Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Hi Chen, I had a similar condition before (reservoir solution with 10% dioxane) and an oil-based cryoprotectant (66.5% w/w paraton-N, 28.5% paraffin oil and 5% glycerol) worked for me. Please check this paper out: http://scripts.iucr.org/cgi-bin/paper?wd5052 In addition, have you tried cryoprotection without removing the sealing membrane? I did it by punching a hole on the membrane with a needle, taking away the reservoir solution with a syringe, and adding the cryo solution to the reservoir. You can then wash the crystals withy the cryo solution, seal the membrane and let it equilibrate before looping the crystal. Also I think it better to change the reservoir solution in small steps (like from 30% dioxane -- 25% dioxane plus 5%EG -- 20%dioxane plus 10%EG ... until dioxane is 0%). To be honest I never tried it with a dioxane-containing condition, but it worked pretty well with a condition containing 20% isopropanol.
[ccp4bb] Looking for open_EPMR
Thanks for your replies! Does anyone know where to find free open_EPMR software? It seems that website is no longer reachable. Chao
[ccp4bb] A dimer as a MR search model?
Dear CCP4 community: I am trying to solve a structure using Molecular Replacement. We obtained several crystal forms of the same molecule, which is a hetero-trimer(18KD+16kD+15kD). We have solved the structure of one crystal form(form_1), which has space group P 42 22, 1 molecule per Asymmetric Unit, solvent content = 60%. It has resolution 2.7A and both R values around 0.25. Now I am trying to solve the structure of a second crystal form(form_2), which has space group P 41 22 or P 422, 2 or 3 molecules per ASU. The data has resolution around 3.3A; at 9-7A shell Chi2 around 2.5, Rlinear=0.054 and Rsquare=0.057. Completeness and redundancy are fine. However, when I tried Molecular Replacement using the solved form_1 structure as the search model, I did not get any solutions, after trying AmoRe, Molrep and Phaser. I think that probably the two (or three) molecules in one Asymmetric Unit of form_2 crystal have structural differences, and these differences are so big that MR using one single molecule just can not work (I know this sounds weird; pardon me I am a beginner). With this in mind and following other people's advice, I tried the following steps: 1) Look at the solved form_1 structure in Coot; Identify all possible combinations of two molecules that pack against each other; 2) Combine these combinations into separate new pdb files, each containing two molecules; 4) Use the new pdb files as search model for MR. I think that, if I use two molecules as a search model, probably the structural differences between the two molecules will not be so significant, because the model's size is twice increased compared to using one molecule. However, I tried all possible combinations of two molecules in AmoRe, Phaser and MolRep and still got no solutions. Cutting resolution range to 15-4A did not help either. Now I am thinking that in the Asymmetric Unit of my new form_2 crystal, maybe the two molecules' geometric relationship is not found in my solved form_1 crystal. So my question is: 1) Is there any way to find the possible geometry of two molecules in form_2 Asymmetric Unit, combine them into new pdb files and use them as MR models? 2) If I continue to use the single molecule as a MR model, should I remove some possibly flexible regions? Or just remove one or two whole monomer(s)? 3) Is it possible that the same molecule in different crystal forms have structures so different as to preclude MR trials? I appreciate any advice. Have a nice Thanksgiving. Chao
[ccp4bb] Help request: Failed MR using the same molecule as model
