Re: [ccp4bb] truncate ignorance
This is a very educational thread but I should remind you that the assumed distributions are NOT reliable when either a) the data is very anisotropic, or b) the data is very incomplete or c) there is a non-crystallographic translation vector in the structure or d) the data is twinned. I for one dont really know what to do about this, but remember the Is are as measured and are in these cases safer reflections of the experiment.. Eleanor Ian Tickle wrote: Having read the remainder of the paper more carefully I note that the authors do go into an extensive discussion about Jeffreys (which they don't recommend) and Wilson priors, which indeed overcome my objection to the use of the improper prior. They conclude that the simpler expression is adequate for their purposes. George Sheldrick's objection would be valid for their simple prior since the effect on intensities in a shell where the true average intensity was zero would be to give a non-zero positive and hence biased average intensity. However I don't think it's valid to conclude without more careful analysis that their simple prior is also adequate in the single crystal case, since the kinds of errors encountered (namely from deconvoluting overlapping reflections) are quite different. -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: 08 September 2008 22:20 To: Jacob Keller Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] truncate ignorance I would also recommend reading of the following paper: D.S. Sivia W.I.F. David (1994), Acta Cryst. A50, 703-714. A Bayesian Approach to Extracting Structure-Factor Amplitudes from Powder Diffraction Data. Despite of the title, most of the analysis presented in this paper applies equally well to single-crystal data (see especially sections 3 and 5). If you are not interested in the specific powder-diffraction problems (i.e. overlapping peaks), you can simply skip sections 4 and 6. A few interesting points from this paper : (1) The conversion from I's to F's can be done (in a Bayesian way) by applying two simple formula (equations 11 and 12 in the paper), which, for all practical purposes, are as valid as the more complicated French Wilson procedure (see discussion in section 5). (2) Re. the use of I's rather than F's : this is discussed on page 710 (final part of section 5). The authors seem to be more in favor of using F's. Marc Schiltz Quoting Jacob Keller [EMAIL PROTECTED]: Does somebody have a .pdf of that French and Wilson paper? Thanks in advance, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Ethan Merritt [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 08, 2008 3:03 PM Subject: Re: [ccp4bb] truncate ignorance On Monday 08 September 2008 12:30:29 Phoebe Rice wrote: Dear Experts, At the risk of exposing excess ignorance, truncate makes me very nervous because I don't quite get exactly what it is doing with my data and what its assumptions are. From the documentation: ... the truncate procedure (keyword TRUNCATE YES, the default) calculates a best estimate of F from I, sd(I), and the distribution of intensities in resolution shells (see below). This has the effect of forcing all negative observations to be positive, and inflating the weakest reflections (less than about 3 sd), because an observation significantly smaller than the average intensity is likely to be underestimated. = But is it really true, with data from nice modern detectors, that the weaklings are underestimated? It isn't really an issue of the detector per se, although in principle you could worry about non-linear response to the input rate of arriving photons. In practice the issue, now as it was in 1977 (FrenchWilson), arises from the background estimation, profile fitting, and rescaling that are applied to the individual pixel contents before they are bundled up into a nice Iobs. I will try to restate the original French Wilson argument, avoiding the terminology of maximum likelihood and Bayesian statistics. 1) We know the true intensity cannot be negative. 2) The existence of Iobs0 reflections in the data set means that whatever we are doing is producing some values of Iobs that are too low. 3) Assuming that all weak-ish reflections are being processed equivalently, then whatever we doing wrong for reflections with Iobs near zero on the negative side surely is also going wrong for their neighbors that happen to
[ccp4bb] interface energetics
