Hi Vincent, To calculate the best possible (self)rotation function, you want to have as many as possible intramolecular vectors (within the search molecule) and as little as possible intramolecular vectors (between molecules in the crystal). These latter vectors are meaningless (noise) as long as the orientation of your search molecule has not been found and you are not calculating a translation function.
Even with a small Patterson radius, you will get some intramolecular vectors near crystal contacts, but not many. With a large Patterson radius, you will get many intramolecular vectors which may obscure your rotation function solution. So in difficult cases, you have to find the optimal radius: if it is too small, you won’t get much signal, if the radius is too large, you will get too much noise and you won’t see the correct solution either. The number of intramolecular vectors you get depend on the crystal packing and the shape of your molecule and there are no universally applicable hard rules. In your case it might be a good idea to run the (self)rotation function with different radii, too see which radius gives the best signal to noise ratio. Hope this explains things a little, Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von vincent Chaptal Gesendet: Freitag, 2. Juni 2017 12:05 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] help needed to interpret a SRF Hi Michael, thank you for your explanations. On 01/06/2017 15:41, R. Michael Garavito wrote: Vincent, I see a few of problems with your SRF (the maps) which would impact the interpretation. First you say that both crystals are processed as P21, which you would expect a very strong peak (100% of your origin peak) on kappa/chi=180 at the b* axis (one of the major axes of your map); this arises from the crystallographic symmetry. Where this axis is depends on what the conventions are of the program you are using. Your SRF has a major peak along Z, but is that the b* axis? (My b* axis is always placed North-South or your X, á la the Rossmann conventions. I don’t know the conventions for Molrep) Then you see major off-axial peaks. The sad thing about your off-axial peaks is that they are badly split. The datasets were processed by XDS in primitive monoclinic P2 (space group 3), the reindexed to P21 by Aimless as the most probable space group. I just double checked that it is indeed P21, you made me doubt. I tried to look for the conventions for Molrep but couldn't find them, I'm sorry. Following your 3rd comment bellow, I reproduced the maps with a radius of 30A as you suggested (see attached maps). I guess I can see the same features on both maps, with a peak along Y that would correspond to the 2 fold axis (if the convention is turned 90° compared to yours), and 2 off origin peaks along X. The maps from the crystal without additive is an awful mess. I should add that my monomer has an internal 2fold (pseudo)symmetry. Could the two main peaks mean that I have 2 monomers, each with his own 2 fold symmetry? I'm puzzled by this radius of 30A as I read in the documentation that is corresponds to the approximate radius of the protein. My protein is about 130Ax40A in the closed form to 130Ax70A in the open form according to other structures available. Shouldn't I compute maps with a radius of 70A or more? I'm not posting here these results here as they look more "artsy". All the best Vincent The second problem is that the second crystal shows very strong peak (1001% of your origin peak) on kappa/chi=180, but at different position (along Y). The control peak is the expected peak which arises from the P21 crystallographic symmetry. If the data sets were processed as P21, they should be indexed with the 2-fold axis along b/b*, thus the control peak is the expected peak which arises from the P21 crystallographic symmetry. Once that peak appears in the same place on each map, you can then compare the maps with more confidence. The third problem is that all your maps have the “appearance" of mirror symmetry across the x-axis, which would be expected as it is the consequence of the P2(1) symmetry and you are looking at a hemisphere. But it suggests that the 2-fold axis is along Y (b/b*). Then map 1 is missing the expected peak on kappa/chi=180 at the b* axis. Final comment is that the maps labeling suggest that the radius of integration is 62-66 Å, which is way, way too large, even for an empty unit cell. If it is, reduce it down to 20-30 Å to avoid intermolecular cross-peaks and see if the maps become clearer. I have attached a SRF map from a P21 crystal form (radius of integration = 20 Å, resol. = 2.8 Å) with the 2-fold axis indexed along b/b* (Y) from polarrfn; note the “appearance" of mirror symmetry perpendicular to across the b/b*-axis (North-South or y-axis). Cheers, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com<mailto:garav...