1520 2.56 0.073 5348 1.076 0.988 54690.053 0.997 0.053 14 0.1642 2.47 -0.009 5486 0.991 0.987 56110.057 0.996 0.058 15 0.1764 2.38 0.032 5698 1.032 0.985 58270.061 0.995 0.062 16 0.1885 2.30 0.091 5945 1.095 0.985 60670.063 0.993 0.066 17 0.2007 2.23 0.038 6002 1.039 0.985 61180.064 0.991 0.070 18 0.2128 2.17 0.086 6331 1.089 0.983 64450.068 0.987 0.075 19 0.2250 2.11 0.075 6411 1.078 0.983 65560.070 0.983 0.079 20 0.2372 2.05 0.097 6608 1.102 0.983 67560.070 0.977 0.083 21 0.2493 2.00 0.141 6712 1.152 0.981 68800.078 0.968 0.088 22 0.2615 1.96 0.104 6556 1.110 0.974 71260.093 0.957 0.092 23 0.2737 1.91 0.141 5595 1.152 0.953 70540.121 0.943 0.096 24 0.2858 1.87 0.149 4659 1.160 0.936 66520.151 0.923 0.101 25 0.2980 1.83 0.125 3630 1.133 0.909 59360.184 0.898 0.105 26 0.3101 1.80 0.138 2640 1.148 0.874 51180.219 0.866 0.109 27 0.3223 1.76 0.111 1793 1.116 0.851 40350.250 0.825 0.114 28 0.3345 1.73 0.040 1040 1.041 0.782 28570.305 0.776 0.118 29 0.3466 1.70 0.094 300 1.097 0.713 14740.371 0.717 0.122 30 0.3588 1.67 - 2 - 0.589 2280.497 0.649 0.127 $$ Overall:0.027 122024 1.027 0.997 1385260.048 CCanomNanom RCRanom CC1/2 NCC1/2 Rsplit CCfit CCanomfit MR solution: SOLU SET RFZ=3.9 TFZ=7.1 PAK=0 LLG=38 RFZ=3.7 TFZ=8.9 PAK=0 LLG=75 RFZ=4.2 TFZ=8.2 PAK=0 LLG=40 LLG=160 RFZ=3.2 TFZ=11.2 PAK=0 LLG=249 LLG=262 RFZ=4.2 TFZ=9.4 PAK=0 LLG=331 LLG=339 RFZ=4.1 TFZ=11.3 PAK=0 LLG=405 LLG=418 RFZ=3.9 TFZ=11.6 PAK=0 LLG=487 LLG=494 RFZ=4.2 TFZ=8.9 PAK=0 LLG=539 LLG=554 RFZ=3.3 TFZ=8.6 PAK=0 LLG=612 LLG=638 RFZ=3.1 TFZ=8.7 PAK=0 LLG=679 LLG=691 RFZ=2.6 TFZ=12.4 PAK=0 LLG=910 LLG=926 RFZ=3.0 TFZ=9.2 PAK=0 LLG=987 LLG=1001 RFZ=2.7 TFZ=9.8 PAK=3 LLG=1018 LLG=1043 RFZ=2.9 TFZ=9.1 PAK=5 LLG=1039 -Original Message- From: dom.bell...@diamond.ac.uk [mailto:dom.bell...@diamond.ac.uk] Sent: Thursday, February 05, 2015 10:28 AM To: Keller, Jacob; ccp4bb@jiscmail.ac.uk Subject: RE: Resolve Domain Sequence Ambiguities Dear Jacob, Are you sure your MR solution is correct? What is your resolution? Best, D -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: 05 February 2015 15:21 To: ccp4bb Subject: [ccp4bb] Resolve Domain Sequence Ambiguities Hi All, I have 14 identical domains placed in my ASU with reasonable MR scores, but the problem is that there should really be 7+7 or 8+8 of domains A and B (which are structurally similar). Can anyone think of a great
Re: [ccp4bb] Resolve Domain Sequence Ambiguities
Did you try ShelxE autotrace? At that resolution it should work nicely, or at least be able to distinguish between the two sequences. D -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: 05 February 2015 15:49 To: ccp4bb Subject: Re: [ccp4bb] Resolve Domain Sequence Ambiguities Sorry--should have included a bit more info: A huge caveat: data is tetartohedrally twinned, which makes everything an issue. But, I have managed to get Rfree down to ~32% in refmac with intensity-based twin refinement. I know--R's with de-twinning are illusory, but I still think it's solved, since I also can see anomalous signal in partial-model LLG maps from phaser at places predicted by the structure (and I don't think that anomalous info could be encoded in the MR phases (can it?)). So, that was a nice confirmatory moment... Resolution is good, but cut off by detector limit on home system (set at max resolution w/o 2theta) N 1/d^2Dmid CCanomNanom RCRanom CC1/2 NCC1/2 Rsplit CCfit CCanomfit $$ $$ 1 0.0061 12.82 -0.018 938 0.982 0.998 9890.028 1.000 0.001 2 0.0182 7.40 0.026 1831 1.026 0.998 18800.027 1.000 0.006 3 0.0304 5.73 0.062 2334 1.064 0.995 23850.032 1.000 0.010 4 0.0426 4.85 0.048 2806 1.049 0.995 28810.033 1.000 0.014 5 0.0547 4.27 0.036 3164 1.036 0.995 32430.034 1.000 0.019 6 0.0669 3.87 0.025 3524 1.026 0.994 36060.035 1.000 0.023 7 0.0791 3.56 0.011 3758 1.011 0.993 38730.038 1.000 0.027 8 0.0912 3.31 0.021 4111 1.021 0.993 42060.041 0.999 0.032 9 0.1034 3.11 -0.003 4340 0.997 0.991 44410.044 0.999 0.036 10 0.1155 2.94 -0.040 4602 0.961 0.989 47240.049 0.999 0.040 11 0.1277 2.80 0.050 4864 1.051 0.991 49680.048 0.999 0.045 12 0.1399 2.67 0.051 4996 1.052 0.988 51210.052 0.998 0.049 13 0.1520 2.56 0.073 5348 1.076 0.988 54690.053 0.997 0.053 14 0.1642 2.47 -0.009 5486 0.991 0.987 56110.057 0.996 0.058 15 0.1764 2.38 0.032 5698 1.032 0.985 58270.061 0.995 0.062 16 0.1885 2.30 0.091 5945 1.095 0.985 60670.063 0.993 0.066 17 0.2007 2.23 0.038 6002 1.039 0.985 61180.064 0.991 0.070 18 0.2128 2.17 0.086 6331 1.089 0.983 64450.068 0.987 0.075 19 0.2250 2.11 0.075 6411 1.078 0.983 65560.070 0.983 0.079 20 0.2372 2.05 0.097 6608 1.102 0.983 67560.070 0.977 0.083 21 0.2493 2.00 0.141 6712 1.152 0.981 68800.078 0.968 0.088 22 0.2615 1.96 0.104 6556 1.110 0.974 