Re: [ccp4bb] Challenging Mol.Rep. Problem
Generally fab geometry presents problems for mr due to variable interdomain angle. Assuming your sg is correct and this is not a twin, i would try searching at 4 or even 5 a resolution. I would not reject the chance that you have high solvent content, so you might have fewer molecules in au than expected from vm estimates. We recently solved a whole bunch of structures with large unit cell dimensions and high solvent content in the 75% or even 82% range. All had issues with resolution, freezing, and twinning - similar to shat you describe. In a few cases we screened over 35 crystals (of the same protein variant, same condition, just repeats) to find one that diffracted beyond 4a. Don't give up! May the pork be with you. Artem On Dec 9, 2014 12:32 PM, Mo Wong mowon...@gmail.com wrote: Hi all, I’m stuck on a rather complex molecular replacement problem. The crystals are of an antigen-Fab complex totaling ~67 kDa (waiting to confirm using PAGE gel). They diffract to ~3.5A at the synchrotron and process in C2 with dimensions 220x130x230 and beta at 103 so it looks like there are round 9to12-ish copies in the ASU. The overall Rmerge is high at ~25% with I/sig cutoff ~2 and redundancy of 5; however, at 4.5A this drops to ~15%. Furthermore, processing in P1 gives similar Rmerge values too. Self-Patterson doesn’t suggest translational symmetry, but the self-rotation function (SRF) suggests high NCS (see below/attached). I’m hoping the SRF might be helpful in trying to confirm/dismiss C2 is the likely space group, and perhaps suggest a logical assembly with the ASU (I see strong 2-fold and 3-fold NCS operators suggesting to me dimeric trimers or vice versa - however, I’ve never had to really analyze SRFs in the absence of a mol. rep. solution so my interpretation could be wrong). Anyway, any help to bringing a molecular replacement solution closer to reality would be appreciated. Thanks! [image: Inline image 1]
Re: [ccp4bb] Challenging Mol.Rep. Problem
Thank you cell phone typing for the inadvertent prophanity. Sorry! On Dec 9, 2014 1:00 PM, Artem Evdokimov artem.evdoki...@gmail.com wrote: Generally fab geometry presents problems for mr due to variable interdomain angle. Assuming your sg is correct and this is not a twin, i would try searching at 4 or even 5 a resolution. I would not reject the chance that you have high solvent content, so you might have fewer molecules in au than expected from vm estimates. We recently solved a whole bunch of structures with large unit cell dimensions and high solvent content in the 75% or even 82% range. All had issues with resolution, freezing, and twinning - similar to shat you describe. In a few cases we screened over 35 crystals (of the same protein variant, same condition, just repeats) to find one that diffracted beyond 4a. Don't give up! May the pork be with you. Artem On Dec 9, 2014 12:32 PM, Mo Wong mowon...@gmail.com wrote: Hi all, I’m stuck on a rather complex molecular replacement problem. The crystals are of an antigen-Fab complex totaling ~67 kDa (waiting to confirm using PAGE gel). They diffract to ~3.5A at the synchrotron and process in C2 with dimensions 220x130x230 and beta at 103 so it looks like there are round 9to12-ish copies in the ASU. The overall Rmerge is high at ~25% with I/sig cutoff ~2 and redundancy of 5; however, at 4.5A this drops to ~15%. Furthermore, processing in P1 gives similar Rmerge values too. Self-Patterson doesn’t suggest translational symmetry, but the self-rotation function (SRF) suggests high NCS (see below/attached). I’m hoping the SRF might be helpful in trying to confirm/dismiss C2 is the likely space group, and perhaps suggest a logical assembly with the ASU (I see strong 2-fold and 3-fold NCS operators suggesting to me dimeric trimers or vice versa - however, I’ve never had to really analyze SRFs in the absence of a mol. rep. solution so my interpretation could be wrong). Anyway, any help to bringing a molecular replacement solution closer to reality would be appreciated. Thanks! [image: Inline image 1]
Re: [ccp4bb] Challenging Mol.Rep. Problem
