[ccp4bb] Mutated biological binding interactions as crystal contacts
Hi all - I am looking for examples of structures of protein-protein complexes (homo or hetero) wherein the crystallized proteins had mutations within their binding interfaces, and yet the same (weakened) interactions were still recapitulated in crystal contacts. Any leads would be much appreciated. Thanks, Owen
Re: [ccp4bb] How to detect the concentration of detergent?
Could anybody tell me how to detect the concentration of detergent? From Butler et al. (2004) J Mol Biol 340: 797-808 The concentration of DDM was determined by a colorimetric assay that detects the sugar component of the detergent, which has given results identical with the standard procedure using radioactive DDM (T. Warne, unpublished data). A 60 μl sample containing 4–16 μg of detergent was mixed briefly with 300 μl of 5% (w/v) phenol and then 720 μl of concentrated sulphuric acid was added followed immediately by vortex mixing for five seconds. The samples were left to cool for 30 minutes and then the absorbance at 490 nm was measured. A typical standard curve contained six samples containing between 0 μg and 20 μg of DDM, which gave a straight line (r20.97). HTH, Owen On Oct 4, 2010, at 10:42 AM, Van Den Berg, Bert wrote: Hi YB, For membrane protein crystallization it is common practice (although not always necessary) to dialyze the protein after the final concentration step (against GF buffer). The problem with DDM is that dialysis is slow due to the low cmc, and in general it is advisable to finish the prep and set up drops as quickly as possible. I would not dialyze more than 1 x O/N, although if your protein is really stable you could try longer. You could also try 100 kDa cutoff concentrators, as these may allow passage of empty DDM micelles. As for measuring the detergent concentration, in the case of DDM and other sugar-containing detergents you could do a sugar (Fehling’s) kind of assay. I’m not sure if anything has been published, but it should be fairly easy to do. You could also try TLC, but this may be less accurate. Also, 10 mg/ml is not high at all (although its a good starting point), and you should try much higher if most drops are clear: try 50 mg/ml and see what happens. Good luck, Bert On 10/4/10 10:28 AM, yybbll yyb...@gmail.com wrote: Dear all, I want to crystallize a symport transporter, which contains 12 transmembrane alpha-helices. We used Ni-resin column firstly, and then size exclusion. After size exclusion, only one peak, it is very nice. the final condition is 10 mM mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, and 0.02% DDM. The CMC of DDM is about 0.008%. However, when we concentrate protein using a concentrator with 50 kDa cutoff, detergent all was concentrated. So final the concentration of detergent should be very high (10 times more than CMC). We don't know how to detect the concentration of detergent. We used these samples to grow crystal. We found almost drops are clear, and the final concentration of protein is about 10 mg/ml. For membrane protein, I think this concentration is high. But for us, we can obtain so high concentration easily. Could anybody tell me how to detect the concentration of detergent? And how to dilute detergent? Thanks all. Y.B. Lin 2010-10-04 yybbll
Re: [ccp4bb] Translational pseudosymmetry?