71260.093 0.957 0.092 23 0.2737 1.91 0.141 5595 1.152 0.953 70540.121 0.943 0.096 24 0.2858 1.87 0.149 4659 1.160 0.936 66520.151 0.923 0.101 25 0.2980 1.83 0.125 3630 1.133 0.909 59360.184 0.898 0.105 26 0.3101 1.80 0.138 2640 1.148 0.874 51180.219 0.866 0.109 27 0.3223 1.76 0.111 1793 1.116 0.851 40350.250 0.825 0.114 28 0.3345 1.73 0.040 1040 1.041 0.782 28570.305 0.776 0.118 29 0.3466 1.70 0.094 300 1.097 0.713 14740.371 0.717 0.122 30 0.3588 1.67 - 2 - 0.589 2280.497 0.649 0.127 $$ Overall:0.027 122024 1.027 0.997 1385260.048 CCanomNanom RCRanom CC1/2 NCC1/2 Rsplit CCfit CCanomfit MR solution: SOLU SET RFZ=3.9 TFZ=7.1 PAK=0 LLG=38 RFZ=3.7 TFZ=8.9 PAK=0 LLG=75 RFZ=4.2 TFZ=8.2 PAK=0 LLG=40 LLG=160 RFZ=3.2 TFZ=11.2 PAK=0 LLG=249 LLG=262 RFZ=4.2 TFZ=9.4 PAK=0 LLG=331 LLG=339 RFZ=4.1 TFZ=11.3 PAK=0 LLG=405 LLG=418 RFZ=3.9 TFZ=11.6 PAK=0 LLG=487 LLG=494 RFZ=4.2 TFZ=8.9 PAK=0 LLG=539 LLG=554 RFZ=3.3 TFZ=8.6 PAK=0 LLG=612 LLG=638 RFZ=3.1 TFZ=8.7 PAK=0 LLG=679 LLG=691 RFZ=2.6 TFZ=12.4 PAK=0 LLG=910 LLG=926 RFZ=3.0 TFZ=9.2 PAK=0 LLG=987 LLG=1001 RFZ=2.7 TFZ=9.8 PAK=3 LLG=1018 LLG=1043 RFZ=2.9 TFZ=9.1 PAK=5 LLG=1039 -Original Message- From: dom.bell...@diamond.ac.uk [mailto:dom.bell...@diamond.ac.uk] Sent: Thursday, February 05, 2015 10:28 AM To: Keller, Jacob; ccp4bb@jiscmail.ac.uk Subject: RE: Resolve Domain Sequence Ambiguities Dear Jacob, Are you sure your MR solution is correct? What is your resolution? Best, D -Original Message- From: CCP4 bulletin board
Re: [ccp4bb] Resolve Domain Sequence Ambiguities
Hi Jacob, I would superpose domain B onto each domain A in turn, and run refinement in Phaser. If the LLG goes up, exchange that domain A for a domain B, otherwise keep. This requires 14 refinement calculations. This procedure may not give you what you are hoping for if your models are distant, since in this case domain A may be a better model for both domains. However, you could still improve your solution this way, and is therefore probably worth it. BW, Gabor On 2015-02-05 15:20, Keller, Jacob wrote: Hi All, I have 14 identical domains placed in my ASU with reasonable MR scores, but the problem is that there should really be 7+7 or 8+8 of domains A and B (which are structurally similar). Can anyone think of a great and easy way of resolving the ambiguity? I was thinking potentially: -change one domain to polyAla -SA omit map of that one -rebuild/refine -iterate through all domains, noting scores Seems it might be pretty low reliability and a fair amount of work though. Otherwise, could try to go back to a small SAD signal, use partial model phases to find HAs, then phase without the model, rebuild from there. Any thoughts or similar experiences? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org *** -- ## Dr Gabor Bunkoczi Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital Hills Road Cambridge CB2 0XY ##
Re: [ccp4bb] proton scattering by X-rays
What about the bogey of Fourier truncation ripples--I have heard many have been fooled by the into thinking they were seeing orbitals. How does one tell the difference? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phil Jeffrey Sent: Thursday, February 05, 2015 3:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] proton scattering by X-rays Mark, In the small-molecule crystal structures I work with it's relatively common to see localized difference electron density along covalent bonds or in the places you'd expect to see lone pairs during refinement after you've fit and modeled the atoms reasonably well and the phases are pretty good. It's usually not as strong as difference density for hydrogens, before you put them in, but it's often pretty clearly visible once you have. (I use SHELXLE as an interface for small molecule refinements because of a somewhat Coot-like experience in viewing maps). Phil Jeffrey Princeton What you CAN do in fact is appropriately subtract spherical electron density from the experimental density and see what is left (i.e. directional ED that is 'surplus'). I tried to quickly find a paper on that, they exist, and they show that experimental density does confirm what we learn in chemistry class, orbitals are not imaginary. Mark
Re: [ccp4bb] proton scattering by X-rays
What about the bogey of Fourier truncation ripples--I have heard many have been fooled by the into thinking they were seeing orbitals. How does one tell the difference? Indeed, there are such dangers. Hints are here: On the possibility of observation of valence electron density for individual bonds in proteins in conventional difference maps. (2004). Acta Cryst., D60, 260-274. Have a look at Figure 4. Then compare with Figure 1 here: J. Biol. Chem. (1999) 274, 20753-29755. Bottom line is: be careful with unbalanced Fourier syntheses, balanced ones may be a safer option. Pavel
