Re: [ccp4bb] SeMet data
Hi Lindsey, I do agree with the others on improving your experimental strategy to enhance the anomalous signal in your data, but did you try to solve the structure with the data you already have? I have seen cases where there is only a pathetic anomalous signal from fully Se-Met incorporated crystals in any experimental setting and those could eventually be solved. You probably have to deal with a significant number of false positives in your substructure but if the anomalous signal is observed accurately you are quite likely able to solve the structure with weak data alone. How much anomalous signal did you get out of those frames and how much did you expect to get? Have you tried merging data from several crystals collected at the same wavelength ? Best, Stefan To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] align structure in Pymol based on dsDNA helix
Dear Xiao Lei, You might want to consider UCSF Chimera for this task https://www.cgl.ucsf.edu/chimera/docs/ContributedSoftware/matchalign/matchalign.html Best, Stefan
Re: [ccp4bb] Interesting DNA contamination
Pramod, You already got good suggestions on how to handle DNA contamination in protein preparations. Let me point out briefly that you haven't demonstrated yet that your contamination is DNA. I had the same observation when purifying UvsX. A very persistent and strong contamination in all my preps at ~500kb. To test weather it was DNA or RNA I boiled the protein 30 minutes and incubated it with DNAase and RNAse and result was the same. I concluded it was neither RNA nor DNA and continued as if nothing had happened. This publication is reporting the same observation: Formosa and Alberts (1986) Purification and characterization of the T4 bacteriophage uvsX protein. J Biol Chem. 1986 May 5;261(13):6107-18. If you ever find out what it is that runs like 500kb DNA on Agarose, please let me know. S.
Re: [ccp4bb] Interesting DNA contamination
Correction, I meant to say 0.5kb, not 500kb sorry for that. S.
Re: [ccp4bb] experimental phasing at low resolution
Hi, I doubt you have a TaBr cluster bound in your soaks. You'd expect a strong anomalous signal from cluster compounds (that's what they are made for) and you can easily get a low resolution anomalous signal from a few dislocated HA in the solvent channels, especially if you don't counter soak the crystals (expose them to HA free buffer briefly prior to freezing to leech the solvent channels from excess HA). I wouldn't collect MAD data on cluster compound soaks anyway. If they are present, you should be able to solve it with SAD. Alternatively, try the LIII and LII edges at the respective peak wavelength instead of collecting data at wavelength around the same edge. I don't think that's your problem because I don't think the TaBr bound at all in your crystals. In your case, I'd recommend trying a different HA soak first. If you cannot express your protein with selenium but you can get your hands on xenon derivatization equipment, try xenon!! Mercury is always worth a try IMHO. Non-isomorphous data from various soaks is not very helpful, that single SAD dataset with that little bit of extra signal IS very helpful. Optimize your cryo protectant and data collection strategy (inverse wedges, kappa goni, etc.). If you want to try MAD, try collecting all wavelength data from the same crystal. Don't do MAD if you have radiation sensitive non-isomorphous crystals. An non interpretable MR solution will help you locate the HA sites as long it is correct. So, if you have one, try to use it to phase an anomalous difference map. You indicated that you have several datasets from two compounds. Try merging the 'identical' datasets and see if that works better. SHARP is really good for MAD data, but you need to collect and process the data right, which is difficult. SHELX is great for low resolution/low signal SAD/SIR. I'd always use SHELX first because it was the only thing working for me below 5A so far (sample size=1, anecdotal evidence, handle with care!). good luck, S.
[ccp4bb] A crystal symmetry broken beyond repair?
I have three brief questions (number two should be the easy one).
1. Where could an extra electron density of an (physically impossible)
additional molecule in an otherwise perfect map come from? (even in a wrong
solution in a {probably} too high space group?)
2. I would like to see how my data looks like in a RvR plot. How can I make
one? (Acta Cryst.D62, 83-95.)
3. Is it possible to have an order/disorder crystal defect with a pure
rotational disorder, e.g. the unit cell translations are not affected by
the disorder, only the orientations of the molecules in separate layers?