@gmail.com> **************************************************************** On Jun 1, 2017, at 4:51 AM, vincent Chaptal <vincent.chap...@ibcp.fr<mailto:vincent.chap...@ibcp.fr>> wrote: Dear Manfred, attached are the postcript files. Vincent On 01/06/2017 10:19, Manfred S. Weiss wrote: Dear Vincent, it would be good if you post the postscript file as well. It is called molrep_rf.ps or something like that. Cheers, Manfred Am 01.06.2017 um 10:12 schrieb vincent Chaptal: Thank you for your email. the anisotropic resolutions of the datasets are 5.6-7.1A for the best and worst diffracting directions of the crystal without additive, and 4.0-5.8A for the crystal with additive. The two crystals come from the same prep and same drop setup, only differ from the presence of the additive during crystallogenesis. They are indeed two different crystals, I would be curious to know more how to compare these two datasets together as I thought it was not possible with such different cell parameters. On 31/05/2017 17:12, Eleanor Dodson wrote: Well - you dont give details o f resolution but the sovent content and the peak of 0.51 would suggest a possible dimer in crystal 1 Crystal 2 is so different it might well have a dimer in a different orientation. Yes, it could very well be the case. But wouldn't there be a peak as well in the SRF? I would try to see if there was a relationship between Xtal 1 and Xtal 2 - can tell you how I would do that if you are interested.. But doesnt mass spec or some such technique suggest whether there is a dimer or not? I am not aware of a way to test by Mass Spec or other techniques the content of the ASU, I would be very interested if anyone can further my knowledge on this. All the best Vincent Eleanor Self Rotation Functions are a) hard to interpret and b) often misleading! On 31 May 2017 at 14:18, vincent Chaptal <vincent.chap...@ibcp.fr<mailto:vincent.chap...@ibcp.fr>> wrote: Dear all, I need help interpreting results from a SRF; I am very naïve at interpreting them and would appreciate any pointer... I have 2 crystals, before and after additive during crystallogenesis. They have different cell parameters, and I am wondering if I have a monomer or a dimer in the ASU, and if the additive changed this. crystal w/o additive: P21 114,5 107,6 134,6 beta=95,74 1monomer in the ASU = 80% solvent, 1 dimer in the ASU = 61% solvent. Note that this high solvent content agrees well with the fact that this is a membrane protein, and diffracts both to low resolution and very anisotropic... SRF from Molrep: +------------------------------------------+ | theta phi chi P(i)/P(0)| +------------------------------------------+ | 1 0.00 0.00 0.00 1.00 | | 2 148.48 0.00 180.00 0.51 | | 3 161.29 0.00 180.00 0.31 | | 4 105.34 180.00 180.00 0.30 | | 5 74.78 -56.26 179.58 0.27 | | 6 15.14 -163.78 180.00 0.25 | | 7 67.61 -40.16 180.00 0.24 | | 8 134.54 -180.00 180.00 0.19 | | 9 72.33 -37.82 180.00 0.18 | | 10 69.72 38.92 175.62 0.18 | | 11 110.28 -141.08 175.62 0.18 | | 12 96.58 101.90 179.79 0.18 | | 13 67.04 -15.02 179.71 0.18 | | 14 62.76 -15.52 179.45 0.17 | | 15 117.26 164.48 179.45 0.17 | | 16 68.39 -18.92 179.86 0.17 | | 17 70.52 -16.51 180.00 0.17 | | 18 24.05 -162.02 179.98 0.17 | | 19 78.61 -36.36 180.00 0.17 | | 20 83.17 78.42 173.34 0.16 | | 21 96.83 -101.58 173.34 0.16 | | 22 81.25 -75.03 179.73 0.16 | | 23 81.81 -17.73 180.00 0.16 | | 24 11.02 -142.22 180.00 0.16 | | 25 76.14 -16.64 179.71 0.16 | | 26 76.56 -16.93 180.00 0.16 | | 27 162.29 32.44 180.00 0.16 | | 28 10.03 -136.82 180.00 0.15 | | 29 68.67 -58.10 179.49 0.15 | | 30 111.33 121.90 179.49 0.15 | | 31 62.99 -20.19 180.00 0.14 | | 32 97.24 145.00 179.78 0.14 | | 33 99.56 165.86 179.74 