Re: [ccp4bb] off topic: rmsf in simulation
Different proteins do different things. Some adopt fewer conformations and a more rigid structure after binding a ligand, and others do the opposite. Haemoglobin is a nice example of a protein that becomes a lot more flexible after picking up ligands. For any reaction of the kind P + L - PL there is an entropy cost of making one molecule from two. For the protein to activate low frequency modes in the complex is one way to compensate for this by increasing the entropy of the bound form. The paper by Sturtevant (PNAS 74, 2236, 1977) is worth a read, as is Cooper and Dryden (Eur Biophys J, 11, 103, 1984), if you are interested in relating fluctuations to thermodynamics. All too often people attempt direct comparisons of structural models and affinities without realising that the so-called angstroms to calories problem often frames the question in a form that cannot be answered sensibly. For example, imagine a protease which is produced as a zymogen. Both forms may have essentially identical crystal structures even though the zymogen is more flexible. The protease can be activated by loss of vibrational modes in the unbound state which are re-awakened in the complex with substrate; hence the zymogen will have lower substrate binding and activity. You might be interested in a review by Homans (ChemBioChem 6, 1585, 2005) which discusses the use of NMR to look at entropy changes in protein-ligand binding reactions. It is by no means unusual for a residue's entropy to increase in the bound state, although in your case it seems to be the whole protein! On Dec 9, 2012, at 1:05 PM, anita p wrote: Hi All, I am trying to understand the mechanism of protein-peptide interaction in two complexes (protein-pepA and protein-pepB). While trying to perform some simulation experiments, I find that the root mean square fluctuation (RMSF) by residues of protein in the complex is higher than that of the protein alone. Please refer the figure attached to this email. pepA binds with higher affinity (in uM-range) than pepB according to invitro studies. Does this happen normally?? Please advice. Thanks in advance Anita RMSF.png
Re: [ccp4bb] off topic: rmsf in simulation
to complement the very nice description by jeremy, you may wish to try and decompose the vibrational modes to get this sense by focussing on the origins of the red shift in the vibrational spectrum and this accounts largely for the increased vibrational entropies upon complexation. This paper may be used as a guide (Dissecting the vibrational entropy change on protein/ligand binding: burial of a water molecule in bovine pancreatic trypsin inhibitor J Phys Chem B 2001 105 8050-8055) There is some very nice work by Olano Rick in JACS 2004 126:7991 on Hydration free energies and entropies for water in protein interiors. and by carol post on how the increased entropies upon complexation are the origin of the mechanism of some drugs. (for example Influence of an Antiviral Compound on the Temperature Dependence of Viral Protein Flexibility and Packing: a Molecular Dynamics Study J. Mol. Biol. (1998) 276: 331-337) Quoting Jeremy Tame jt...@tsurumi.yokohama-cu.ac.jp: Different proteins do different things. Some adopt fewer conformations and a more rigid structure after binding a ligand, and others do the opposite. Haemoglobin is a nice example of a protein that becomes a lot more flexible after picking up ligands. For any reaction of the kind P + L - PL there is an entropy cost of making one molecule from two. For the protein to activate low frequency modes in the complex is one way to compensate for this by increasing the entropy of the bound form. The paper by Sturtevant (PNAS 74, 2236, 1977) is worth a read, as is Cooper and Dryden (Eur Biophys J, 11, 103, 1984), if you are interested in relating fluctuations to thermodynamics. All too often people attempt direct comparisons of structural models and affinities without realising that the so-called angstroms to calories problem often frames the question in a form that cannot be answered sensibly. For example, imagine a protease which is produced as a zymogen. Both forms may have essentially identical crystal structures even though the zymogen is more flexible. The protease can be activated by loss of vibrational modes in the unbound state which are re-awakened in the complex with substrate; hence the zymogen will have lower substrate binding and activity. You might be interested in a review by Homans (ChemBioChem 6, 1585, 2005) which discusses the use of NMR to look at entropy changes in protein-ligand binding reactions. It is by no means unusual for a residue's entropy to increase in the bound state, although in your case it seems to be the whole protein! On Dec 9, 2012, at 1:05 PM, anita p wrote: Hi All, I am trying to understand the mechanism of protein-peptide interaction in two complexes (protein-pepA and protein-pepB). While trying to perform some simulation experiments, I find that the root mean square fluctuation (RMSF) by residues of protein in the complex is higher than that of the protein alone. Please refer the figure attached to this email. pepA binds with higher affinity (in uM-range) than pepB according to invitro studies. Does this happen normally?? Please advice. Thanks in advance Anita RMSF.png