Hey there, I am working on (the theoretical side of) a protein complex whose structure has been solved. The protein homo-dimerizes, mediated primarily by two long helices. Using sequencing alignment and the WHAT IF server, I built monomeric hybrid models containing the bulk of the known structure and the dimerization helices of homologous proteins. Naturally, I want to know how likely they are to form dimers. To look at the energetics, I've run the phenix geometry regularization algorithm to minimize clashes and side chain energies. The backbone conformation only changes minimally. Next I calculated in Rosetta the energetic scores of the models before and after regularization and compared with that of the native structure. This gave me some numbers that are not inconsistent with experiments. Before I sit down and write this up, I wanted to ask the community if what I've done makes sense and if there are alternative methods for minimizing and calculating interface energies. I don't necessarily need docking algorithms as the interface is known. I just want to get an energetic description. Thank you. Andreas -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London
Re: [ccp4bb] applied optics--LCD projectors
Dear All, a very late reaction, but we have a Dell proyector that elicit the same reaction as yours did. In another building there is a BenQ MP622c, which is much, much better. Mark This is way off-topic, but that's never stopped me before. And what group is better qualified to pontificate about matters lying at the intersection of computers and optics than this one? The LCD projector in our departmental seminar room was stolen over the weekend (!), and I have been asked to look into what we should buy to replace it. The missing projector was a Dell 3300MP, and IMHO it sucked. If I had a nickel for every seminar speaker who said, Well, you can't see this, but on my laptop it's very clear that..., I'd never need to write another grant. Alas, I know very little about what's available and what performs well. Perhaps you can save me hours of careening around the internet researching this question. Do any of you good folks have experience with particular projectors that you like/don't like? Or perhaps have a reasoned opinion (or even a wild irrational idée fixe) about the sorts of specifications a good projector should exhibit? The room in question is not large (about 8 x 10 m, seating a max of ca. 40 people for a talk). I should mention that we're NOT looking for any kind of stereographic projection here (cool as that would be); just plain, vanilla, projection of the image shown on the laptop's screen. Many thanks for any info you can contribute. Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED] Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/
[ccp4bb] postdoctoral position in Greifswald, Germany
Institute for Biochemistry, University of Greifswald, Germany Dear All, In the group of Prof. Hinrichs a postdoctoral position is available for initially 3 years. The position involves research and teaching, usually also for undergraduates. Some knowledge in German is thus preferred, you should test your skills with the official posting of the university regarding this position: http://www.uni-greifswald.de/informieren/stellenausschreibung/oeffentlich/wissenschaftliches- personal/institut-fuer-biochemie-nr-08b67.html Questions and applications should be sent to Prof. Hinrichs ([EMAIL PROTECTED] greifswald.de) before end of September. See also our homepage (http://www.chemie.uni-greifswald.de/~biochem/endex. htm) or http://www.rigaku.com/protein/sotm-002.html for an example of current equipment and research in the lab. Dr. Gottfried Palm Ernst-Moritz-Arndt-Universität Inst. für Biochemie (MNF) Abt. Biochemie I Felix-Hausdorff-Straße 4 17489 Greifswald email [EMAIL PROTECTED] === WEB-Mailer der Uni-Greifswald ( http://www.uni-greifswald.de/ ) ===
Re: [ccp4bb] truncate ignorance
-Original Message- From: Bart Hazes [mailto:[EMAIL PROTECTED] Sent: 08 September 2008 23:44 To: Ian Tickle Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] truncate ignorance How a seemingly innocent question can explode ... Well there seems to be a widespread misunderstanding of the French Wilson (Truncate) procedure! I actually thought I understood this but little of what has been discussed matches my mental picture of the truncate process. Truncate can do multiple things, but the truncate part I believe really just deals with converting I to F and the inherent problems due to experimental error and mathematical problems in deriving SigF from SigI when I is near zero. This only depends on how close I is to zero (relative to SigI), and not on the Wilson distribution itself. Not sure what you mean here: in the French Wilson procedure as currently implemented by Truncate the corrected F sigF will depend on the values of I, sigI and the Wilson distribution parameter, Sigma. This is assumed not to be a constant for the whole data set, as is implied by a linear Wilson plot, but instead is obtained by spline fitting to the log(average I) in resolution shells. This is fully described in the FW paper itself of course, but I find that here is a more succinct summary of the procedure: http://xtal.sourceforge.net/man/bayest-desc.html . This also has the advantage that it covers the case of obtaining the corrected I sigI which FW (or Truncate itself) doesn't. I did some illustrative calculations (see attachment) using