Re: [ccp4bb] Methylation of macromolecular complexes
Hi All, Many thanks for your reply. I am quite sure that my crystals are the complex. I made the complex by 1:1.1 ratio with the small component excess, then did the methylation, followed by gel filtration to remove the small reductant. The SDS-PAGEs for gel filtration fractions and crystals showed two bands.Both indice from XDS and mosflm gave P222 with cell of a=30.13, b=47.38, c=119.6 and apha=beta=gama=90 degree. Mathews calculation gave 42.37% solvent content with molecular weight of 20 kDa (the complex).The space group was also checked by pointless that gave P222 as well. However, phaser gave P22121, RFZ=3.7, TFZ=4.8, park=4, LLG=26, LLG=24. Thanks peter Date: Tue, 24 Nov 2009 20:37:01 -0500 From: mjvdwo...@netscape.net Subject: Re: [ccp4bb] Methylation of macromolecular complexes To: CCP4BB@JISCMAIL.AC.UK There is a reasonable experiment you can do: Take the unit cell of your (presumed) crystal complex and put the search model into the until cell in a way that seems reasonable (use coot, or your favorite program). Make structure factors (with CCP4 or your favorite program) and throw away the phases. Then rotate your MR search model randomly and put the search model and structure factors into the MR program. See if it finds a solution. You can repeat it with structure factor data to which noise has been added. You can use this method to optimize your MR search parameters. Don't be surprised if you cannot find a MR solution at all (even if you know for sure it should exist in your artificial problem). Once upon a time I tried to solve the structure of a small poly-peptide and with the method above I proved to myself that it was not possible to find a solution (with the programs that were available at that time, when Dinosaurs still roamed the Earth). I found that MR is very finicky when applied to small peptides. At least you will be able to determine what the optimal parameters are (resolution, search radius) and whether it can succeed at all. To pursue Se-Met is smart. Small peptide crystals resist heavy atom soaking. If your resolution is high enough, you can also try direct methods. On such a small peptide that should be easy (provided your crystals are well-behaved). Frequently the diffraction resolution of small peptide crystals is high enough that direct methods work very well. Mark -Original Message- From: Sean Seaver To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, Nov 24, 2009 5:13 pm Subject: Re: [ccp4bb] Methylation of macromolecular complexes >I have a small complex, one component is 13 kDa with structure available and the other is 7 kDa, which could not be able to grew crystals after lots of efforts. It grew crystals after methylation and diffracted to 2.3 A, however, I could not be able to solve the structure using the structure of the big component as a template for molecular replacement, and heavy atoms soaking was not successful. I plan to do selenomethionine expression next. Does the methylation change protein structures a lot? otherwise, why does molecular replacement not work? I would much appreciate any idea and suggestions how to solve the structure using the data and the template avaliable. --- A couple of questions that I would consider in regards to the molecular replacement not working: Is the binding between the proteins 1:1? Does the Matthews coefficient reflect this? MR maybe difficult if you have a number of 7 kDa proteins binding to a 13 kDa protein. Hope that Helps, Sean _ Windows Live Hotmail: Your friends can get your Facebook updates, right from Hotmail®. http://www.microsoft.com/middleeast/windows/windowslive/see-it-in-action/social-network-basics.aspx?ocid=PID23461::T:WLMTAGL:ON:WL:en-xm:SI_SB_4:092009
Re: [ccp4bb] Help request: Failed MR using the same molecule as model