Stefan
[ccp4bb] Triclinic solution for a dataset with pseudo-translational symmetry, possible pseudo-centering and possible pseudo-merohedral twinning. How to proceed?
Dear experts, This is a messy question concerning a messy dataset and I try to put it correctly. I have a severely anisotropic dataset up to 2.3A that merges quite fine in all 4/mmm. Automatic indexing is primitive tetragonal but I can push it to a centered tetragonal cell if I want to. The crystals and the diffraction images are highly reproducible. There is one non-origin peak of ~45% origin peak height with vector 0.5 0.5 0.46. Intensity statistics do not indicate twinning. Now I have a good MR solution with a 99% model in P1 that reveals four proteins in the cell. Dimer A,C and Dimer B,D with pseudo-translation A to B and C to D and a twofold between A,C and B,D. The twofold lies almost perfectly within a=b and B,C mimic a center at half c. Each protein (A,B,C,D) is a pseudo-octameric homo-oligomer. It is also still possible that the orientation of the multimers is off by one rotation in my MR solution and there actually exists a higher symmetry, although there are no convincing MR solutions in C2, P2, P2(1), yet. Initial refinement in P1 stalled at R=0.35 and R-free 0.40. There are seven possible pseudo-merohedral twin laws and all of them decrease the R-factors to R~0.30 and R-free ~0.35 in refinement, so I can't tell which one is the correct one or if it is twinned at all. What should I try next? Is there a good advice how to refine such a type of structure? How can I determine if it is twinned or not ? Should I omit anisotropic scaling in this case and cut back the resolution to preserve the weak reflections on the original scale? feeling a little lost here with all the pseudo symmetries , Stefan Gajewski
Re: [ccp4bb] Resolution, R factors and data quality
Jim, This is coming from someone who just got enlightened a few weeks ago on resolution cut-offs. I am asked often: What value of CC1/2 should I cut my resolution at? The KD paper mentioned that the CC(1/2) criterion loses its significance at ~9 according to student test. I doubt that this can be a generally true guideline for a resolution cut-off. The structures I am doing right now were cut off at ~20 to ~80 CC(1/2) You probably do not want to do the same mistake again, we all made before, when cutting resolution based on Rmerge/Rmeas, do you? What should I tell my students? I've got a course coming up and I am sure they will ask me again. This is actually the more valuable insight I got from the KD paper. You don't use the CC(1/2) as an absolute indicator but rather as an suggestion. The resolution limit is determined by the refinement, not by the data processing. I think I will handle my data in future as follows: Bins with CC(1/2) less than 9 should be initially excluded. The structure is then refined against all reflections in the file and only those bins that add information to the map/structure are kept in the final rounds. In most cases this will probably be more than CC(1/2) 25. If the last shell (CC~9) still adds information to the model, process the images again, e.g. till CC(1/2) drops to 0, and see if some more useful information is in there. You could also go ahead and use CC(1/2) 0 as initial cut-off, but I think that will rather increase computation time than help your structure in most cases. So yes, I would feel comfortable with giving true resolution limits based on the refinement of the model, and not based on any number derived from data processing. In the end, you can always say I tried it and this was the highest resolution I could model vs. I cut at _numerical value X of this parameter_ because everybody else does so.
Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ?