the equations at the BAYEST site so you can see the effect of varying the WDP and the measured intensity. I used the numerical integration routine DQAGI (http://www.netlib.org/slatec/src/dqagi.f) to do the integrals (which cannot unfortunately be expressed in closed form), and for simplicity and to keep the number of variables to a minimum I assumed that sigI = 1 throughout (this does not imply any loss of generality since you can simply replace I by I/sigI in the table). My mental picture is as follows: Visualize a gaussian distribution representing I and its standard deviation, with I being close to zero (either positive or negative). Part of the gaussian will stretch into negative-I territory, which is fine for the experimental I (because of experimental error) but not the true I. Given this prior knowledge you can re-estimate I by TRUNCATEing the negative tail of the gaussian and integrating just the positive part to find the new mean and standard deviation. As a result any reflection will become positive (including those starting out with negative I). The extend to which the method affects the intensity depends on how much of a negative tail it has, so nearly no effect on I/SigI=2 reflections and not really that much on even I/SigI=2 reflections. I actually think this is a very elegant solution. The only thing better, is to use I directly and avoid the entire issue. I personally think you want to use the experimental I without correcting it as explained above since it will introduce bias and the refinement procedure should take proper care of random experimental error, unless you mess around with it. However, when you need amplitudes, truncate is the way to go. The procedure you describe (which is the same as the one Peter Zwart described for phenix.reflection_file_converter and the one used in the Sivia David paper) will indeed introduce bias in both I and F because it uses an improper prior, i.e. it implicitly assumes that an infinite intensity is as likely as any other, whereas in reality of course an infinite intensity is physically impossible; this is taken care of by the Wilson distribution (for acentrics) P(I) = exp(-I/S). In practice what this will mean is that the correction added to I is always positive (all I's are shifted towards the average prior I at +infinity). So in fact one can do a lot better and use the FW/Truncate solution instead (which I think is indeed elegant since it produces exactly the result we intuitively expect). As can be seen from the table no bias is introduced for the case where the WDP and hence Itrue is exactly zero: the corrected intensity is also exactly zero. The correction added to I is always positive for I 0 as it must be, but it's negative for large I's, so the net effect is that average I is unbiased. -- Ian Bart Ian Tickle wrote: But there's a fundamental difference in approach, the authors here assume the apparently simpler prior distribution P(I) = 0 for I 0 P(I) = const for I = 0. As users of Bayesian priors well know this is an improper prior since it integrates to infinity instead of unity. This means that, unlike the case I described for the French Wilson formula based on the Wilson distribution which gives unbiased estimates of the true I's and their average, the effect on the corrected intensities
Re: [ccp4bb] truncate ignorance
For comparison I repeated my previous calculation using the flat (non-Wilson) prior that you suggested (you would get the same results using the FW method in the limit of an infinite WDP). So now the results are indeed totally independent of the WDP - but IMO the results are also completely counter-intuitive, i.e. you get I 0 even when you know a priori (from the Wilson plot) that I = 0 !! Imeas I1cI2cI1aI2a FmeasF1c F2cF1aF2a -5.00.1870.1810.1870.181 0.0000.384 0.1970.3840.197 -4.00.2260.2160.2260.216 0.0000.423 0.2150.4230.215 -3.00.2830.2660.2830.266 0.0000.475 0.2390.4750.239 -2.00.3730.3380.3730.338 0.0000.549 0.2690.5490.269 -1.00.5250.4460.5250.446 0.0000.656 0.3070.6560.307 0.00.7980.6030.7980.603 0.0000.822 0.3490.8220.349 1.01.2880.7941.2880.794 1.0001.070 0.3771.0700.377 2.02.0550.9422.0550.942 1.4141.388 0.3581.3880.358 3.03.0040.9933.0040.993 1.7321.706 0.3041.7060.304 4.04.0001.0004.0001.000 2.0001.983 0.2581.9830.258 5.05.0001.0005.0001.000 2.2362.224 0.2282.2240.228 -- Ian Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] truncate ignorance
