Dear All, Sorry for the late reply. I was out of town in last two weeks.
Again, thanks for your kindly comments, and here are the summary for the additional comments and my responses: 1. Summary: The high value of the R-merge might be due to the wrong orientation matrix (beam center), anisotropic, potential sample vibration, or radiation damage. The suggestions are checking the orientation matrix and beam position, and use pointless "centre" option in XDS to correct the center, use only partially frames to do integration, or avoid sample vibration. 2. Responses to the comments: (1). Dr. Klaus Futterer suspected the data has large variation during the lattice parameters refinement or orientation matrix coefficients during integration, and suggested to determine the orientation matrix in XDS with all frames or only part (10-20%) of the frames with clear diffraction spots, and then integrate the frames without floating the lattice parameters. Response: Thanks for the suggestion, I will work on this soon and see what is going to happen. (2). Dr. Eric Montemayor suspected the data is anisotropic, and suggested to submit the data to the staraniso server. Response: I submit the data to the anisotropic server ( http://services.mbi.ucla.edu/anisoscale/), and you can see in the attachment that my data only has low and mild anisotropy. [image: anisotropy.png] (3). Dr. James Holton suspected that current results could be caused by sample vibration due to particular crystal and loop shape (reference: J. Appl. Cryst., 2008, 41, 1122). (I) Ways to diagnose sample vibration: (a) Look at the highest intensity bin to see Rmerge for the brightest spots; (b) Take a series of "stills" or at least the same narrow rotation about 3-10 times in a row from the same crystal with the same starting phi each time, integrate the images as usual, and look at the variation (Rmerge) for each hkl individually. Then plot the variation vs. location on the detector. If we see swaths of reciprocal space with high variation while others low, and the orientation of the swaths is not related to the phi axis, then we know we've got vibration issues. (c) Look at other data sets from the same instrument. If the low-angle Rmerge in P1 of the dataset stands out, then it was probably sample vibration. (II) Ways to solve this issue (a) adjust the flow rate of the cold stream; (b) shore-up the mounts; (c) coat the twisty loop neck with epoxy before mounting the crystal; (d) use loop and base (curved) from MiTeGen (MiTeGen must pay the advertising fee!). (III) If Rmerge is high like I am seeing here I should definitely inform whomever is maintaining the instrument I used that there is a problem. Response: Thanks for the suggestion. Actually, I have discussed with the beamline stuff to argue this potential issue. However, (a) after collecting this set of data, I do collect data from other crystals grown from different protein, and the R-merge looks fine. (b) I have collected four sets of data again four months later, and all of them show similar high R-merge value. The stuff thought the high R-merge value of the crystals from only this type of protein should not due to the hardware of the synchrotron nor the loop itself. (4). Dr. Thomas Cleveland mentioned that he had a similar situation before, and this could be caused by a misindexing of the diffraction origin by +/- 1 due to a slightly incorrect beam center. He suggested to use the pointless "centre" option in XDS to do a search for the correct center just like the example at https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Pointless, and then use the INDEX_ORIGIN parameter to specify the correct origin, and reintegrate the data. Response: Thanks for the suggestion, I will work on this soon and see what is going to happen. (5). Dr. Kajander, Tommi A asked that if I can see R-merge difference if I only process a sector from different place or lower the given symmetry for the redundancy issue, and if I have tried XSCALE after XDS and crystal name for correction to look at low resolution binds. He suggested that I need to check the direct beam position as well as the radiation damage. Response: I do the integration under P1 or only scale part of the frame, but the R-merge increased to over 45% very soon. Regarding to the radiation damage issue, I do see high radiation damage. However, due to the weak diffraction, I have to increase the exposure time to get the resolution. Anyway, I will try to use XDS to correct the beam position and use partial frames to redo the integration. Thank you for your kindly help again, and I will update once I process the data by using XDS to correct the beam position later! Best, Liang Kajander, Tommi A <tommi.kajan...