Hi Mahesh, First of all, I risk going out on a limb here since I have no demonstrable experience concerning your topic. I was trying to solve a non-merohedral dataset for years, and I failed (like failure as defined in the dictionary). That is hardly a reference, but I got to read a little on the topic and maybe I got some comments that can be useful for you. So, if you data would be twinned by non-merohedry, you would not have a valid solution by now. You would have only a fraction of the reflections indexed and a significant fraction in that index would be overlaps. The others would be partial overlaps/splits and the rest fully split reflections (singlets). Solving a non-merohedrally twinned structure requires developing an independent crystal rotation matrix for each twin domain, integrating in the correct point group, leaving all related reflections unmerged and with the original index, solving the structure and identifying the twin law (the one that describes the superposition of one twin domain on top of the other one) during structure solution/refinement. According to my knowledge, this has not been done with a novel structure as of now. (As for anything I say here, someone please educate me if I am wrong) The really mean part with non-merohedral data is, that you cannot even index the data properly, although a variety of indexing choices will give you reasonably well integration/merging statistics and you might not notice it in the beginning. It is very hard to identify the correct cell. A rough guideline is, that in many cases, crystals twinned by non-merohedry display a relationship of a~2b (2a~b, depending on choice) so you can try to double the axis for either a or b in a tetragonal cell and see if your predictions for a primitive cell with those dimensions overlap better with your reflections. The true space group of the lattice will emerge at a later point of time. The best way to do the indexing is supposedly using programs for small-molecule crystallography. When (=if) you have the two matrices, you can forward them to your protein x-ray crystallography software and integrate twice using one matrix at a time. A much better way to deal with non-merohedry is to make different crystals. Or to optimize the cryo. If your data is twinned by pseudo-merohedry, most of the reflections will be overlaps with a very few splits, rather in higher resolution bins than in lower ones. In case your data is really twined, I suspect that is what you are dealing with here by looking at the diffraction patterns you showed before. This means you can solve your structure by integrating your data with only one crystal rotation matrix. However, your data might appear to be not only in a higher point group but also in a higher lattice system than it actually is. Your data then emulates a P422 but your structure does not generate the crystal by that symmetry relationship. Since there is no twin law for P422, phenix.xtriage can not help you there with merged data. Treating twinning by pseudo-merohedry is pretty much straight forward. You must process your data in the correct point group and later identify and apply the twin law. Start with P1 and work up if you have enough frames. Also, if you have more than one molecule in the ASU, you might have simply overestimated the symmetry. I have seen this once in a monoclinic cell with two copies in the ASU where one angle was ~90.08, but not 90.00. It still merged reasonably well as an orthorombic cell, but it was not twined. In either case, try processing your images in a lower point group first, and look carefully at your data. Did your predicted reflections cover all the observed reflections? Did you predict reflections (entire lunes?) that are not there? How does your native Patterson map look like if you process your data in P1? good luck, s.
Re: [ccp4bb] Where to cut the data in this medium resolution dataset
Nat, What do correct B-factors look like? What refinement strategy did you use for them? 1) If I see strong positive density in the Fo-Fc map along the backbone of two turns of an correctly placed alpha helix, therefore the B-factors are too high in that region. The model after refinement suggest less scattering in that region than is observed which is most likely explained by incorrectly high B-factors. 2) xyz, TLS and individual B-factors (no grouping). SS and ramachandran restraints on coordinates and NCS restraints on coordinates and B-factors. Note that the R-free value in the 3.4A shell is lower than the R-work (and also the Rpim in that shell!) which clearly indicates this refinement was not stable. I don't think it indicates anything about the stability of refinement - my guess would be that the NCS is biasing R-free. I suppose it could also indicate that the data in the 3.6-3.4 range are basically noise, although if the maps look better then that would suggest the opposite. I think the refinement is not parametrized correctly. Thank you, S.