0.14 | | 34 84.27 -82.68 180.00 0.14 | | 35 84.51 72.76 174.42 0.13 | +------------------------------------------+ Crystal with additive: P21 CELL 117.2840 110.3330 155.6880 90.0000 93.4280 90.0000 1 monomer in the ASU = 84% solvent, 1 dimer = 68% solvent. SRF from Molrep: +------------------------------------------+ | theta phi chi P(i)/P(0)| +------------------------------------------+ | 1 0.00 0.00 0.00 1.00 | | 2 59.55 0.00 180.00 0.36 | | 3 156.11 0.00 180.00 0.29 | | 4 10.00 -165.58 180.00 0.26 | | 5 110.22 -180.00 180.00 0.26 | | 6 12.97 -169.15 180.00 0.23 | | 7 166.21 -9.06 180.00 0.22 | | 8 81.64 -9.61 179.74 0.21 | | 9 86.02 80.72 178.22 0.20 | | 10 93.98 -99.28 178.22 0.20 | | 11 5.96 -54.00 180.00 0.19 | | 12 67.40 -10.81 180.00 0.19 | | 13 87.10 -94.56 179.91 0.19 | | 14 147.13 47.70 10.71 0.19 | | 15 147.13 -47.70 10.71 0.19 | | 16 87.45 91.04 155.74 0.19 | | 17 92.55 -88.96 155.74 0.19 | | 18 5.33 -63.00 180.00 0.19 | | 19 104.60 169.61 179.59 0.19 | | 20 88.01 -85.32 179.05 0.18 | | 21 92.02 94.64 179.68 0.18 | | 22 87.61 91.31 153.16 0.18 | | 23 92.39 -88.69 153.16 0.18 | | 24 6.65 -46.80 180.00 0.18 | | 25 153.26 36.01 21.16 0.18 | | 26 153.26 -36.01 21.16 0.18 | | 27 63.11 -11.30 179.51 0.18 | | 28 76.82 -5.09 179.61 0.18 | | 29 87.69 91.37 148.72 0.18 | | 30 92.31 -88.63 148.72 0.18 | | 31 86.74 -9.44 179.90 0.17 | | 32 85.83 80.82 148.49 0.17 | | 33 94.17 -99.18 148.49 0.17 | | 34 175.25 57.61 180.00 0.17 | | 35 4.63 -90.00 180.00 0.17 | +------------------------------------------+ Can I say that the peak at 0.51 in the crystal without additive is significant, concluding that there is probably a dimer in the ASU in that crystal, in contrast to the crystal with additive where no peak stands out, which would lean towards a monomer in the ASU? Thank you for your help. Best Vincent -- Vincent Chaptal, PhD Institut de Biologie et Chimie des Protéines Drug Resistance and Membrane Proteins Laboratory 7 passage du Vercors 69007 LYON FRANCE +33 4 37 65 29 01<tel:+33%204%2037%2065%2029%2001> http://www.ibcp.fr<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibcp.fr_&d=DQMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=HRnlpRSDU2rjBs8ME1uAPcTPFo8iHmIhHxCcHjlgR0s&s=wMDi6x5DZBrJBwz59jhEnBIra67xjlP7phX_hz07ltI&e=> -- Vincent Chaptal, PhD Institut de Biologie et Chimie des Protéines Drug Resistance and Membrane Proteins Laboratory 7 passage du Vercors 69007 LYON FRANCE +33 4 37 65 29 01 http://www.ibcp.fr<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibcp.fr_&d=DQMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=HRnlpRSDU2rjBs8ME1uAPcTPFo8iHmIhHxCcHjlgR0s&s=wMDi6x5DZBrJBwz59jhEnBIra67xjlP7phX_hz07ltI&e=> -- Dr. Manfred S. Weiss Macromolecular Crystallography Helmholtz-Zentrum Berlin Albert-Einstein-Str. 15 D-12489 Berlin Germany ________________________________ Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V. Aufsichtsrat: Vorsitzender Dr. Karl Eugen Huthmacher, stv. Vorsitzende Dr. Jutta Koch-Unterseher Geschäftsführung: Prof. Dr. Bernd Rech (kommissarisch), Thomas Frederking Sitz Berlin, AG Charlottenburg, 89 HRB 5583 Postadresse: Hahn-Meitner-Platz 1 D-14109 Berlin http://www.helmholtz-berlin.de<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.helmholtz-2Dberlin.de_&d=DQMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=HRnlpRSDU2rjBs8ME1uAPcTPFo8iHmIhHxCcHjlgR0s&s=kWLIM_uoWVVXgLhWYk6BibNL_U-tQzcaT--8ja8Icts&e=> -- Vincent Chaptal, PhD Institut de Biologie et Chimie des Protéines Drug Resistance and Membrane Proteins Laboratory 7 passage du Vercors 69007 LYON FRANCE +33 4 37 65 29 01 http://www.ibcp.fr<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibcp.fr_&d=DQMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=HRnlpRSDU2rjBs8ME1uAPcTPFo8iHmIhHxCcHjlgR0s&s=wMDi6x5DZBrJBwz59jhEnBIra67xjlP7phX_hz07ltI&e=> <crystal-with-additive_rf.pdf><crystal-without-additive_rf.pdf> -- Vincent Chaptal, PhD Institut de Biologie et Chimie des Protéines Drug Resistance and Membrane Proteins Laboratory 7 passage du Vercors 69007 LYON FRANCE +33 4 37 65 29 01 http://www.ibcp.fr<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibcp.fr&d=DQMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=HRnlpRSDU2rjBs8ME1uAPcTPFo8iHmIhHxCcHjlgR0s&s=jjzLhbxU8fxh2hQhlNOQ7cOin1PZc4e2KTa_KP_PUZg&e=>