Thanks for all the interesting answers so far! The anisotropy issue is one that got me worrying about truncate for data from DNA-containing crystals in particular - and the fact that since its a default in ccp4i, new people have stopped worrying about whether or not they should use it. The DNAs usually stack end-to-end, and thus are very often aligned with a particular axis. Since all those nice flat bases are ~3.4A apart, there are often whomping spots in only one direction at ~3.4A (even if the DNA isn't even half the total scattering mass). So even if the overall diffraction limits are roughly isotropic, in certain resolution shells isotropy is still a bad assumption. Phoebe Original message Date: Tue, 9 Sep 2008 09:59:58 +0100 From: Eleanor Dodson [EMAIL PROTECTED] Subject: [SPAM:#] Re: [ccp4bb] truncate ignorance To: CCP4BB@JISCMAIL.AC.UK This is a very educational thread but I should remind you that the assumed distributions are NOT reliable when either a) the data is very anisotropic, or b) the data is very incomplete or c) there is a non-crystallographic translation vector in the structure or d) the data is twinned. I for one dont really know what to do about this, but remember the Is are as measured and are in these cases safer reflections of the experiment.. Eleanor Ian Tickle wrote: Having read the remainder of the paper more carefully I note that the authors do go into an extensive discussion about Jeffreys (which they don't recommend) and Wilson priors, which indeed overcome my objection to the use of the improper prior. They conclude that the simpler expression is adequate for their purposes. George Sheldrick's objection would be valid for their simple prior since the effect on intensities in a shell where the true average intensity was zero would be to give a non-zero positive and hence biased average intensity. However I don't think it's valid to conclude without more careful analysis that their simple prior is also adequate in the single crystal case, since the kinds of errors encountered (namely from deconvoluting overlapping reflections) are quite different. -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: 08 September 2008 22:20 To: Jacob Keller Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] truncate ignorance I would also recommend reading of the following paper: D.S. Sivia W.I.F. David (1994), Acta Cryst. A50, 703- 714. A Bayesian Approach to Extracting Structure-Factor Amplitudes from Powder Diffraction Data. Despite of the title, most of the analysis presented in this paper applies equally well to single-crystal data (see especially sections 3 and 5). If you are not interested in the specific powder- diffraction problems (i.e. overlapping peaks), you can simply skip sections 4 and 6. A few interesting points from this paper : (1) The conversion from I's to F's can be done (in a Bayesian way) by applying two simple formula (equations 11 and 12 in the paper), which, for all practical purposes, are as valid as the more complicated French Wilson procedure (see discussion in section 5). (2) Re. the use of I's rather than F's : this is discussed on page 710 (final part of section 5). The authors seem to be more in favor of using F's. Marc Schiltz Quoting Jacob Keller [EMAIL PROTECTED]: Does somebody have a .pdf of that French and Wilson paper? Thanks in advance, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Ethan Merritt [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 08, 2008 3:03 PM Subject: Re: [ccp4bb] truncate ignorance On Monday 08 September 2008 12:30:29 Phoebe Rice wrote: Dear Experts, At the risk of exposing excess ignorance, truncate makes me very nervous because I don't quite get exactly what it is doing with my data and what its assumptions are. From the documentation: ... the truncate procedure (keyword TRUNCATE YES, the default) calculates a best estimate of F from I, sd (I), and the distribution of intensities in resolution shells (see below). This has the effect of forcing all negative observations to be positive, and inflating the weakest reflections (less than about 3 sd), because an observation significantly smaller than the average intensity is likely to be underestimated. = But is it really true, with data from nice modern
Re: [ccp4bb] truncate and anisotropy
On Sep 9, 2008, at 8:12 AM, Phoebe Rice wrote: Thanks for all the interesting answers so far! The anisotropy issue is one that got me worrying about truncate for data from DNA-containing crystals in particular - and the fact that since its a default in ccp4i, new people have stopped worrying about whether or not they should use it. The DNAs usually stack end-to-end, and thus are very often aligned with a particular axis. Since all those nice flat bases are ~3.4A apart, there are often whomping spots in only one direction at ~3.4A (even if the DNA isn't even half the total scattering mass). So even if the overall diffraction limits are roughly isotropic, in certain resolution shells isotropy is still a bad assumption. Phoebe We have a similar phenomenon with RNA, even if it is more globular. If I refine with phenix.refine on I's rather than F's, and avoid truncate, I have noticed the maps look slightly different. In one case the Mn cluster at 2.0 Å resolution is better defined in the sigmaA-weighted 2Fo-Fc map made from truncate/refmac than it is in the sigmaA-weighted 2Fo-Fc map made from phenix.refine. I wonder if truncate is actually helping more than hurting in this case?