Re: [ccp4bb] proton scattering by X-rays
Mark, In the small-molecule crystal structures I work with it's relatively common to see localized difference electron density along covalent bonds or in the places you'd expect to see lone pairs during refinement after you've fit and modeled the atoms reasonably well and the phases are pretty good. It's usually not as strong as difference density for hydrogens, before you put them in, but it's often pretty clearly visible once you have. (I use SHELXLE as an interface for small molecule refinements because of a somewhat Coot-like experience in viewing maps). Phil Jeffrey Princeton What you CAN do in fact is appropriately subtract spherical electron density from the experimental density and see what is left (i.e. directional ED that is 'surplus'). I tried to quickly find a paper on that, they exist, and they show that experimental density does confirm what we learn in chemistry class, orbitals are not imaginary. Mark
Re: [ccp4bb] proton scattering by X-rays
This may be useful reading on charge density crystallography: http://journals.iucr.org/a/issues/1998/05/00/by0157/by0157.pdf I like the phrase ‘gourmet crystallographers’ Best, BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark van der Woerd Sent: Thursday, February 05, 2015 9:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] proton scattering by X-rays Not very well. When you look in the originally quoted article, there is really not much difference in the ED for H+ and H- (which are conveniently both shown in Fig 2). You build a model that is consistent with 'book knowledge' (i.e. normal hydrogen has only one bond) and take it from there. You have a point that the resolution isn't very good at 0.89A (sorry!) but I suspect that the number of recorded reflections is much more important than the nebulous number of 0.89A (i.e. you can start to see these little details because you finally have an over-determined system parameters). What you CAN do in fact is appropriately subtract spherical electron density from the experimental density and see what is left (i.e. directional ED that is 'surplus'). I tried to quickly find a paper on that, they exist, and they show that experimental density does confirm what we learn in chemistry class, orbitals are not imaginary. Mark -Original Message- From: Doug Ohlendorf oh...@umn.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Mon, Feb 2, 2015 11:29 am Subject: Re: [ccp4bb] proton scattering by X-rays But, how with x-rays can one experimentally tell the difference between a hydrogen and a filled orbital (say of N)? I will grant that the electron density for a bound H should extend farther from the heavy atom but I believe you would need resolution better than 0.89 Ang to see this difference. Doug Douglas H. Ohlendorf Phone: 612-624-8436 Professor FAX:612-624-5121 Dept. of Biochemistry, Molecular Biology Biophysics Twin Cities Campus, University of Minnesota Lab web site: http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK? ] On Behalf Of Colin Nave Sent: Monday, February 02, 2015 9:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] proton scattering by X-rays “As you say the proton itself is invisible to X-rays.” Not quite! The ratio of scattering between electrons and protons should go as the inverse square of the masses. Ratio of mass 1:1860, ratio of scattering 1:3459600. A small correction but doubtless it has been incorporated in to SHELX. Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK? ] On Behalf Of Ian Tickle Sent: 02 February 2015 13:35 To: ccp4bb Subject: Re: [ccp4bb] proton scattering by X-rays Peter, if it's a covalently-bonded H atom it surely can't be a bare proton, it must have at least some partial electron around it for the (possibly partial) covalent bond, enough to diffract X-rays anyway. As you say the proton itself is invisible to X-rays. Cheers -- Ian On 2 February 2015 at 13:08, Peter Moody pcem1bigfi...@gmail.commailto:pcem1bigfi...@gmail.com mailto:pcem1bigfi...@gmail.com? wrote: Dear BB I have (again) realised how limited by understanding of our subject is. In Nature’s online site http://www.nature.com/nature/journal/vaop/ncurrent/full/nature14110.html?WT.ec_id=NATURE-20150129 there is a paper describing an X-ray structure determined with sub-atomic data (nice!). The figures show density for H+ as well as H-. In my simple way I had assumed that any X-ray scattering from the nucleus was negligible, and that the electrons are responsible for this. I would expect a proton (i.e. H+) alone to be invisible to X-rays, and certainly not to look similar to a hydride (with two electrons in (electron density) maps. What have I missed? Could someone please explain, or point me to a suitable reference? Best wishes, Peter (please use peter.mo...@le.ac.ukmailto:peter.mo...@le.ac.uk mailto:peter.mo...@le.ac.uk? to reply directly) http://www2.le.ac.uk/departments/biochemistry/staff/moody -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability
[ccp4bb] absolute-scale and noise maps
Dear all, I do apologize for the slightly off-topic subject but, how could you calculate ccp4 absolute-scale and noise maps in ccp4?. Basically I need e/A3 and noise maps to discard that some maps contoured at certain sigmas are not just noise. I do know END/RAPID, but I have some problems trying to run RAPID (I am hoping that they would be included sometime in Phenix). Thanks a lot, Isabel Isabel Garcia-Saez PhD Institut de Biologie Structurale Viral Infection and Cancer Group (VIC)-Cell Division Team 71, Avenue des Martyrs CS 10090 38044 Grenoble Cedex 9 France Tel.: 00 33 (0) 457 42 86 15 e-mail: isabel.gar...@ibs.fr FAX: 00 33 (0) 476 50 18 90 http://www.ibs.fr
[ccp4bb] Resolve Domain Sequence Ambiguities
Hi All, I have 14 identical domains placed in my ASU with reasonable MR scores, but the problem is that there should really be 7+7 or 8+8 of domains A and B (which are structurally similar). Can anyone think of a great and easy way of resolving the ambiguity? I was thinking potentially: -change one domain to polyAla -SA omit map of that one -rebuild/refine -iterate through all domains, noting scores Seems it might be pretty low reliability and a fair amount of work though. Otherwise, could try to go back to a small SAD signal, use partial model phases to find HAs, then phase without the model, rebuild from there. Any thoughts or similar experiences? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
[ccp4bb] 2nd Announcement and etc - the 5th Winter School on Soft X-rays in MX
Posted on behalf of the Organizing Committee: John Rose and Bi-Cheng Wang (University of Georgia, Athens, Georgia, U.S.A.) Manfred Weiss (Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin, Germany) Christoph Mueller-Dieckmann (ESRF, Grenoble, France)2nd AnnouncementandNewAdditions to theProgramThe 5thWinter School on Soft X-Rays in Macromolecular Crystallography Location: Georgia Center,TheUniversity of Georgia, Athens, Georgia, USA 30602 Dates: March 1 - 4, 2015 Registration: Online at: http://wssxmc.bmb.uga.edu.We announced theWinterSchool on the CCP4 bulletin board on December 19, 2014. Since then,we have addeda new Session on Detector Considerations on Monday evening, March 2 (pleasesee the attached Program Outline) and updated thecourseinformation on thethreehands-on workshopson Wednesday, March 4.The aim of the School is to bring together experts and young scientists interested in using the anomalous signal from light atoms (e.g. S, P, Ca, K, Mg) for macromolecular structure determination employing soft(lambda 1.5Å) X-ray techniques.The previous four Schools were held in Europe:1st Winter School: Bressanone/Brixen, Italy; Feb. 25-27, 20032ndWinter School: Seefeld, Austria; March 22-25, 20063rdWinter School: Berlin, Germany; February 18-20, 20094thWinter School: Grenoble, France, February 6-8, 2012There will also be hands-on workshops on Wednesday March 4.Optimizing Native-SAD Data Collection - (John Rose Albert Fu) will demonstrate features thatmaybe used to optimize data quality.Getting The Most Out of Data Processing - (Albert Fu John Rose) will demonstrate data processing using XDS, Kylin and HKL2000.Experimental Phasing With SHELX C/D/E -(George Sheldrick) will demonstrate the use of the SHELX suite for experimental phasing of SAD data.Participants are encouraged to bring their laptops (Windows, Mac or Linux) so that theymaywork through the test data during the workshop. The required software and data sets will be provided via the web (directions will be provided at the Winter School) or via USB drive at the meeting.Limited travel bursaries arestillavailable to young investigators (students and postdoc) who wish to attend. You may submit your application online athttp://wssxmc.bmb.uga.edu/Travel_Bursary.htmlor contact John Rose (jpr...@uga.edu).Online registration at: http://wssxmc.bmb.uga.edu.An updated Program Outline is attached. 5th Winter School Program Outline_150204.pdf Description: Adobe PDF document
Re: [ccp4bb] proton scattering by X-rays
