Re: [ccp4bb] Two Equally Good MR Solutions Found by Phaser
Hi All, Thanks for all the replies, I would like to add more information, after reindex it to P21212, the cell parameter is a=88.71, b=116.26, c=55.12, the molecule is a long rod like head to head dimer with a length of 110Å (55Å long for each monomer) and we used the dimer to search the orthorhombic data, two solutions were found as shown in the original thread which can be both refined to almost the same very good R/Rfree, coot was used to generated the symmetry related molecules from both solutions and the two solutions had the same packing but translated along the b axis by 31.9Å, thus clashed into each other and made a mess in the overlap region, so I think they are not crystallographically the same solutions, unless the origin of the cell in P21212 can be placed on b-axis arbitrarily, and I think Phaser will also prune the crystallographically same solutions. Interestingly, the most prominent pseudo-translational peak (1/3 of the origin peak), has a fractional vector 0.5000 0.2741 0.5000, and the fraction on b axis = 0.2741*116.26 = 31.8755Å, and that is exactly how long the two solutions translated along the b-axis, I don't know what that means, does these information verify this is the second case Eleanor suggested? if so should I keep the messy overlapped region and set the rest 0 occupancy to check the density? Thanks for all the suggestions so far! Xiaoli From the Pattersn peak it seems very likely that you have two molecules in the asymmetric unit seperated by the very vector that seperates your two MR solutions, and both MR solutions are correct? Or is that not possible? Is there no room for 2 molecules in the asymmetric unit, and the Patterson peak isa function of the "highly repetitive dimer" Eleanor If that is so you need to set the occupancy of any differences in the solution to 0.00 and check from the maps after refinement if you can see which copy of the molecule fits the difference density best - it would nice if you had a TRP/ALA pair of residues or something very distinctive.. Eleanor X Xiong, Cellular & Molecular Medicine wrote: Dear Crystallographers, We got a highly repetitive dimeric protein solved by SeMet-SAD in P21 crystal form, and I am now trying to solve a dataset collected from a non-reproducible orthorhombic crystal of the same protein using the structure refined from P21 data. From the Scala statistics, the orthorhombic crystal diffracted to 2.2Å with an I/sigma of 3.1 at outmost shell; 98% complete overall, 89% complete 43.4-7.0Å, 99% complete 2.32-2.2Å, no twinning was detected. Due to the incompleteness at low resolution, it was hard to determine which orthorhombic space group it is in so data was scaled in P222. Very strong pseudo-translational symmetry has been detected by self-Patterson, as shown for reindexed data P21212 (space group later found by Phaser): Order No. Site Height/RmsGrid Fractional coordinates Orthogonal coordinates 111 128.24 0 0 0 0. 0. 0. 0.00 0.00 0.00 2 13 13 57.5160 44 38 0.5000 0.2741 0.5000 44.37 31.88 27.55 322 33.75 0 7 0 0. 0.0414 0. 0.00 4.82 0.00 4 14 14 16.0960 50 38 0.5000 0.3150 0.5000 44.37 36.63 27.55 5 12 12 15.7560 37 38 0.5000 0.2324 0.5000 44.37 27.03 27.55 633 12.28 0 13 0 0. 0.0836 0. 0.00 9.72 0.00 7 1507.0660 57 38 0.5000 0.3574 0.5000 44.37 41.56 27.55 8446.18 0 72 0 0. 0.4503 0. 0.00 52.36 0.00 9995.68 5 0 5 0.0410 0. 0.0683 3.64 0.00 3.76 10555.36 2 20 2 0.0142 0.1254 0.0206 1.26 14.59 1.14 11 11 115.3358 31 38 0.4852 0.1909 0.5000 43.06 22.20 27.55 12663.98 5 0 2 0.0435 0. 0.0286 3.86 0.00 1.58 13773.82 2 27 3 0.0168 0.1659 0.0334 1.49 19.30 1.84 14883.68 0 0 5 0. 0. 0.0722 0.00 0.00 3.98 15 10 103.4160 64 37 0.5000 0.4007 0.4872 44.37 46.59 26.84 Phaser was used to test all possible alternative space groups to find MR solution using the structure from P21 data: # Phaser_P222_MosFLM_all_spacegroup SPACegroup HALL P 2bc 2 #P 2 21 21 SOLU SET RFZ=9.1 TFZ=24.3 PAK=0 LLG=2545 LLG=3718 SOLU 6DIM ENSE ensemble1 EULER 273.0971.162 88.144 FRAC -0.03394 0.50659 -0.22125 SOLU SET RFZ=9.1 TFZ=25.0 PAK=0 LLG=2496 LLG=3622 SOLU 6DIM ENSE ensemble1 EULER 91.4910.850 89.812 FRAC 0.03435 -0.00618 0.00352 and it found 2 solutions with very similar Z-scores and LLG gains, If I am right they are not crystallographic equivalent, and Phaser checks that as well. I reindexed the data to P21212 and Phaser found the same solutions: # Phaser_Reindexed_P21212_2_solutions SPACegroup HALL P 2 2ab #P 21 21 2 SOLU SET