A few things to try (or double-check): 1. If you ran phaser with SGALTERNATIVE ALL, make sure the mtz file you gave to refmac has the same screw axes as the MR solution. If this is off, it'll lead to higher R-factors. 2. As Fred mentioned, try with a single copy of the protein, and check the FWT (sigma_a weighted 2Fo-Fc) maps. If the solution is reasonably correct, you should be able to see some density for the other components. Also, run some controls. Delete a portion of your search model before MR, and insure that density for that region is present in maps phased with the positioned model. 3. Use your seleniums. If your MR solution is correct, then you should see peaks in model-phased anomalous difference maps. But confirm this with your form_1 data. In my experience, these are not very sensitive to model completeness (but that's with a different protein). Since you've mentioned you're relatively new at this, you should re-merge (scala or scalepack) as anomalous for anomalous difference maps if you haven't done so already (normally I'd assume this was the case for dealing with anomalous data and not mention it...sorry if I'm telling you things you already know). Good luck, pm Chao Quan wrote: 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 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
[ccp4bb] Bad geometry for alt. conformation refined in Refmac5
Hello All, We are trying to refine ARG residues with two conformations in Refmac5, and the refined atom positions in the output PDB file are all over the place, as if the geometry restraints are not well defined. We've tried several different formats for the input file, based on previous postings to the bulletin board and the PDB standard (two examples below), but the result is always the same. We are using Refmac5 in CCP4 Suite 6.1.2, GUI 2.0.5 on Mac OSX. We'd appreciate any suggestions. Thanks. -John Examples of ARG format: 1) ATOM 1472 N ARG A 1 -5.737 26.887 38.372 1.00 29.53 CN ATOM 1473 CA ARG A 1 -5.445 25.560 37.882 1.00 30.24 CC ATOM 1474 CB ARG A 1 -5.314 24.548 39.036 1.00 30.63 CC ATOM 1475 CG ARG A 1 -5.426 23.052 38.627 1.00 34.81 CC ATOM 1476 CD AARG A 1 -4.827 22.075 39.644 0.50 37.09 CC ATOM 1477 CD BARG A 1 -4.301 22.419 39.279 0.50 37.09 CC ATOM 1478 NE AARG A 1 -3.430 21.777 39.304 0.50 42.71 CN ATOM 1479 NE BARG A 1 -4.482 21.902 40.627 0.50 42.71 CN ATOM 1480 CZ AARG A 1 -2.998 20.868 38.402 0.50 44.91 CC ATOM 1481 CZ BARG A 1 -3.648 22.142 41.638 0.50 44.91 CC ATOM 1482 NH1AARG A 1 -3.841 20.117 37.678 0.50 45.20 CN ATOM 1483 NH1BARG A 1 -2.584 22.912 41.464 0.50 45.20 CN ATOM 1484 NH2AARG A 1 -1.688 20.715 38.210 0.50 44.99 CN ATOM 1485 NH2BARG A 1 -3.878 21.619 42.831 0.50 44.99 CN ATOM 1486 C ARG A 1 -6.518 25.176 36.830 1.00 29.97 CC ATOM 1487 O ARG A 1 -7.675 25.501 36.971 1.00 31.02 CO 2) ATOM 44 N AARG A 1 26.671 62.112 46.990 0.50 30.13 AN ATOM 45 CA AARG A 1 26.970 63.346 47.667 0.50 30.65 AC ATOM 46 CB AARG A 1 27.172 64.495 46.676 0.50 31.07 AC ATOM 47 CG AARG A 1 27.152 65.897 47.322 0.50 34.20 AC ATOM 48 CD AARG A 1 27.993 66.976 46.599 0.50 37.16 AC ATOM 49 NE AARG A 1 27.726 67.425 45.342 0.50 42.06 AN ATOM 50 CZ AARG A 1 28.315 67.639 44.168 0.50 44.78 AC ATOM 51 NH1AARG A 1 29.525 67.160 43.918 0.50 45.37 AN ATOM 52 NH2AARG A 1 27.690 68.340 43.240 0.50 45.67 AN ATOM 53 C AARG A 1 25.839 63.640 48.622 0.50 30.37 AC ATOM 54 O AARG A 1 24.690 63.377 48.340 0.50 31.24 AO ATOM 55 N BARG A 1 26.667 62.080 47.010 0.50 30.13 AN ATOM 56 CA BARG A 1 26.921 63.329 47.640 0.50 30.65 AC ATOM 57 CB BARG A 1 27.108 64.390 46.581 0.50 31.07 AC ATOM 58 CG BARG A 1 27.138 65.756 47.103 0.50 34.20 AC ATOM 59 CD BARG A 1 28.447 66.452 46.933 0.50 37.16 AC ATOM 60 NE BARG A 1 28.377 67.657 47.707 0.50 42.06 AN ATOM 61 CZ BARG A 1 29.349 68.269 48.373 0.50 44.78 AC ATOM 62 NH1BARG A 1 30.572 67.836 48.373 0.50 45.37 AN ATOM 63 NH2BARG A 1 29.066 69.368 49.034 0.50 45.67 AN ATOM 64 C BARG A 1 25.822 63.636 48.638 0.50 30.37 AC ATOM 65 O BARG A 1 24.698 63.372 48.403 0.50 31.24 AO John Pascal, PhD Thomas Jefferson University Department of Biochemistry & Molecular Biology 233 South 10th Street, BLSB 804 Philadelphia, Pennsylvania 19107 ph 215.503.4596 fx 215.923.2117