Re: [ccp4bb] refining against weak data and Table I stats
Le Vendredi 7 Décembre 2012 18:48 CET, Gerard Bricogne g...@globalphasing.com a écrit: May I add something to Gerard's comment. In the same vein, provided one does consider two sets of terms with zero mean (which corresponds to the proviso mentioned by Gerard), one can define an R-factor R as the sine of the same angle leading to a correlation coefficient C and one has R^2 + C^2 = 1. Thus, in some way, on a practical ground, an R-factor is a sensitive criterion for higly correlated data, whereas a correlation coefficient is better suited for poorly correlated data. Likely, I just rephrased here ideas that have been written long time ago in well-known papers. Did I ? Philippe Dumas Dear Zbyszek, That is a useful point. Another way of making it is to notice that the correlation coefficient between two random variables is the cosine of the angle between two vectors of paired values for these, with the proviso that the sums of the component values for each vector add up to zero. The fact that an angle is involved means that the CC is independent of scale, while the fact that it is the cosine of that angle makes it rather insensitive to small-ish angles: a cosine remains close to 1.0 for quite a range of angles. This is presumably the nature of correlation coefficients you were referring to. With best wishes, Gerard. -- On Fri, Dec 07, 2012 at 11:14:50AM -0600, Zbyszek Otwinowski wrote: The difference between one and the correlation coefficient is a square function of differences between the datapoints. So rather large 6% relative error with 8-fold data multiplicity (redundancy) can lead to CC1/2 values about 99.9%. It is just the nature of correlation coefficients. Zbyszek Otwinowski Related to this, I've always wondered what CC1/2 values mean for low resolution. Not being mathematically inclined, I'm sure this is a naive question, but i'll ask anyway - what does CC1/2=100 (or 99.9) mean? Does it mean the data is as good as it gets? Alan On 07/12/2012 17:15, Douglas Theobald wrote: Hi Boaz, I read the KK paper as primarily a justification for including extremely weak data in refinement (and of course introducing a new single statistic that can judge data *and* model quality comparably). Using CC1/2 to gauge resolution seems like a good option, but I never got from the paper exactly how to do that. The resolution bin where CC1/2=0.5 seems natural, but in my (limited) experience that gives almost the same answer as I/sigI=2 (see also KK fig 3). On Dec 7, 2012, at 6:21 AM, Boaz Shaanan bshaa...@exchange.bgu.ac.il wrote: Hi, I'm sure Kay will have something to say about this but I think the idea of the K K paper was to introduce new (more objective) standards for deciding on the resolution, so I don't see why another table is needed. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Douglas Theobald [dtheob...@brandeis.edu] Sent: Friday, December 07, 2012 1:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] refining against weak data and Table I stats Hello all, I've followed with interest the discussions here about how we should be refining against weak data, e.g. data with I/sigI 2 (perhaps using all bins that have a significant CC1/2 per Karplus and Diederichs 2012). This all makes statistical sense to me, but now I am wondering how I should report data and model stats in Table I. Here's what I've come up with: report two Table I's. For comparability to legacy structure stats, report a classic Table I, where I call the resolution whatever bin I/sigI=2. Use that as my high res bin, with high res bin stats reported in parentheses after global stats. Then have another Table (maybe Table I* in supplementary material?) where I report stats for the whole dataset, including the weak data I used in refinement. In both tables report CC1/2 and Rmeas. This way, I don't redefine the (mostly) conventional usage of resolution, my Table I can be compared to precedent, I report stats for all the data and for the model against all data, and I take advantage of the information in the weak data during refinement. Thoughts? Douglas ^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^` Douglas L. Theobald Assistant Professor Department of Biochemistry Brandeis University Waltham, MA 02454-9110 dtheob...@brandeis.edu http://theobald.brandeis.edu/ ^\ /` /^. / /\