Dear Jacob, there was a nice small computation project by Anne Bochow published in the CCP4 Newsletters (2005), http://www.ccp4.ac.uk/newsletters/newsletter42/content.html, communication 9, illustrating how strong and how confusing the ripples may be indeed; if you want I may send it you off list the corresponding pdf file. Please make a look the figures, they are very impressive. And Pavel just commented how to distinguish such the ripples from the peaks for the deformation bond density. With best regards, Sacha Urzhumtsev De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Keller, Jacob [kell...@janelia.hhmi.org] Envoyé : jeudi 5 février 2015 21:53 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] proton scattering by X-rays What about the bogey of Fourier truncation ripples--I have heard many have been fooled by the into thinking they were seeing orbitals. How does one tell the difference? JPK
[ccp4bb] Superposition of select residues
Hi all, Does anyone know of a program to use to superpose only selected protein residues, e.g. enzyme active site residues. I solved my structure using molecular replacement now I want to compare its active site to those of homologous structures. I have used the DALI server before but I had to upload the whole model. Thank you for your advise Best Regards, Oarabile M. Kgosisejo, MSc. Candidate University of Saskatchewan o.kgosis...@usask.ca
Re: [ccp4bb] Superposition of select residues
The program chimera will align homologous structures from which you can generate a sequence alignment (based on the structural alignment) http://www.cgl.ucsf.edu/chimera/docs/ContributedSoftware/matchalign/matchalign.html ~JH On 05.02.2015 14:41, Kgosisejo, Oarabile wrote: Hi all, Does anyone know of a program to use to superpose only selected protein residues, e.g. enzyme active site residues. I solved my structure using molecular replacement now I want to compare its active site to those of homologous structures. I have used the DALI server before but I had to upload the whole model. Thank you for your advise BEST REGARDS, Oarabile M. Kgosisejo, MSc. Candidate University of Saskatchewan o.kgosis...@usask.ca
Re: [ccp4bb] Superposition of select residues
The free ICM-Browser from MolSoft can do this - http://www.molsoft.com/icm_browser.html It will calculate the Ca-atom, backbone and heavy atom differences between multiple structures and superimpose. To do this: 1. Download and install ICM-Browser here (Win, Mac or Linux) http://www.molsoft.com/getbrowser.cgi 2. Read in the PDB structures using the search tab or File/Open 2. Select the residues in the proteins you wish to superimpose 3. Click on the superimpose button in the display tab - you can select one structure to be static. http://www.molsoft.com/gui/protein-structure-tutorials.html#protein-structure-tutorials-superimpose Thanks, Andrew -- Andrew Orry Ph.D. Senior Research Scientist MolSoft LLC 11199 Sorrento Valley Road San Diego, CA 92121 -- Tel: 858-625-2000 x108 Fax: 858-625-2888 www.molsoft.com www.twitter.com/MolSoft On 2/5/2015 2:41 PM, Kgosisejo, Oarabile wrote: Hi all, Does anyone know of a program to use to superpose only selected protein residues, e.g. enzyme active site residues. I solved my structure using molecular replacement now I want to compare its active site to those of homologous structures. I have used the DALI server before but I had to upload the whole model. Thank you for your advise *Best Regards,* * * *Oarabile M. Kgosisejo, MSc. Candidate University of Saskatchewan * *o.kgosis...@usask.ca*
Re: [ccp4bb] Superposition of select residues
Dear Andrew, Thank you very much for the detailed instructions. I am downloading it Best Regards, Oarabile M. Kgosisejo o.kgosis...@usask.ca From: Andrew Orry [a...@molsoft.com] Sent: February 5, 2015 4:57 PM To: Kgosisejo, Oarabile; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Superposition of select residues The free ICM-Browser from MolSoft can do this - http://www.molsoft.com/icm_browser.html It will calculate the Ca-atom, backbone and heavy atom differences between multiple structures and superimpose. To do this: 1. Download and install ICM-Browser here (Win, Mac or Linux) http://www.molsoft.com/getbrowser.cgi 2. Read in the PDB structures using the search tab or File/Open 2. Select the residues in the proteins you wish to superimpose 3. Click on the superimpose button in the display tab - you can select one structure to be static. http://www.molsoft.com/gui/protein-structure-tutorials.html#protein-structure-tutorials-superimpose Thanks, Andrew -- Andrew Orry Ph.D. Senior Research Scientist MolSoft LLC 11199 Sorrento Valley Road San Diego, CA 92121 -- Tel: 858-625-2000 x108 Fax: 858-625-2888 www.molsoft.comhttp://www.molsoft.com www.twitter.com/MolSofthttp://www.twitter.com/MolSoft On 2/5/2015 2:41 PM, Kgosisejo, Oarabile wrote: Hi all, Does anyone know of a program to use to superpose only selected protein residues, e.g. enzyme active site residues. I solved my structure using molecular replacement now I want to compare its active site to those of homologous structures. I have used the DALI server before but I had to upload the whole model. Thank you for your advise Best Regards, Oarabile M. Kgosisejo, MSc. Candidate University of Saskatchewan o.kgosis...@usask.camailto:o.kgosis...@usask.ca