Re: [ccp4bb] Bad geometry for alt. conformation refined in Refmac5
Could you please try the version from York: www.ysbl.york.ac.uk/refmac/latest_refmac.html I think probkem you mention is related with compilation or something. At least I cannot repat it on my computer regards Garib On 25 Nov 2009, at 16:18, John Pascal wrote: Hello All, We are trying to refine ARG residues with two conformations in Refmac5, and the refined atom positions in the output PDB file are all over the place, as if the geometry restraints are not well defined. We've tried several different formats for the input file, based on previous postings to the bulletin board and the PDB standard (two examples below), but the result is always the same. We are using Refmac5 in CCP4 Suite 6.1.2, GUI 2.0.5 on Mac OSX. We'd appreciate any suggestions. Thanks. -John Examples of ARG format: 1) ATOM 1472 N ARG A 1 -5.737 26.887 38.372 1.00 29.53 CN ATOM 1473 CA ARG A 1 -5.445 25.560 37.882 1.00 30.24 CC ATOM 1474 CB ARG A 1 -5.314 24.548 39.036 1.00 30.63 CC ATOM 1475 CG ARG A 1 -5.426 23.052 38.627 1.00 34.81 CC ATOM 1476 CD AARG A 1 -4.827 22.075 39.644 0.50 37.09 CC ATOM 1477 CD BARG A 1 -4.301 22.419 39.279 0.50 37.09 CC ATOM 1478 NE AARG A 1 -3.430 21.777 39.304 0.50 42.71 CN ATOM 1479 NE BARG A 1 -4.482 21.902 40.627 0.50 42.71 CN ATOM 1480 CZ AARG A 1 -2.998 20.868 38.402 0.50 44.91 CC ATOM 1481 CZ BARG A 1 -3.648 22.142 41.638 0.50 44.91 CC ATOM 1482 NH1AARG A 1 -3.841 20.117 37.678 0.50 45.20 CN ATOM 1483 NH1BARG A 1 -2.584 22.912 41.464 0.50 45.20 CN ATOM 1484 NH2AARG A 1 -1.688 20.715 38.210 0.50 44.99 CN ATOM 1485 NH2BARG A 1 -3.878 21.619 42.831 0.50 44.99 CN ATOM 1486 C ARG A 1 -6.518 25.176 36.830 1.00 29.97 CC ATOM 1487 O ARG A 1 -7.675 25.501 36.971 1.00 31.02 CO 2) ATOM 44 N AARG A 1 26.671 62.112 46.990 0.50 30.13 AN ATOM 45 CA AARG A 1 26.970 63.346 47.667 0.50 30.65 AC ATOM 46 CB AARG A 1 27.172 64.495 46.676 0.50 31.07 AC ATOM 47 CG AARG A 1 27.152 65.897 47.322 0.50 34.20 AC ATOM 48 CD AARG A 1 27.993 66.976 46.599 0.50 37.16 AC ATOM 49 NE AARG A 1 27.726 67.425 45.342 0.50 42.06 AN ATOM 50 CZ AARG A 1 28.315 67.639 44.168 0.50 44.78 AC ATOM 51 NH1AARG A 1 29.525 67.160 43.918 0.50 45.37 AN ATOM 52 NH2AARG A 1 27.690 68.340 43.240 0.50 45.67 AN ATOM 53 C AARG A 1 25.839 63.640 48.622 0.50 30.37 AC ATOM 54 O AARG A 1 24.690 63.377 48.340 0.50 31.24 AO ATOM 55 N BARG A 1 26.667 62.080 47.010 0.50 30.13 AN ATOM 56 CA BARG A 1 26.921 63.329 47.640 0.50 30.65 AC ATOM 57 CB BARG A 1 27.108 64.390 46.581 0.50 31.07 AC ATOM 58 CG BARG A 1 27.138 65.756 47.103 0.50 34.20 AC ATOM 59 CD BARG A 1 28.447 66.452 46.933 0.50 37.16 AC ATOM 60 NE BARG A 1 28.377 67.657 47.707 0.50 42.06 AN ATOM 61 CZ BARG A 1 29.349 68.269 48.373 0.50 44.78 AC ATOM 62 NH1BARG A 1 30.572 67.836 48.373 0.50 45.37 AN ATOM 63 NH2BARG A 1 29.066 69.368 49.034 0.50 45.67 AN ATOM 64 C BARG A 1 25.822 63.636 48.638 0.50 30.37 AC ATOM 65 O BARG A 1 24.698 63.372 48.403 0.50 31.24 AO John Pascal, PhD Thomas Jefferson University Department of Biochemistry & Molecular Biology 233 South 10th Street, BLSB 804 Philadelphia, Pennsylvania 19107 ph 215.503.4596 fx 215.923.2117
Re: [ccp4bb] Help request: Failed MR using the same molecule as model