Dear CCP4 community: I am a beginner to crystallography and therefore my apologies if this question is too simple. Basically we obtained several crystal forms of the same molecule, which is a hetero- trimer containing protein A(18kD), protein B(16kD) and a RNA segment(40nt or about 15kD). We have solved the structure of one crystal form(form_1); its information is as follows: space group = P 42 22; unit cell = 126.514 126.514 76.766 90.00 90.00 90.00 resolution = 2.7A; Rwork = 0.25, Rfree = 0.265; solvent content = 60%; Number of molecules per Asymmetric Unit = 1; Data redundancy = 5; Data Completeness = 94%; I am now trying to solve the structure of form_2 crystal using molecular replacement. So far the information I know about form_2 is as follows: space group = I 422 or I 41 22; unit cell = 180.096 180.096 152.530 90.00 90.00 90.00 (unit cell is about 4 times the size of form_1) resolution = 3.3A; (which is low) Number of molecules per Asymmetric Unit = 2 or 3; Data redundancy = 4; Data Completeness = 92%; There is no twinning(as shown by Sfcheck); As shown inAnalyse Data for MR, the first peak is 100 and second is 68.92; I am not sure if this indicates translation in a Asymmetric Unit; The problem is, I can not get a good solution by MR using Phaser (both I422 and I4122 are tried). When I searched for 3 molecules per Asymmetric Unit, Phaser did not give solutions at all. When I used 2/ASU instead, I was able to get some solutions, with typical statistics as follows: RFZ=14, TFZ=35.9, PAK=0, LLG=1036; However, these solutions had high R values(like Rwork=0.59 and Rfree=0.58), which indicated that they are not solutions at all. Still, I tried refinement using refmac5, but R values did not go down even after 50 rounds; sometimes they even increased after refinement. Besides, the RMS values bond length, bond angle and chiral center were all 0 as show by refmac5. I tried limiting resolution range to 15-4A in Phaser, which did not help either. Now I am completely stuck. Could anyone give me some advice? I know this situation is very strange, because I am using the SAME molecule for MR but can not can a solution. Thanks a lot, P.S. 1) Both form_1 and form_2 crystals were grown using Selenomethionine-containing samples. There are 3 Sel_Met in protein A and 1 in protein B. 2) A 10-aa internal segment of protein B is missing in the solved structure, which may indicate high flexibility. Chao
[ccp4bb] Help request: Failed MR using the same molecule as model
Dear CCP4 community: Sorry if there is a duplicate post. I am a beginner to crystallography and therefore my apologies if this question is too simple. Basically we obtained several crystal forms of the same molecule, which is a hetero- trimer containing protein A(18kD), protein B(16kD) and a RNA segment(40nt or about 15kD). We have solved the structure of one crystal form(form_1); its information is as follows: space group = P 42 22; unit cell = 126.514 126.514 76.766 90.00 90.00 90.00 resolution = 2.7A; Rwork = 0.25, Rfree = 0.265; solvent content = 60%; Number of molecules per Asymmetric Unit = 1; Data redundancy = 5; Data Completeness = 94%; I am now trying to solve the structure of form_2 crystal using molecular replacement. So far the information I know about form_2 is as follows: space group = I 422 or I 41 22; unit cell = 180.096 180.096 152.530 90.00 90.00 90.00 (unit cell is about 4 times the size of form_1) resolution = 3.3A; (which is low) Number of molecules per Asymmetric Unit = 2 or 3; Data redundancy = 4; Data Completeness = 92%; There is no twinning(as shown by Sfcheck); As shown in Analyse Data for MR, the first peak is 100 and second is 68.92; I am not sure if this indicates translation in a Asymmetric Unit; The problem is, I can not get a good solution by MR using Phaser (both I422 and I4122 are tried). When I searched for 3 molecules per Asymmetric Unit, Phaser did not give solutions at all. When I used 2/ASU instead, I was able to get some solutions, with typical statistics as follows: RFZ=14, TFZ=35.9, PAK=0, LLG=1036; However, these solutions had high R values(like Rwork=0.59 and Rfree=0.58), which indicated that they are not solutions at all. Still, I tried refinement using refmac5, but R values did not go down even after 50 rounds; sometimes they even increased after refinement. Besides, the RMS values bond length, bond angle and chiral center were all 0 as show by refmac5. I tried limiting resolution range to 15-4A in Phaser, which did not help either. Now I am completely stuck. Could anyone give me some advice? I know this situation is very strange, because I am using the SAME molecule for MR but can not can a solution. Thanks a lot, P.S. 1) Both form_1 and form_2 crystals were grown using Selenomethionine-containing samples. There are 3 Sel_Met in protein A and 1 in protein B. 2) A 10-aa internal segment of protein B is missing in the solved structure, which may indicate high flexibility. Chao