Try othercell to look for other indexing - C2 is a possible alternate SG to H32. And have you run pointless to check on symmetry operators? You certainly have 4 independent 3 fold rotations which would generate 12 mols per ASU. But first think about higher symmetry SGs - that will make the whole problem easier! Eleanor On 9 December 2014 at 19:00, Artem Evdokimov artem.evdoki...@gmail.com wrote: Generally fab geometry presents problems for mr due to variable interdomain angle. Assuming your sg is correct and this is not a twin, i would try searching at 4 or even 5 a resolution. I would not reject the chance that you have high solvent content, so you might have fewer molecules in au than expected from vm estimates. We recently solved a whole bunch of structures with large unit cell dimensions and high solvent content in the 75% or even 82% range. All had issues with resolution, freezing, and twinning - similar to shat you describe. In a few cases we screened over 35 crystals (of the same protein variant, same condition, just repeats) to find one that diffracted beyond 4a. Don't give up! May the pork be with you. Artem On Dec 9, 2014 12:32 PM, Mo Wong mowon...@gmail.com wrote: Hi all, I’m stuck on a rather complex molecular replacement problem. The crystals are of an antigen-Fab complex totaling ~67 kDa (waiting to confirm using PAGE gel). They diffract to ~3.5A at the synchrotron and process in C2 with dimensions 220x130x230 and beta at 103 so it looks like there are round 9to12-ish copies in the ASU. The overall Rmerge is high at ~25% with I/sig cutoff ~2 and redundancy of 5; however, at 4.5A this drops to ~15%. Furthermore, processing in P1 gives similar Rmerge values too. Self-Patterson doesn’t suggest translational symmetry, but the self-rotation function (SRF) suggests high NCS (see below/attached). I’m hoping the SRF might be helpful in trying to confirm/dismiss C2 is the likely space group, and perhaps suggest a logical assembly with the ASU (I see strong 2-fold and 3-fold NCS operators suggesting to me dimeric trimers or vice versa - however, I’ve never had to really analyze SRFs in the absence of a mol. rep. solution so my interpretation could be wrong). Anyway, any help to bringing a molecular replacement solution closer to reality would be appreciated. Thanks! [image: Inline image 1]
Re: [ccp4bb] Challenging Mol.Rep. Problem
Hi, We’ve had good luck with Phaser in placing many copies of proteins or domains, if the model is reasonably good. Because of the variable elbow angle, you probably want to place the domains separately. However, as soon as you find one convincing assembly you can switch to searching for the whole Fab. Alternatively, you can try a bunch of different models and hope that one is right. Something that might be useful is a new “MR_GYRE feature in Phaser. This is a likelihood-based version of the PC refinement that can be done in CNS, to optimise the relative orientations of domains between the rotation and translation searches. If you get stuck, we might be able to give a hand — we haven’t had much experience ourselves with using this on unsolved structures. Of course, the advice you’ve had from others to be certain first of the space group is very good! Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 9 Dec 2014, at 18:31, Mo Wong mowon...@gmail.com wrote: Hi all, I’m stuck on a rather complex molecular replacement problem. The crystals are of an antigen-Fab complex totaling ~67 kDa (waiting to confirm using PAGE gel). They diffract to ~3.5A at the synchrotron and process in C2 with dimensions 220x130x230 and beta at 103 so it looks like there are round 9to12-ish copies in the ASU. The overall Rmerge is high at ~25% with I/sig cutoff ~2 and redundancy of 5; however, at 4.5A this drops to ~15%. Furthermore, processing in P1 gives similar Rmerge values too. Self-Patterson doesn’t suggest translational symmetry, but the self-rotation function (SRF) suggests high NCS (see below/attached). I’m hoping the SRF might be helpful in trying to confirm/dismiss C2 is the likely space group, and perhaps suggest a logical assembly with the ASU (I see strong 2-fold and 3-fold NCS operators suggesting to me dimeric trimers or vice versa - however, I’ve never had to really analyze SRFs in the absence of a mol. rep. solution so my interpretation could be wrong). Anyway, any help to bringing a molecular replacement solution closer to reality would be appreciated. Thanks! SELF_RF.gif