I would also try Open-EPMR as well as Phaser. It can sometimes find solutions that Phaser or Amore cannot, especially for multiple chains and low resolution. EPMR is especially good at handling high copy numbers of search models. Cheers. Pete Meyer wrote: A few things to try (or double-check): 1. If you ran phaser with SGALTERNATIVE ALL, make sure the mtz file you gave to refmac has the same screw axes as the MR solution. If this is off, it'll lead to higher R-factors. 2. As Fred mentioned, try with a single copy of the protein, and check the FWT (sigma_a weighted 2Fo-Fc) maps. If the solution is reasonably correct, you should be able to see some density for the other components. Also, run some controls. Delete a portion of your search model before MR, and insure that density for that region is present in maps phased with the positioned model. 3. Use your seleniums. If your MR solution is correct, then you should see peaks in model-phased anomalous difference maps. But confirm this with your form_1 data. In my experience, these are not very sensitive to model completeness (but that's with a different protein). Since you've mentioned you're relatively new at this, you should re-merge (scala or scalepack) as anomalous for anomalous difference maps if you haven't done so already (normally I'd assume this was the case for dealing with anomalous data and not mention it...sorry if I'm telling you things you already know). Good luck, pm Chao Quan wrote: 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 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 -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu
Re: [ccp4bb] Help request: Failed MR using the same molecule as model
also try MOLREP (CCP4i) - we had a case where AMORE and PHASER did not work (in our hands), but MOLREP did, using the internal MOLREP sequence alignment/unequal amino acid trimming feature. This was a case of a P1 cell with 12 copies of the same molecule, 2.3A resolution, not very high-quality data, see J Virol. 2008 November; 82(22): 11208–11216. Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ researcherID: B-3678-2009 On 25 Nov 2009, at 17:42, Roger Rowlett wrote: I would also try Open-EPMR as well as Phaser. It can sometimes find solutions that Phaser or Amore cannot, especially for multiple chains and low resolution. EPMR is especially good at handling high copy numbers of search models. Cheers. Pete Meyer wrote: A few things to try (or double-check): 1. If you ran phaser with SGALTERNATIVE ALL, make sure the mtz file you gave to refmac has the same screw axes as the MR solution. If this is off, it'll lead to higher R-factors. 2. As Fred mentioned, try with a single copy of the protein, and check the FWT (sigma_a weighted 2Fo-Fc) maps. If the solution is reasonably correct, you should be able to see some density for the other components. Also, run some controls. Delete a portion of your search model before MR, and insure that density for that region is present in maps phased with the positioned model. 3. Use your seleniums. If your MR solution is correct, then you should see peaks in model-phased anomalous difference maps. But confirm this with your form_1 data. In my experience, these are not very sensitive to model completeness (but that's with a different protein). Since you've mentioned you're relatively new at this, you should re- merge (scala or scalepack) as anomalous for anomalous difference maps if you haven't done so already (normally I'd assume this was the case for dealing with anomalous data and not mention it...sorry if I'm telling you things you already know). Good luck, pm Chao Quan wrote: 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 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 -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu
Re: [ccp4bb] Help request: Failed MR using the same molecule as model
To complicate things some more, a couple of things could be going on. 1. You could have pseudosymmetry, where you true SG is I4 and the additional NCS operators make the crystal look I422. 2. You could have twinning. If merohedral, could be I4. If pseudomerohedral, could be any of the subgroups. 3. You could have both pseudosymmetry and twinning in which case the intensity statistics may not show the presence of twinning. I would recommend reprocessing in lower symmetry and running phenix.xtriage on the data. It is really good at finding pseudosymmetry/twinning and may help you find the correct SG. Peter or Andrey will probably chime in here if this is your problem. HTH, Jon Schuermann -- Jonathan P. Schuermann, Ph. D. Beamline Scientist NE-CAT, Building 436E Advanced Photon Source (APS) Argonne National Laboratory 9700 South Cass Avenue Argonne, IL 60439 email: schue...@anl.gov Tel: (630) 252-0682 Fax: (630) 252-0687 Chao Quan wrote: 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 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