[ccp4bb] Are cell parameter shifts real?
AW: [ccp4bb] truncate ignoranceI have had a general crystallogrphy question for quite a while, upon which perhaps the CCP4BB members can shed some light: Are small shifts in cell parameters real? I know that datasets at cryo vs. rt, for example, are often the same spacegroup, but have smaller cell dimensions by (several?) angstroms, with accompanying lower B-factors, so the reality of changes seems pretty plausible, but I find it difficult to imagine how all of the units cells composing a crystal can shrink in harmony, without cracking the crystal or otherwise. The *mechanism* of cell changes is hard to imagine, even in the case of two similar crystals, perhaps even from the same drop. Do all of the unit cells have to change at exactly the same time, making one macro-macromolecular motion? And that is only the case of translations, but what about the observed phenomenon of molecules rotating slightly in unit cells throughout a crystal? Must that also be concerted? What would the intermediate state look like? Is packing really not very specific? But if so, how is it that we get such nice diffraction spots, as it should be the case that some molecules should be in one orientation, others in another? Again, we fit cell parameters in indexing, but perhaps for crystals from exactly the same conditions, this parameter should be fixed at some best value, and other experimental parameters massaged to make up the difference? It seems to me that detector distance et al would be more likely to be difference between two crystals than that there should be two solutions to the crystal packing problem under identical conditions. Also, shouldn't unit cell parameters perhaps be refined during the model building process, or is the experimental error so small that it would make no difference? The bottom line questions are: 1. given that there really are cases of cell shifts, and that there are also probably experimental artifactual changes, how is one to decide what to do? 2. Can there be (or what is?) a plausible mechanism for these shifts? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] ***
Re: [ccp4bb] Are cell parameter shifts real?
How would you tell the difference between a unit cell shift and a wavelength shift when collecting diffraction data at a synchrotron beamline? Well, all the cell length would scale by the wavelength, so that would be one hint that the wavelength changed. If a got longer and c got shorter, then it would be less likely to be a wavelength shift. The crystal-to-detector distance can easily change if the crystal rotation device (i.e. goniometer) rotates the crystal without keeping it at the exact same crystal-to-detector distance. This could easily happen if the crystal is not centered at the rotation point or if more crystal volume rotated into the beam during your experiment. However in a typical experiment the crystal is not going to move by 5 mm and remain in the X-ray beam, so you would not expect your distance to change by 5 mm. Jim _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Tuesday, September 09, 2008 3:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Are cell parameter shifts real? . The bottom line questions are: 1. given that there really are cases of cell shifts, and that there are also probably experimental artifactual changes, how is one to decide what to do? 2. Can there be (or what is?) a plausible mechanism for these shifts? Jacob Keller
Re: [ccp4bb] Are cell parameter shifts real?