Re: [ccp4bb] Two Equally Good MR Solutions Found by Phaser
Hi, Thanks for all the replies. Paul said the two solutions are the same by crystallographical symmetry, does that mean the origin of the unit cell has changed and P21212 can have arbitrary origin of the cell along the b-axis? As I have generated the symmetry related pairs from each solution in Coot, they do not overlap each other but partly clashed into each other along the b-axis. Another question is I thought Phaser can automatically prune the solutions that are cryptographically the same (as suggested in its log file ), did Phaser fail that in my case? For the comments on incompleteness at low resolution, I thought it was crystal mounting problem, there was no overloads as crystal is tiny and diffracted weakly, but it is very strange that two of the axes are very incomplete at low resolution, I don't know any explanation for that, and due to the incompleteness at low res, systematic absences can not be observed, so space group can not determined by looking at them. Thank for all the advices so far, and I would like more suggestions to clarify my doubts. cheers -- Xiaoli Xiong PhD Candidate Department of Cellular and Molecular Medicine School of Medical Sciences University of Bristol University Walk Bristol BS8 1TD, UK x.l.xi...@bristol.ac.uk
[ccp4bb] Two Equally Good MR Solutions Found by Phaser
Dear Crystallographers, We got a highly repetitive dimeric protein solved by SeMet-SAD in P21 crystal form, and I am now trying to solve a dataset collected from a non-reproducible orthorhombic crystal of the same protein using the structure refined from P21 data. From the Scala statistics, the orthorhombic crystal diffracted to 2.2Å with an I/sigma of 3.1 at outmost shell; 98% complete overall, 89% complete 43.4-7.0Å, 99% complete 2.32-2.2Å, no twinning was detected. Due to the incompleteness at low resolution, it was hard to determine which orthorhombic space group it is in so data was scaled in P222. Very strong pseudo-translational symmetry has been detected by self-Patterson, as shown for reindexed data P21212 (space group later found by Phaser): Order No. Site Height/RmsGrid Fractional coordinates Orthogonal coordinates 111 128.24 0 0 0 0. 0. 0. 0.00 0.00 0.00 2 13 13 57.5160 44 38 0.5000 0.2741 0.500044.37 31.88 27.55 322 33.75 0 7 0 0. 0.0414 0. 0.00 4.82 0.00 4 14 14 16.0960 50 38 0.5000 0.3150 0.500044.37 36.63 27.55 5 12 12 15.7560 37 38 0.5000 0.2324 0.500044.37 27.03 27.55 633 12.28 0 13 0 0. 0.0836 0. 0.00 9.72 0.00 7 1507.0660 57 38 0.5000 0.3574 0.500044.37 41.56 27.55 8446.18 0 72 0 0. 0.4503 0. 0.00 52.36 0.00 9995.68 5 0 5 0.0410 0. 0.0683 3.64 0.00 3.76 10555.36 2 20 2 0.0142 0.1254 0.0206 1.26 14.59 1.14 11 11 115.3358 31 38 0.4852 0.1909 0.500043.06 22.20 27.55 12663.98 5 0 2 0.0435 0. 0.0286 3.86 0.00 1.58 13773.82 2 27 3 0.0168 0.1659 0.0334 1.49 19.30 1.84 14883.68 0 0 5 0. 0. 0.0722 0.00 0.00 3.98 15 10 103.4160 64 37 0.5000 0.4007 0.487244.37 46.59 26.84 Phaser was used to test all possible alternative space groups to find MR solution using the structure from P21 data: # Phaser_P222_MosFLM_all_spacegroup SPACegroup HALL P 2bc 2 #P 2 21 21 SOLU SET RFZ=9.1 TFZ=24.3 PAK=0 LLG=2545 LLG=3718 SOLU 6DIM ENSE ensemble1 EULER 273.0971.162 88.144 FRAC -0.03394 0.50659 -0.22125 SOLU SET RFZ=9.1 TFZ=25.0 PAK=0 LLG=2496 LLG=3622 SOLU 6DIM ENSE ensemble1 EULER 91.4910.850 89.812 FRAC 0.03435 -0.00618 0.00352 and it found 2 solutions with very similar Z-scores and LLG gains, If I am right they are not crystallographic equivalent, and Phaser checks that as well. I reindexed the data to P21212 and Phaser found the same solutions: # Phaser_Reindexed_P21212_2_solutions