[ccp4bb] Where to cut the data in this medium resolution dataset
Hey! I was reading a lot lately on data processing and the ongoing debate in the community on how to submit Table 1. Here is an example of medium resolution data integrated with XDS, merged in SCALA and preliminarily refined in phenix. Overall InnerShell OuterShell Low resolution limit 49.06 49.06 3.58 High resolution limit 3.40 10.74 3.40 Rmerge 0.224 0.050 1.324 Rmerge in top intensity bin0.049- - Rmeas (within I+/I-) 0.235 0.052 1.391 Rmeas (all I+ I-)0.235 0.052 1.391 Rpim (within I+/I-)0.067 0.014 0.407 Rpim (all I+ I-) 0.067 0.014 0.407 Fractional partial bias0.000 0.000 0.000 Total number of observations 275312 8491 39783 Total number unique21137 765 3016 Mean((I)/sd(I)) 10.2 31.2 2.3 Completeness98.4 95.7 98.4 Multiplicity13.0 11.1 13.2 r_work=0.2461 (0.3998) r_free= 0.2697 (0.3592) SigmaA highest shell = 0.78 scale factor highest shell (phenix.refine) = 0.87 Overall InnerShell OuterShell Low resolution limit 49.59 49.59 3.80 High resolution limit 3.60 11.39 3.60 Rmerge 0.231 0.048 0.819 Rmerge in top intensity bin0.045- - Rmeas (within I+/I-) 0.242 0.050 0.860 Rmeas (all I+ I-)0.242 0.050 0.860 Rpim (within I+/I-)0.069 0.014 0.249 Rpim (all I+ I-) 0.069 0.014 0.249 Fractional partial bias0.000 0.000 0.000 Total number of observations 230945 6997 33402 Total number unique17794 646 2531 Mean((I)/sd(I)) 11.5 30.7 3.7 Completeness98.3 95.5 98.7 Multiplicity13.0 10.8 13.2 r_work= 0.2372 (0.3585) r_free= 0.2663 (0.3770) SigmaA highest shell = 0.79 scale factor highest shell (phenix.refine) = 0.95 XSCALE gives significantly lower average Rrim and Rmerge for both integrations (~18%) and CC(1/2) is above 0.7 in all bins The diffraction pattern looks great, the 3.4A reflections are visible by eye and the edge of the detector is about 2.8A. The crystals were 10x20x50 um in size and spacegroup is P6522. The maps shows signs of over fitting, the B-factors do not look correct in my opinion. Note that the R-free value in the 3.4A shell is lower than the R-work (and also the Rpim in that shell!) which clearly indicates this refinement was not stable. The structure contains no beta sheets and refinement also profits greatly from very rigid high-order NCS. The maps are very detailed, in fact better than some 2.8A maps I've seen before. The 0.2A in question here are actually quite helpful to increase the map quality, so I keep wondering if I should deposit the structure with them or keep them only for my own interpretation. Before I continue optimizing the integration/refinement I would like to hear suggestions from the experts where to make the resolution cut-off in this case? Do I have all information I need to make that decision? What arguments should I present when dealing with the reviewers? I mean, the Rrim/Rmerge values are really very high. Thank you for your input, Stefan Gajewski
[ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?
Hey! I was just wondering, do you know of any recent (~10y) publication that presented a structure solution solely based on MIR? Without the use of any anomalous signal of some sort? When was the last time you saw a structure that was solved without the use of anomalous signal or homology model? Is there a way to look up the answer (e.g. filter settings in the RCSB) I am not aware of? Thanks, S. (Disclaimer: I am aware that isomorpous data is a valuable source of information)
Re: [ccp4bb] Expression of Viral proteins for crystallography
Rubén, the previous answer probably addresses your problem accurately. However. If your protein of interest modifies DNA/RNA, it is quite common that your pET constructs will mutate rapidly in E.coli. Lac operons tend to leak quite a bit, which is not enough to detect the protein prior to induction but can definitely drive the selection for mutated plasmids in E.coli. We saw that behavior in a nuclease cloned from homogeneous genomic DNA. Every colony had a different mutant when grown on LB. Also mutants that do not appear to affect the enzyme activity judged by the structure. We assumed, that any mutation that slightly reduces the toxicity for E.coli will be selected for. So, if you experience the same issue again with synthetic DNA, or have the patience to repeat, try adding ~1% glucose to all media, liquid and solid. This will tighten up the Lac operon and worked well for our nuclease project. You still have to sequence every batch you purify. pLysS cells can also work, but we still ended up adding glucose because it is cheap and doesn't cause trouble downstream. Stefan
Re: [ccp4bb] very strange lattice: high anisotropy, 78% solvent content and maybe merohedral twinning