Re: [ccp4bb] Binding constants/kinetics for crystallisation
Good points have been brought up; here's one more to consider from my experience. If you are going to run SEC prior to crystallization, I would highly recommend running a native gel of the peak you collect. Especially if you don't know the stoichiometry or if the stoichiometry is complex. I crystallized a multimeric complex where a large oligomer bound to four smaller proteins with high affinity (sub nM), and even after making the complex in stoichiometric excess, somehow one of the ligands would fall off during SEC (probably the 3-bound and 4-bound mers were in equilibrium on column) and only after I discovered this by running a native gel did I start getting single crystals instead of multi-lattice xtals. Basically the band for the full complex was fuzzy (I.e. A range of stoichiometries) and after titrating in more ligand after SEC did the band look nice. Also, you can run a native gel of your crystals (assuming you obtain some!) by harvesting a number of them in a buffer in which they'll dissolve and then use a sensitive staining method, like silver staining. Good luck! Geoff On 12/7/12 4:00 PM, CCP4BB automatic digest system lists...@jiscmail.ac.uk wrote:
Re: [ccp4bb] off topic: rmsf in simulation
Hi All thanks for your detailed reply. A higher RMSF(as I showed in the png.) *doesnot *mean that the RMSD for Calpha backbone showed be high. Am I correct ?? Because in my case the backbone RMSD for the receptor of the peptide bound structure is lower than the receptor alone. Because I wanted to know if my simulations have gone fine. thanks again in advance. Anita On Sun, Dec 9, 2012 at 6:44 PM, Chandra Verma chan...@bii.a-star.edu.sgwrote: to complement the very nice description by jeremy, you may wish to try and decompose the vibrational modes to get this sense by focussing on the origins of the red shift in the vibrational spectrum and this accounts largely for the increased vibrational entropies upon complexation. This paper may be used as a guide (Dissecting the vibrational entropy change on protein/ligand binding: burial of a water molecule in bovine pancreatic trypsin inhibitor J Phys Chem B 2001 105 8050-8055) There is some very nice work by Olano Rick in JACS 2004 126:7991 on Hydration free energies and entropies for water in protein interiors. and by carol post on how the increased entropies upon complexation are the origin of the mechanism of some drugs. (for example Influence of an Antiviral Compound on the Temperature Dependence of Viral Protein Flexibility and Packing: a Molecular Dynamics Study J. Mol. Biol. (1998) 276: 331-337) Quoting Jeremy Tame jt...@tsurumi.yokohama-cu.ac.**JPjt...@tsurumi.yokohama-cu.ac.jp : Different proteins do different things. Some adopt fewer conformations and a more rigid structure after binding a ligand, and others do the opposite. Haemoglobin is a nice example of a protein that becomes a lot more flexible after picking up ligands. For any reaction of the kind P + L - PL there is an entropy cost of making one molecule from two. For the protein to activate low frequency modes in the complex is one way to compensate for this by increasing the entropy of the bound form. The paper by Sturtevant (PNAS 74, 2236, 1977) is worth a read, as is Cooper and Dryden (Eur Biophys J, 11, 103, 1984), if you are interested in relating fluctuations to thermodynamics. All too often people attempt direct comparisons of structural models and affinities without realising that the so-called angstroms to calories problem often frames the question in a form that cannot be answered sensibly. For example, imagine a protease which is produced as a zymogen. Both forms may have essentially identical crystal structures even though the zymogen is more flexible. The protease can be activated by loss of vibrational modes in the unbound state which are re-awakened in the complex with substrate; hence the zymogen will have lower substrate binding and activity. You might be interested in a review by Homans (ChemBioChem 6, 1585, 2005) which discusses the use of NMR to look at entropy changes in protein-ligand binding reactions. It is by no means unusual for a residue's entropy to increase in the bound state, although in your case it seems to be the whole protein! On Dec 9, 2012, at 1:05 PM, anita p wrote: Hi All, I am trying to understand the mechanism of protein-peptide interaction in two complexes (protein-pepA and protein-pepB). While trying to perform some simulation experiments, I find that the root mean square fluctuation (RMSF) by residues of protein in the complex is higher than that of the protein alone. Please refer the figure attached to this email. pepA binds with higher affinity (in uM-range) than pepB according to invitro studies. Does this happen normally?? Please advice. Thanks in advance Anita RMSF.png