AW: [ccp4bb] truncate ignoranceIn thinking about one or two responses, I am wondering whether mosaicity could be the result of having a distribution of each of the cell parameters within one crystal? Is that what mosaicity really is? That is, if axis a has a broad distribution of lengths, due to being caught in the act of changing to the lower-temp state, the spots will be streaky along a*, and so for the others, resulting in an overall mosaicity value which encompasses all of these distributions, being used mainly as a data-processing expedient? The actual physical phenomenon of mosaicity has always puzzled me, but perhaps this is an answer? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Jim Pflugrath To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, September 09, 2008 3:54 PM Subject: Re: [ccp4bb] Are cell parameter shifts real? How would you tell the difference between a unit cell shift and a wavelength shift when collecting diffraction data at a synchrotron beamline? Well, all the cell length would scale by the wavelength, so that would be one hint that the wavelength changed. If a got longer and c got shorter, then it would be less likely to be a wavelength shift. The crystal-to-detector distance can easily change if the crystal rotation device (i.e. goniometer) rotates the crystal without keeping it at the exact same crystal-to-detector distance. This could easily happen if the crystal is not centered at the rotation point or if more crystal volume rotated into the beam during your experiment. However in a typical experiment the crystal is not going to move by 5 mm and remain in the X-ray beam, so you would not expect your distance to change by 5 mm. Jim -- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Tuesday, September 09, 2008 3:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Are cell parameter shifts real? . The bottom line questions are: 1. given that there really are cases of cell shifts, and that there are also probably experimental artifactual changes, how is one to decide what to do? 2. Can there be (or what is?) a plausible mechanism for these shifts? Jacob Keller
Re: [ccp4bb] Are cell parameter shifts real?
lurk mode The answer I think is yes, but partially. Colin Nave published a very nice paper on this, A description of Imperfections in Protein Crystals in Acta Cryst D54, 848-853 (1998). The 'real' mosaicity is a convolution of the variation in cell dimensions within crystallites or domains making up a crystal (observed with X-ray topography), the misalignment of those domains (measured with very parallel beams and fine phi slicing) and finally the volume variation. The measured mosaicity is typically something else entirely as often, even for many cryocooled samples, the mosaicity of the crystal is masked by the contribution of the geometrical and spectral effects of the instrumentation and beam. This is especially the case on a lab source. /lurk mode Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: [EMAIL PROTECTED] Telepathy: 42.2 GHz Heisenberg was probably here! From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Tuesday, September 09, 2008 5:24 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Are cell parameter shifts real? In thinking about one or two responses, I am wondering whether mosaicity could be the result of having a distribution of each of the cell parameters within one crystal? Is that what mosaicity really is? That is, if axis a has a broad distribution of lengths, due to being caught in the act of changing to the lower-temp state, the spots will be streaky along a*, and so for the others, resulting in an overall mosaicity value which encompasses all of these distributions, being used mainly as a data-processing expedient? The actual physical phenomenon of mosaicity has always puzzled me, but perhaps this is an answer? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Jim Pflugrath mailto:[EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, September 09, 2008 3:54 PM Subject: Re: [ccp4bb] Are cell parameter shifts real? How would you tell the difference between a unit cell shift and a wavelength shift when collecting diffraction data at a synchrotron beamline? Well, all the cell length would scale by the wavelength, so that would be one hint that the wavelength changed. If a got longer and c got shorter, then it would be less likely to be a wavelength shift. The crystal-to-detector distance can easily change if the crystal rotation device (i.e. goniometer) rotates the crystal without keeping it at the exact same crystal-to-detector distance. This could easily happen if the crystal is not centered at the rotation point or if more crystal volume rotated into the beam during your experiment. However in a typical experiment the crystal is not going to move by 5 mm and remain in the X-ray beam, so you would not expect your distance to change by 5 mm. Jim From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Tuesday, September 09, 2008 3:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Are cell parameter shifts real? ... The bottom line questions are: 1. given that there really are cases of cell shifts, and that there are also probably experimental artifactual changes, how is one to decide what to do? 2. Can there be (or what is?) a plausible mechanism for these shifts? Jacob Keller
[ccp4bb] Phoenix robot