Re: [ccp4bb] Help request: Failed MR using the same molecule as model
Actually, another thing could be going on as well. You show a large off-origin peak in the Patterson in I422 so you may have pseudotranslation going on and you processed in the supercell. You could probably try to reindex choosing fewer spots and get your P422 cell. I am sure there is some law that would convert your P422 cell to I422 (or vice versa), but I don't know it off the top of my head. That way you could just look at your cell lengths and see if this is possible. Its probably the same crystal form, except this crystal has PTS. Jon Chao Quan wrote: 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 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 -- Jonathan P. Schuermann, Ph. D. Beamline Scientist NE-CAT, Building 436E Advanced Photon Source (APS) Argonne National Laboratory 9700 South Cass Avenue Argonne, IL 60439 email: schue...@anl.gov Tel: (630) 252-0682 Fax: (630) 252-0687
Re: [ccp4bb] decrease of background with distance?
Thank you all for your informative responses! While examining the effects of unusual beam profiles on data collection due to capillary optics, I had collected a wedge of data on a large, high-quality lysozyme crystal at 8 different sample to detector distances. I restricted the analysis of all data to the resolution range seen at the largest distance, so each data set contained nearly the same reflections with the same redundancy. sig> does indeed improve with distance: starting at 7.89 at 135 mm and reaching its maximum of 10.35 at 614 mm. The total linear R factor for that range also falls from 0.09 down to 0.059. So, even with a 1.8 mrad divergent beam (about as divergent as practical at a beamline), the effect is significant. Surely someone has done this experiment before long ago. I like Jim Plufgrath's way of looking at the problem: reflections and background scatter have different effective source sizes and distances. For a reflection, the source is the same as the beamline source (some place way upstream). For solvent the source is the sample itself, for air, the source is spread out over a range of distances ... thus background is very divergent. Richard
Re: [ccp4bb] decrease of background with distance?
Richard Gillilan wrote: I like Jim Plufgrath's way of looking at the problem: reflections and background scatter have different effective source sizes and distances. For a reflection, the source is the same as the beamline source (some place way upstream). For solvent the source is the sample itself, for air, the source is spread out over a range of distances ... thus background is very divergent. I like Jim's way of looking at the problem too. Especially the part about beer. -James Holton MAD Scientist Jim Plufgrath wrote: So when your synchtrotron buddies talk about all this, be sure to buy them beers to help with their thinking. :)
[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
Re: [ccp4bb] Looking for open_EPMR
On Wednesday 25 November 2009 13:56:38 Chao Quan wrote: > Thanks for your replies! > > Does anyone know where to find free open_EPMR software? It seems that > website is no longer reachable. The web site looks perfectly normal from here: http://www.epmr.info/ -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
[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] Truncation of first two residues "rescued" an insoluble protein?