Jürgen, Have you checked a simple selfrotation function in your currently favored space group ? Yes, both selfrotation function and patterson map do not look suspicious in I422. Do you have sufficient data collected to start out in P1 or C2 ? Then I would start there and systematically look at selfrotation functions in those space groups. Also check the native Patterson for translational NCS. I got ~220 degrees before the radiation damage became significant, so there should be enough data and I will look into it. 4 A is not great for stable refinement of cell parameters, which program did you use and which parameters did you fix? HKL2000 without fixing parameters. mosflm can't hold on the lattice and I haven't tried d*trek, yet. Did you use main.ncs=true in the SA approach ? yes Pointless or xtriage ? xtriage Why does it take a year to grow those crystals ? Well, other crystals don't diffract and the protein is quite stable in solution. That's how it is, I guess? Out of curiosity, how did you collect on this crystal without overlapping reflections ? I got away with 1 second exposure and 0.75 degree oscillation at 650mm detector distance. there are some predicted overlaps but they are in those regions that are empty due to anisotropy. Our cryo condition gives well separated small spots of nice, round shape, mosaicity is ~0.6. The pattern itself looks great, although the beamstop shadow is quite big on the frames. Thank You, Stefan
[ccp4bb] very strange lattice: high anisotropy, 78% solvent content and maybe merohedral twinning
I am looking at a highly unusual crystal lattice right now and can't figure out what is going on, so I decided to ask the experts. I recently got data on a oligomeric protein with many highly correlated NCS units (4.0A resolution, linear R-sym is 0.16-0.21 in I4, I422, F222, C2 and 0.12 in P1) with severe anisotropic diffraction (according to diffraction anisotropy server, the F/sigma drops below 3 at a=6.1 b=6.1 c=never, suggested isotropic B-sharpening -125A^2) This lattice has a problem. The apparent unit cell is rather huge (roughly 180 180 620 / 90 90 90) The unit cell dimensions are almost perfectly I4 and the presence of systematic absent reflections 50 I/s in I41 and I4122 suggest no screw axis. I used a very closely related structure solved at 4.2A as molecular replacement model and got a solution from the anisotropy corrected data in I422 space group with two oligomers in the asymmetric unit cell. Confidence of the MR solution is quite high since (a)the MR replacement put one model one NCS raster off the true position resulting in a clash with the second one in an empty region of the map and additional electron density on the other side which corresponds perfectly to the wrongly positioned monomer, and (b) after rotating the model in the right position I could refine the structure to R-work=0.31. R-free=0.35 in one run of rigid body refinement followed by NCS restrained simulated annealing refinement (phenix.refine), which is in my opinion really good at such an early stage of refinement given the low overall resolution and even lower completeness of strong reflections in a and b due to high anisotropy (observables to atoms ratio is about 3:1) . I can even see clear density for some of the bulky sidechains which were not included in the model. Now here is the baffling thing. The unit cell is almost empty with an apparent solvent content of 78%. The molecules cluster around the c-axis and at the origin with an empty gap in a and b of at least 15A and up to 165A(!) in the longest dimension. There is no sign of electron density that would indicate a missing protein in that region and ~98% of my model is already accounted for by the density in the 2Fo-Fc map, making a contact of disordered protein regions across the ASUs unlikely. In fact, the protein density is well defined at the closest gap and no mainchain atom is unaccounted for in that region. The oligomer has a magnitude of ~105A x 70A. I heavily doubt that a crystal lattice with such little contacts and holes as huge as these can exist and therefore think that: (a) the R-factors are misleading me to think the solution is correct and complete (b) I must have been doing something really wrong Since proteins from this family have a well established history of producing twinned crystals I had a look at that possibility. Analyzing the anisotropy corrected I4 data for twinning (Padilla Yeates method) revealed a 2-fold twin law with a twin fraction of 0.42 which would make the discrimination between an almost perfectly merohedral twin in I4 and a (non twinned ?) I422 extremely difficult (to me). MR with anisotropy corrected I4 data gave the same crystal packing and hence the same void solvent region. MR in lower point groups was not successful so far although I haven't pursued that idea vigorously. The same data in I422 has no indication for twinning and in C2 three 2-fold twin laws. Anomalous data is not easily available since those crystals grow in about one year and getting another crystal is also not very likely because this IS the other crystal. I am clueless now on how to proceed here and would appreciate advice from experienced crystallographers on what to try first. Am I worrying too much about the packing? Is it even possible to have such an enormously huge solvent region in a protein crystal? What is the recommended protocol when dealing with many and very strongly correlated NCS units, putative twinning and severe anisotropy all at the same time? Stefan Gajewski