@helsinki.fi> 于2018年10月7日周日 上午3:42写道： > on the redundancy issue - if you lower it given high symmetry or process > only a sector from different place - no difference? > > i think one suggestion was misindexing by 1 in some direction - did you > check your direct beam is correct? > > and did you check radiation damage actually per frame does the > diffraction seem to fade out towards the end (if you have high redundancy > just try with less)? > > There is something systematically wrong when you get such a high low res > Rmerge - i would think - amazing that you can refine it that well….. > > if you look at the really low resoution (below its 50-6 in the first > bin..) what does it look like below 10 Å resolution - also did you try > XSCALE after XDS and > crystal_name for correction… at least you can look at low resolution binds > in more detal if you run it through there… though i am not the specialist > to talk to > on what XDS can do for corrections…. > > > Best, > Tommi > > > On 29 Sep 2018, at 10:58, Zhang Foggy <foggyc...@gmail.com > <foggyc...@gmail.com>> wrote: > > Dear All, > > Thanks for your kindly comments. Here are the summary and my responses: > > 1.Summary: > > The high value of the R-merge might be due to the weak diffraction, as > well as the collection method (low dose per image and high redundancy). > The suggetions is ignore the R-merge value, and condider the Rpim, CC1/2 > and I/sigI value instead. > > 2. Responses to the comments: > > (1) Dr. Herman Schreuder suggested that I can use ADXV to look through my > data and see if there is some bad regions, overload (scale only the first > 30-40 frames) or ice rings, and I also can use other software to scale the > data (xds, mosfilm etc.) > Response: Actually I have tried these alternative ways. Indeed, the > diffraction of my crystals is pretty good (you can see the image from > attached jpg file), there is no ice rings, no significant radiation damage, > no bad regions through the entire frames. I have also tried to use xds to > scale it, unfortunately, the R-merge is still high (~50%). Additionally, I > also tried to only scale part of the frames, however, the R--merge is to > ~48%. > > (2) Dr. Shepard William suggested to try mosfilm or xds, and asked the > multiplicity of the data. > Response: I have tried to scale with XDS, but there is no improvement. > The space group is P43. I can refine the structure to R-value 0.19, R-free > 0.23, indicating that the space group should be correct. I have also tried > to scale to P21 or P1, and there is no improve in R-merge. > > (3) Dr. Phil Evans mentioned that Rmerge is a terrible criterion (Science, > 2012, 336,1030), and CC(1/2) should be generally considered as the best > criterion. In my case, both of the Rmerge (1.59) and CC(1/2) (0.645) in > the outer shell are acceptable. However, the Rmerge (0.284) and CC(1/2) > (0.975) in the inner shell looks not perfect. I should consider the > radiation damage. > Response: Thanks a lot for the comments. As you can see from the attached > figure, the diffraction is sharp, and I do not see any significant > radiation damage > > (4) Dr. Ditlev Egeskov Brodersen suggested to double check the space group > and process part of the data. > Response: as I mentioned in (1) and (2), I have tried to only scale part > of the frames, however, the R--merge is to ~48%; I can refine the > structure to R-value 0.19, R-free 0.23 under the current space group P43. > Moreover, scale to P21 or P1,can not improve the R-merge significantly. > > (5) Dr. Remy Loris mentioned that a high value of R-merge indicates a > wrong symmetry or very weak data. from my data, the reason could be the > weak data as well as high redundancy. > Response: I agree. from the attached image, I can see the the diffraction > is sharp but weak. However, increse the exposure time will introduce more > radiation damage.... > > (6) Dr. Edward A. Berry mentioned that my data has rather high redundancy > as Rpim is much lower than Rmeas value. It could be caused by collecting > low dose per image and making up for it with high redundancy, Dr. Edward A. > Berry suggeted to Look instead at CC1/2 and I/sigI which seem fine. > Response: Thanks for the comments, and I agree. > > (7). Dr. Rajesh Kumar raj suggested me to consider Rpim, CC1/2 and I/sigI > for cutting the data as Rmerge is old approach and it is data redundancy > dependent. > > Thank you for your kindly help again! > > Best, > > Liang > <Diffraction image.jpg> > > > > Rajesh Kumar <rajesh.p...