Dear CCP4BBs, I post the question for one of the staff in our lab: Hi everyone Does anyone have the experience of using Phoenix robot automatically setup the crystal? Which plate the Phoenix could use such as 96-well sitting drop or hanging drop? If available could you also tell me the CAT NO. of hampton!!! Thank you very much for help! Best Hao
Re: [ccp4bb] interface energetics
Andreas, Here's my $0.02. Would you mind clarifying a few things for me? I am working on (the theoretical side of) a protein complex whose structure has been solved. The protein homo-dimerizes, mediated primarily by two long helices. So you have a structure of a homodimer... Using sequencing alignment and the WHAT IF server, I built monomeric hybrid models containing the bulk of the known structure and the dimerization helices of homologous proteins. Naturally, I want to know how likely they are to form dimers. Could you explain what you mean by monomeric hybrid? I'm guessing you want to thread two copies of monomer B onto the backbones of homodimer A. To look at the energetics, I've run the phenix geometry regularization algorithm to minimize clashes and side chain energies. I've never used phenix and I don't know what sort of search function it uses. If it's a deterministic algorithm, like dead end elimination, you'll get the global minimum energy conformation with one run (if it converges, that is). If it's a stochastic algorithm, like Monte Carlo, you'll never know if you're at the global minimum. Your best bet is to run multiple independent minimizations, say 50-100 for starters, and pick the conformation with the lowest energy score. I'm betting its the latter. The backbone conformation only changes minimally. Next I calculated in Rosetta the energetic scores of the models before and after regularization and compared with that of the native structure. This gave me some numbers that are not inconsistent with experiments. The following assumes I correctly stated your design problem. Rosetta does not account for conformational entropy, so the closer the backbones are between the homodimer A and modeled homodimer B to one another, the better. You might want to consider fixing the backbones during minimization. Also, I don't understand the purpose of calculating the energy of the non-optimized structure. I would be more interested in the change in binding energy between the bound and unbound state of the minimized structure. Rosetta can calculate that in -interface mode. There's a flag to keep Rosetta from performing any design calculations; I think its -ddg_only or something like that. Note that this calculation assumes the monomers behave like rigid bodies. Finally, I would minimize homodimer A the same way you minimize modeled homodimer B as a control, then use Rosetta to calculate its change in binding energy. Side chain flips of His, Asn, and Gln will make a big difference. This will give you a number to compare to your modeled dimer. Before I sit down and write this up, I wanted to ask the community if what I've done makes sense and if there are alternative methods for minimizing and calculating interface energies. I don't necessarily need docking algorithms as the interface is known. I just want to get an energetic description. If it were me, I would create the homology model using MODELLER (it uses simulated annealing by default), minimize/relax the structure using Rosetta, then calculate the change in binding energy with Rosetta. Remember to repeat stochastic processes. The 50-100 time guideline was given to me by Deanne Sammond, as in: Sammond DW, Eletr ZM, Purbeck C, Kimple RJ, Siderovski DP, Kuhlman B. Structure-based protocol for identifying mutations that enhance protein-protein binding affinities. J Mol Biol. 2007 Aug 31;371(5):1392-404. Epub 2007 Jun 8. PMID: 17603074 [PubMed - indexed for MEDLINE] Thank you. No worries. Does any one else have any suggestions or corrections? I've only had 7 hours of sleep since Saturday. ~Steve -- Steven Darnell Univeristy of Wisconsin-Madison Madison, WI USA [EMAIL PROTECTED]
Re: [ccp4bb] Phoenix robot
Dear Hao -- On 10 Sep 2008, at 02:00, jxqi wrote: Does anyone have the experience of using Phoenix robot automatically setup the crystal? yes Which plate the Phoenix could use such as 96-well sitting drop or hanging drop? 96 sitting-drop If available could you also tell me the CAT NO. of hampton!!! ?? You might want to have a look there: http://www.rigaku.com/automation/phoenix.html HTH Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] http://personalpages.manchester.ac.uk/staff/leonard.chavas/
Re: [ccp4bb] Phoenix robot
Does anyone have the experience of using Phoenix robot automatically setup thecrystal? We have had a Phoenix in our lab for about 2 years now, and have been very pleased with the performance. Other users I spoke with have also expressed the same satisfaction.Which plate the Phoenix could use such as 96-well sitting drop or hanging drop? Unless there was some new innovation, only a sitting drop format is possible. We tested almost every plate on the market, we found the Intelliplate was the best for crystal retrieval, drop mixing and imaging (using a Formulatrix imaging system).If you're interested in a hanging-drop setup, the Tecan liquid handlers can setup such experiments, though I do not think they can pipette nL volumes.Hope this helps,Jason