Re: [ccp4bb] Superposition of select residues
Hi Oarabile, Coot allows you to align structures by selected atoms: https://www2.mrc-lmb.cam.ac.uk/Personal/pemsley/coot/docs/coot-faq.html#How-do-I-do-a-superposition-on-just-a-part-of-my-structure_003f I guess what you are trying to do can be accomplished by the “multi-range LSQ superposition” method. Note that the commands shown in the above page are in Guile/Scheme format, which is now not supported in Coot (if I am correct). You need to write the commands in python format like this: superpose_with_atom_selection (0, //B/1-100”, 1, //B/1-100,0) -basically: i)replace all ‘-’with ‘_’in function name, ii) put all parameters in a pair of brackets and iii) separate them with commas. For python formatting see https://www2.mrc-lmb.cam.ac.uk/Personal/pemsley/coot/web/docs/coot.html#Python You can either make a little txt file “scripts.py” with these commands and let Coot run it, or you can type them one by one in Calculate-Scripting-python. Zhijie From: Kgosisejo, Oarabile Sent: Thursday, February 05, 2015 5:41 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Superposition of select residues Hi all, Does anyone know of a program to use to superpose only selected protein residues, e.g. enzyme active site residues. I solved my structure using molecular replacement now I want to compare its active site to those of homologous structures. I have used the DALI server before but I had to upload the whole model. Thank you for your advise Best Regards, Oarabile M. Kgosisejo, MSc. Candidate University of Saskatchewan o.kgosis...@usask.ca
Re: [ccp4bb] off topic pymol
Hi Almudena, Have a look in your PDB file in the second to last colum, if it looks something like the example below then delete the As from the fine and it should connect up. I've had this before, not sure where it came from but removing it fixed the problem. ATOM371 CA PRO A 48 -11.300 -19.315 -35.308 1.00 91.62 C ATOM372 CB PRO A 48 -10.546 -20.584 -35.726 1.00 90.06 C ATOM373 CG PRO A 48 -9.106 -20.254 -35.548 1.00 91.26 C ATOM374 CD PRO A 48 -8.992 -19.119 -34.570 1.00 91.06 C ATOM375 C PRO A 48 -12.669 -19.668 -34.722 1.00 85.38 C ATOM376 O PRO A 48 -13.686 -19.209 -35.228 1.00 79.05 O ATOM377 N ARG A 49 -12.683 -20.461 -33.658 1.00 91.80 AN ATOM378 CA ARG A 49 -13.936 -20.872 -33.022 1.00101.75 AC ATOM379 CB ARG A 49 -13.717 -22.064 -32.077 1.00109.37 AC ATOM380 CG ARG A 49 -15.008 -22.749 -31.637 1.00106.33 AC ATOM381 CD ARG A 49 -14.933 -23.259 -30.207 1.00104.16 AC Hope that helps, Sam From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Almudena Ponce Salvatierra [maps.fa...@gmail.com] Sent: 04 February 2015 17:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic pymol Dear all, does anyone know why if I open a pdb in pymol (that appears normal and fully connected in coot) it appears as if it was broken into pieces? Thanks, Almudena. -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
[ccp4bb] Job Advert
Please see the recent job advert for a position with AstraZeneca in Cambridge. http://jobs.astrazeneca.com/jobs/details/l1rRD1248-senior-scientist-crystallography Chris Phillips Associate Director, Protein Structure, UK _ AstraZeneca RD | Innovative Medicines | Discovery Sciences 50S38, Mereside, Alderley Park, Macclesfield, Cheshire, UK, SK10 4TG T: +44 (0)1625 513316 chris.phill...@astrazeneca.commailto:rick.dav...@astrazeneca.com P Please consider the environment before printing this e-mail AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
Re: [ccp4bb] Protein-Ligand Crystallization
Hi Monica, Your Tm results don't suggest any binding of your ligand to the protein. Your ligand concentrations are quite high so I am not convinced of even weak binding. The destabilization that you are seeing at the highest concentration may be due to ligand precipitation causing protein denaturation/unfolding. If available, I would search for other ligands for co-crystallization. Alternatively, confirm the ligand binding by some other technique before investing a lot of time and protein in crystallization screening. John Sent from my iPad On Feb 5, 2015, at 8:43 AM, Monica Mittal monica.mitta...