SPACegroup HALL P 2 2ab #P 21 21 2 SOLU SET RFZ=10.0 TFZ=23.0 PAK=0 LLG=3266 LLG=3718 SOLU 6DIM ENSE ensemble1 EULER 88.843 90.0631.249 FRAC -0.00661 -0.22126 -0.46598 SOLU SET RFZ=9.9 TFZ=23.2 PAK=0 LLG=3178 LLG=3624 SOLU 6DIM ENSE ensemble1 EULER 270.841 89.977 181.339 FRAC -0.50634 -0.00350 0.46533 The difference between the two solutions seems to be that the second solution translated along the longer 21 axis by about ~32Å, I chose the first solution to re-build and refine, and final R/Rfree I got was 21.6%/26.5%. After that, I hope to solve the ambiguity of which MR solution is right by running Phaser again with the complete model (including H2O): # Phaser_Reindexed_P21212_2_solutions SPACegroup HALL P 2 2ab #P 21 21 2 SOLU SET RFZ=12.8 TFZ=28.4 PAK=0 LLG=6542 LLG=7649 SOLU 6DIM ENSE ensemble1 EULER 180.1560.0000.000 FRAC -0.50060 -0.00065 0.49998 SOLU SET RFZ=12.8 TFZ=32.3 PAK=0 LLG=6036 LLG=7059 SOLU 6DIM ENSE ensemble1 EULER 179.201 180.0000.000 FRAC 0.00227 0.22262 -0.50054 It seems that the previous first solution has become the second solution, while the previous second solution became the first. Refmac refinement was performed on both solutions (H2O removed) came out from Phaser, solution 1 R/Rfree = 24.1/28.9 solution 2 R/Rfree = 24.8/28.7 the previous first solution got slightly worse scores, however, the R-factors for both solutions are so similar and both of them gave very similar electron density that I can not figure out which one is the right solution. I would very grateful for any advices. Thanks in advance, -- Xiaoli Xiong PhD Candidate Department of Cellular and Molecular Medicine School of Medical Sciences University of Bristol University Walk Bristol BS8 1TD, UK x.l.xi...@bristol.ac.uk
[ccp4bb] How to Model Methylated N-terminal Amine in Coot
Dear All, My protein has been reductively methylated, and experimental density shows that lysines and N-terminal amines are methylated, while its is easy to model methylated lysine in coot, I can hardly find a way to model the methylated N-terminal. Does anyone know how to do this? Will the methylated N-terminal amine be OK for Refmac refinement? Thanks in advance for any suggestions! -- Xiaoli Xiong PhD Candidate Department of Cellular and Molecular Medicine School of Medical Sciences University of Bristol University Walk Bristol BS8 1TD, UK x.l.xi...@bristol.ac.uk
Re: [ccp4bb] heavy atom derivative choice
Dear All, We have a very similar problem, I have got a protein of 200 residues predicted to have two free cys (but 14 internal cys!), 1 free histidine, 2 methionines (all predicted to be free). Native crystals diffracted to 2.2 A was obtained in 200 mM KSCN, 20% PEG 3350, 13% glycerol, bis tris propane pH 7.5, 20mM spermine, 50mM DTT(absolutely needed to obtain crystal). We tried iodide soak and sulphur SAD but phasing was not promising. From all the posts I have read, mercury seems to be the best to try to derivatise it but probably needs to remove DTT. My Question is: Does mercury tends to get into the protein core to denature protein or not? For gel-shift assay, do people normally use a special gel tank for heavy metal work? thanks in advance Xiaoli Xiong (Alex) --On 15 July 2009 14:33 +0200 Sebastiano Pasqualato wrote: Hi all, I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 histidines, 2 methionines. We have nice diffraction for the native crystals, that grow in 150 mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. We are crystallising the SeMet derivative, but I'm not completely sure I will be able to have nice crystals by Saturday, when we have tunable time at the ESRF. I was thinking of trying with some heavy atom soaks, but only have like 30 crystals, so limited trials allowed! Which compound would you advice as more likely to work, and thus worth testing? Thanks in advance for the suggestions, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 -- Xiaoli Xiong PhD Candidate Department of Cellular and Molecular Medicine School of Medical Sciences University of Bristol University Walk Bristol BS8 1TD, UK x.l.xi...@bristol.ac.uk