@gmail.com> 于2018年9月28日周五 下午11:41写道： > >> I totally agree with Berry. Please consider Rpim, CC1/2 and I/sigI for >> cutting the data. Rmerge is old approach as it is data redundancy dependent. >> >> Thank you >> Rajesh >> >> ---xxxxx---- >> With regards >> Rajesh K. Harijan, Ph.D. >> Schramm Laboratory >> Albert Einstein College of Medicine >> 1300 Morris Park Ave., Bronx, NY 10461 >> Tel: 718.430.2777 | Fax: 718.430.8565 >> >> >> On Fri, Sep 28, 2018 at 11:32 AM Edward A. Berry <ber...@upstate.edu> >> wrote: >> >>> The fact that chi^2 is approximately 1.0 in all shells says that the >>> deviations are about what is expected from the error model. The fact that >>> Rpim is much lower than Rmeas means that you have rather high redundancy. >>> This would seem to be a case of collecting low dose per image and making up >>> for it with high redundancy, a strategy that has been recommended to ensure >>> a full dataset even in the case of high radiation sensitivity. In my >>> opinion the high Rmerge is nothing to worry about. Look instead at CC1/2 >>> and I/sigI which seem fine. >>> >>> On 09/28/2018 04:09 AM, Zhang Foggy wrote: >>> > Dear All, >>> > >>> > Sorry for the off-topic. >>> > >>> > I recently collected a set of data. The diffraction spots are >>> extremely sharp. However, When I used HKL3000 to scale it, I get a final >>> resolution at 3.1A with overall R-merge ~0.54 (R-merge in the highest >>> 3.2A-3.1A shell: 1.59). Then I solve the structure with final R value 0.19 >>> and R free value 0.24 although I know this Rmerge value is totally >>> unacceptable, and the density looks perfect. >>> > >>> > I also tried to collect other four set of data with different >>> crystals. unfortunately, all of them have same problem. >>> > >>> > I ask one of my friend who is an expert in HKL3000, but he had no idea >>> about it. Does anyone has suggestions? >>> > >>> > Here is the scale information for your review: >>> > Space group: P43 (I also tried P1, the Rmerge value is still similar) >>> > >>> > Shell Lower Upper Average Average Norm. Linear Square >>> > limit Angstrom I error stat. Chi**2 R-fac R-fac >>> Rmeas Rpim CC1/2 CC* >>> > 50.00 6.67 11.6 0.9 0.3 1.165 0.191 0.284 >>> 0.198 0.052 0.975 0.994 >>> > 6.67 5.30 4.5 0.5 0.3 0.952 0.317 0.313 >>> 0.329 0.086 0.971 0.993 >>> > 5.30 4.63 7.3 0.7 0.5 0.961 0.293 0.297 >>> 0.304 0.081 0.975 0.994 >>> > 4.63 4.21 7.0 0.8 0.6 0.986 0.369 0.358 >>> 0.382 0.101 0.969 0.992 >>> > 4.21 3.91 5.6 0.8 0.6 1.040 0.522 0.491 >>> 0.541 0.142 0.955 0.988 >>> > 3.91 3.68 4.6 0.9 0.7 1.064 0.718 0.669 >>> 0.746 0.203 0.929 0.981 >>> > 3.68 3.49 3.5 0.9 0.8 1.092 1.059 0.986 >>> 1.101 0.299 0.882 0.968 >>> > 3.49 3.34 2.6 0.9 0.8 1.092 1.382 1.298 >>> 1.438 0.395 0.829 0.952 >>> > 3.34 3.21 2.1 0.9 0.8 1.084 1.543 1.489 >>> 1.614 0.468 0.772 0.933 >>> > 3.21 3.10 1.6 0.9 0.8 1.070 1.591 1.669 >>> 1.680 0.529 0.645 0.885 >>> > All reflections 5.0 0.8 0.6 1.048 0.538 0.487 >>> 0.559 0.153 >>> > >>> > Thank you. >>> > >>> > Liang >>> > >>> > >>> > >>> ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ >>> > >>> > To unsubscribe from the CCP4BB list, click the following link: >>> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >>> > >>> >>> ######################################################################## >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >>> >> >> ------------------------------ >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >> > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > Tommi Kajander, Ph.D., PI > Structural Biology and Biophysics > Institute of Biotechnology > University of Helsinki > Viikinkaari 1 (P.O. Box 65) > 00014 Helsinki, Finland > p. +358-2-941-58904 / +358-050-4480991 > tommi.kajan...@helsinki.fi > http://www.biocenter.helsinki.fi/bi/kajander/ > > > > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