@gmail.com wrote: Hi all, I am working to crystallize a protein-ligand complex. I did a preliminary melting curve analysis for the protein in the absence and presence of 2 ligands (dissolved in protein buffer). I kept the other controls as buffer an a known standard to confirm instrument performance. All expts done in triplicates. Now the results are like : Tm of protein alone is 56 deg, Tm in the presence of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1, 56.0 and 53.5 respectively !! Tm in the presence of Lig 2 conc. of 0.05mM, 0.1mM, 1.0mM, 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6 respectively !! Although the effective delta Tm for both is different at higher concentration, but both are kind of making protein less stable. So i was wondering, will it be difficult to co-crystallize them !! Any suggestions in this regard are highly appreciated !! Thanks Monica
Re: [ccp4bb] Protein-Ligand Crystallization
Hi Monica, A different option is they both bind but destabilize your protein (as you suggested). This will likely not help much if you are trying to co-crystallize them. But there are couple of examples out there in the literature that show the contrary, despite destabilizing Tm they got structures with bound ligands - I think it was the Beryllium Clan out in Seattle that had such an example but I might be misremembering this. There’s a clear dose-dependency in both ligands, so I would give it a shot (assuming you have enough protein ligands). You could run some ITC or SPR experiments to verify this if you happen to have access to such an instrument. Now if you have an enzymatic assay, that can be tricky if you are looking for inhibition. Yes your ligands may inhibit, great but perhaps your protein is simply destabilized and unfolds etc. and fails to fulfill its normal function - here it would be handy to run some CD spectra to actually se if there is a dose-dependent unfolding of the protein. This will also help you decide how likely it might be to co-crystallize the ligand. Do you have native crystals ? Soak your ligands in and observe if they crack/dissolve. Good luck, Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Feb 5, 2015, at 9:33 PM, John Newitt newit...@gmail.commailto:newit...@gmail.com wrote: Hi Monica, Your Tm results don't suggest any binding of your ligand to the protein. Your ligand concentrations are quite high so I am not convinced of even weak binding. The destabilization that you are seeing at the highest concentration may be due to ligand precipitation causing protein denaturation/unfolding. If available, I would search for other ligands for co-crystallization. Alternatively, confirm the ligand binding by some other technique before investing a lot of time and protein in crystallization screening. John Sent from my iPad On Feb 5, 2015, at 8:43 AM, Monica Mittal monica.mitta...@gmail.commailto:monica.mitta...@gmail.com wrote: Hi all, I am working to crystallize a protein-ligand complex. I did a preliminary melting curve analysis for the protein in the absence and presence of 2 ligands (dissolved in protein buffer). I kept the other controls as buffer an a known standard to confirm instrument performance. All expts done in triplicates. Now the results are like : Tm of protein alone is 56 deg, Tm in the presence of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1, 56.0 and 53.5 respectively !! Tm in the presence of Lig 2 conc. of 0.05mM, 0.1mM, 1.0mM, 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6 respectively !! Although the effective delta Tm for both is different at higher concentration, but both are kind of making protein less stable. So i was wondering, will it be difficult to co-crystallize them !! Any suggestions in this regard are highly appreciated !! Thanks Monica
Re: [ccp4bb] proton scattering by X-rays
Hi Peter, Try to think of it as a quantum chemist: What you call H+ is not H+ floating in space. They are hydrogens bound to the rest of the structure by means of electrons. These electrons can be described by wave functions, which relate to probabilities where they (electrons) might be. If we consider for simplicity water, we learn in a simple model that each O-H bond contains two electrons. On average, they like to be closer to O and than to H. But it does not mean H has none. You need high-quality, high-resolution data to actually visualize this and in small molecule work this is commonly done. In macromolecular work not so much, but my search easily found an example (see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC16211/ for Crambin) where you can see the electrons between N and H in the backbone. The main difference between what you think of as H+ and H- is one and two bonds, respectively. The density in their figures does not look all that different, but says something should be here. We then propose H in the location and show that it adequately explains the experimental data. HTH Mark -Original Message- From: Peter Moody pcem1bigfi...