Dear all, Sorry for the off-topic question, but I am really curious why two terminal residues could make such huge difference. I am working on a all helical domain starting from residue 1 to about 130. It expressed well with N-terminal His tag in both E.Coli and insect cell, but stayed insoluble in both hosts. The fusion of N terminal MBP tag could produce soluble form, but the target protein precipitated immediately after cleavage of MBP. I tried several constructs ending at different C-terminal residues, but none helped. However, when I truncrate the N-terminal residues one by one, the deletion of Met1 and Ala2 turned this protein into almost "completely" soluble (30mg yield per liter E.coli cultue), and it behaved good after cleavage of N-terminal His tag. In homologous structures, the first helix starts from the third reidues. So my question is what property of the protein might have been affected by the first two residues, the surface hydrophobicity, the folding process, or something else?And would this one-by-one truncation of N-terminus be commonly helpful when working on insoluble small proteins? Thank you! Best regards, Tiancen Postdoctoral Fellow Novartis Intitutes for Biomedical Research 250 Mass Ave Cambridge MA 02139 U.S.
Re: [ccp4bb] Truncation of first two residues "rescued" an insoluble protein?
Dear Tiancen, This is perhaps a more extreme example of what many of us have experienced over and over - namely that for some proteins very small changes make a huge difference in expression, solubility, activity or all three :) Long and rambling reply follows - don't read if you are easily bored :) In my experience this phenomenon tends to be particularly prominent for domains where it is fairly common to have less than 1% sequence difference be responsible for a transition between soluble & folded protein and insoluble/aggregated garbage. Over time I have convinced myself that this phenomenon is strongly correlated with folding; on several occasions I've had situations similar to yours and the version of the protein that was insoluble when expressed in E. coli was very nicely behaved in insect cells or yeast (without any changes in sequence!) - my best guess that chaperones, different ribosomes, intracellular conditions, or some other complex features of the transcription/translation cascade are different enough between expression hosts and in some hosts the 'insoluble' version of the protein can be properly folded. Perhaps an even more extreme example of very tiny changes affecting protein folding can be the difference in stability of reduced versus oxidized metalloproteins. Specifically I remember reading a cytochrome research paper where the authors took advantage of the marked difference in stability of the two forms and conducted very elegant nanoscale folding experiments using photo-induced oxidation (or was it reduction?) as trigger for folding. This allowed them to study folding on very short time scales. The cool science aside, this is a good example of how a *single electron* can make or break an entire protein. In practical terms this means that in construct design and expression experiments there's always a need to balance the factors that may (and do) influence folding in specific host(s) of choice. Some systems seem to be more forgiving - in my opinion eukaryotic expression hosts possess more advanced 'assisted folding' systems and therefore tolerate sloppy construct design a lot more than prokaryotic ones. In a pinch (and I mean really, when nothing else works and there are no orthologs left to be tried) I've been known to slice proteins with 5 or even 3-aa step - for E. coli work; whereas we got away with a lot larger window in eukaryotic systems. This is a good thing since most eukaryots are a lot less facile as expression hosts and therefore require more effort per construct. Artem -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tiancen Hu Sent: Wednesday, November 25, 2009 10:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Truncation of first two residues "rescued" an insoluble protein? Dear all, Sorry for the off-topic question, but I am really curious why two terminal residues could make such huge difference. I am working on a all helical domain starting from residue 1 to about 130. It expressed well with N-terminal His tag in both E.Coli and insect cell, but stayed insoluble in both hosts. The fusion of N terminal MBP tag could produce soluble form, but the target protein precipitated immediately after cleavage of MBP. I tried several constructs ending at different C-terminal residues, but none helped. However, when I truncrate the N-terminal residues one by one, the deletion of Met1 and Ala2 turned this protein into almost "completely" soluble (30mg yield per liter E.coli cultue), and it behaved good after cleavage of N-terminal His tag. In homologous structures, the first helix starts from the third reidues. So my question is what property of the protein might have been affected by the first two residues, the surface hydrophobicity, the folding process, or something else?And would this one-by-one truncation of N-terminus be commonly helpful when working on insoluble small proteins? Thank you! Best regards, Tiancen Postdoctoral Fellow Novartis Intitutes for Biomedical Research 250 Mass Ave Cambridge MA 02139 U.S.