Dear Eleanor - That is correct. The pseudo-sg is P6, and the structure has been refined in this sg. The intensity difference between the strong and weak subsets is quite significant that for most data sets, auto- indexing routines will miss the weak spots and pick the pseudo-sg instead. The pseudo-sg is a'=b'=90, c'=56, the true sg is a=b=156, c=56. Note that a = a' * sqrt(3). So, the sg assignment is certain. Owen On May 20, 2010, at 6:33 AM, Eleanor Dodson wrote: This looks a bit strange.. If you have a hexamer in the asymmetric unit, in P3, then that means all symmetry copies lie in the same plane. To generate the Patterson peak, 2/3,1/3,0 the hexamer must be centred at 1/3,1/3, z (with symmetry equivalents 0,-1/3,z and -1/3,0,z ) I would expect ypu to have a pseudo higher symmetry SG - does pointless make any suggestions? Eleanor Jürgen Bosch wrote: Hi Owen, you should also make the following plot with your data: y-axis relative intensity of off-origin peak versus x-axis resolution cutoff used for calculation (30 Å - 4 Å in 2 Å steps). You can have multiple cases of shifts and I would start with a perfect hexamer first, take some random monomer and apply a perfect sixfold, move it along the axis where it should be in your crystal lattice (things get more complicated if you have a top/down hexamer, so keep it simple). Now if you shift your hexamer to 2/3,1/3,0 your plot should yield a straight line and be independent of resolution. Now start rotating the second hexamer relative to the first clockwise with your sixfold, I would use increments of 3 degrees, which will result in 19 models, then recalculate the off- origin peak heights and see if they match up with your data. I should note here, if your real data does not show a strong drop in peak height of the off-origin peak, then you most likely don't have a slight rotational translation in your second hexamer. One other important thing you should look at is the relative orientation of your sixfold axis, is it truly perfectly aligned with one of the cell axis ? If not fix this in your model, otherwise your calculations will be of academic nature. For this particular case the use of GLRF is more helpful than MOLREP (sorry Garib, but maybe Garib can come up with a solution to zoom into certain peaks like you can do in GLRF). When the tilt is fixed you should be able to figure out the rotational translation in your second hexamer. Enjoy your puzzle, Jürgen P.S. P3 is certain ? Check with pointless or by human brain visual inspection (HBVI) On May 15, 2010, at 11:53 PM, Owen Pornillos wrote: Dear ccp4bb – I have questions with regards to crystal disorder that gives rise to translational pseudosymmetry. We have a rotationally hexameric protein that crystallized in P3, with one hexamer in the asu. The local 6-fold axis of the hexamer is non- crystallographic, and is essentially parallel to the crystallographic 3-fold, which gave rise to translational pseudosymmetry. Intensities for the (h,h+/-3n,l) reflections were on average about 8 times stronger than the weak reflections, and the native patterson gave an off-origin peak about 70-80% of origin (depending on the crystal) at fractional coordinates (2/3,1/3,0). We are hypothesizing that the break in local 6-fold symmetry is caused by small rigid-body displacements of each subunit (as opposed to conformational changes in the protein), and we are trying to estimate the magnitude of the displacements in the crystal. To do this, a perfectly symmetric hexamer with the local 6-fold axis parallel to the crystallographic 3-fold was generated, and then shifts were introduced to the atomic coordinates. The direction of the shift was chosen randomly for each atom, and a single magnitude applied to all atoms, which was then changed incrementally. Structure factors were calculated from these models, and their pattersons were examined. The magnitude of the off-origin peak could be reproduced with an atomic shift of say, 1 Å. Because all of these calculations were made with synthetic structure factors, this is not necessarily a reliable estimate. The questions are, how far off are we, and in what direction (i.e., are these shifts underestimates or overestimates)? Is there a way to obtain a reliable estimate? Thanks in advance, Owen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
[ccp4bb] Translational pseudosymmetry?
Dear ccp4bb – I have questions with regards to crystal disorder that gives rise to translational pseudosymmetry. We have a rotationally hexameric protein that crystallized in P3, with one hexamer in the asu. The local 6-fold axis of the hexamer is non- crystallographic, and is essentially parallel to the crystallographic 3-fold, which gave rise to translational pseudosymmetry. Intensities for the (h,h+/-3n,l) reflections were on average about 8 times stronger than the weak reflections, and the native patterson gave an off-origin peak about 70-80% of origin (depending on the crystal) at fractional coordinates (2/3,1/3,0). We are hypothesizing that the break in local 6-fold symmetry is caused by small rigid-body displacements of each subunit (as opposed to conformational changes in the protein), and we are trying to estimate the magnitude of the displacements in the crystal. To do this, a perfectly symmetric hexamer with the local 6-fold axis parallel to the crystallographic 3-fold was generated, and then shifts were introduced to the atomic coordinates. The direction of the shift was chosen randomly for each atom, and a single magnitude applied to all atoms, which was then changed incrementally. Structure factors were calculated from these models, and their pattersons were examined. The magnitude of the off-origin peak could be reproduced with an atomic shift of say, 1 Å. Because all of these calculations were made with synthetic structure factors, this is not necessarily a reliable estimate. The questions are, how far off are we, and in what direction (i.e., are these shifts underestimates or overestimates)? Is there a way to obtain a reliable estimate? Thanks in advance, Owen