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Mon, Feb 2, 2015 11:33 am Subject: [ccp4bb] proton scattering by X-rays Dear BB I have (again) realised how limited by understanding of our subject is. In Nature’s online site http://www.nature.com/nature/journal/vaop/ncurrent/full/nature14110.html?WT.ec_id=NATURE-20150129 there is a paper describing an X-ray structure determined with sub-atomic data (nice!). The figures show density for H+ as well as H-. In my simple way I had assumed that any X-ray scattering from the nucleus was negligible, and that the electrons are responsible for this. I would expect a proton (i.e. H+) alone to be invisible to X-rays, and certainly not to look similar to a hydride (with two electrons in (electron density) maps. What have I missed? Could someone please explain, or point me to a suitable reference? Best wishes, Peter (please use peter.mo...@le.ac.uk to reply directly) http://www2.le.ac.uk/departments/biochemistry/staff/moody
Re: [ccp4bb] proton scattering by X-rays
Not very well. When you look in the originally quoted article, there is really not much difference in the ED for H+ and H- (which are conveniently both shown in Fig 2). You build a model that is consistent with 'book knowledge' (i.e. normal hydrogen has only one bond) and take it from there. You have a point that the resolution isn't very good at 0.89A (sorry!) but I suspect that the number of recorded reflections is much more important than the nebulous number of 0.89A (i.e. you can start to see these little details because you finally have an over-determined system parameters). What you CAN do in fact is appropriately subtract spherical electron density from the experimental density and see what is left (i.e. directional ED that is 'surplus'). I tried to quickly find a paper on that, they exist, and they show that experimental density does confirm what we learn in chemistry class, orbitals are not imaginary. Mark -Original Message- From: Doug Ohlendorf oh...@umn.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Mon, Feb 2, 2015 11:29 am Subject: Re: [ccp4bb] proton scattering by X-rays But, how with x-rays can one experimentally tell the difference between a hydrogen and a filled orbital (say of N)? I will grant that the electron density for a bound H should extend farther from the heavy atom but I believe you would need resolution better than 0.89 Ang to see this difference. Doug Douglas H. Ohlendorf Phone: 612-624-8436 Professor FAX:612-624-5121 Dept. of Biochemistry, Molecular Biology Biophysics Twin Cities Campus, University of Minnesota Lab web site: http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Colin Nave Sent: Monday, February 02, 2015 9:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] proton scattering by X-rays “As you say the proton itself is invisible to X-rays.” Not quite! The ratio of scattering between electrons and protons should go as the inverse square of the masses. Ratio of mass 1:1860, ratio of scattering 1:3459600. A small correction but doubtless it has been incorporated in to SHELX. Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ian Tickle Sent: 02 February 2015 13:35 To: ccp4bb Subject: Re: [ccp4bb] proton scattering by X-rays Peter, if it's a covalently-bonded H atom it surely can't be a bare proton, it must have at least some partial electron around it for the (possibly partial) covalent bond, enough to diffract X-rays anyway. As you say the proton itself is invisible to X-rays. Cheers -- Ian On 2 February 2015 at 13:08, Peter Moody pcem1bigfi...@gmail.commailto:pcem1bigfi...@gmail.com wrote: Dear BB I have (again) realised how limited by understanding of our subject is. In Nature’s online site http://www.nature.com/nature/journal/vaop/ncurrent/full/nature14110.html?WT.ec_id=NATURE-20150129 there is a paper describing an X-ray structure determined with sub-atomic data (nice!). The figures show density for H+ as well as H-. In my simple way I had assumed that any X-ray scattering from the nucleus was negligible, and that the electrons are responsible for this. I would expect a proton (i.e. H+) alone to be invisible to X-rays, and certainly not to look similar to a hydride (with two electrons in (electron density) maps. What have I missed? Could someone please explain, or point me to a suitable reference? Best wishes, Peter (please use peter.mo...@le.ac.ukmailto:peter.mo...@le.ac.uk to reply directly) http://www2.le.ac.uk/departments/biochemistry/staff/moody -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
