Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
Hi Francisco, I'll play devil's advocate, but a measurement without an estimate of its error is closer to theology than to science. The fact that the standard deviations are not used for calculating an electron density map via FFT is only due to the hidden assumption that you have 100% complete, error-free data set, extending to sufficient high (infinite) resolution. When these assumptions do not apply (as is usually the case with physical reality), then the simple-minded FFT is not the correct inversion procedure (and the data do not univocally define a single map). Under these conditions other inversion mathods are needed (such as maximum entropy) for which the standard deviations are actively being used for calculating the map. My twocents, Nicholas On Tue, 22 May 2012, Francisco Hernandez-Guzman wrote: Hello everyone, My apologies if this comes as basic, but I wanted to get the expert's take on whether or not the sigmaF values are required in the calculation of an electron density map. If I look at the standard ED equation, sigma's don't appear to be a requirement but all the scripts that I've looked at do require sigma values. I wanted to calculate the electron density for PDB id: 1HFS but the structure file only lists the Fo's, Fc's and Phases, but no sigmas. Would such structure factor file be considered incomplete? Thank you for your kind explanation. Francisco -- Nicholas M. Glykos, Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620, Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
I may be wrong here (and please by all means correct me), but I think it's not entirely true that experimental errors are not used in modern map calculation algorithm. At the very least, the 2mFo-DFc maps are calibrated to the model error (which can be ideologically seen as the error of experiment if you include model inaccuracies into that). And I quote from Acta D53:240, REFMAC also includes the sigma_E0 in the derivation of these terms (m and D) which usually leads to improved behavior. In fact in several cases when this has not been so it has been shown that the sigma_I0 were wrongly estimated during data processing. Thus, the experimental errors do affect the maps (albeit indirectly). I have not done extensive (or any for that matter) testing, but my evidence-devoid gut feeling is that maps not using experimental errors (which in REFAMC can be done either via gui button or by excluding SIGFP from LABIN in a script) will for a practicing crystallographer be essentially indistinguishable. The reason for this is that model errors as estimated by various maximum likelihood algorithms tend to exceed experimental errors. It may be that these estimates are inflated (heretical thought but when you think about it uniform inflation of the SIGMA_wc may have only proportional impact on the log-likelihood or even less so when they correlate with experimental errors). Or it may be that the experimental errors are underestimated (another heretical thought). Nevertheless, the perceived situation is that our models are not as good as our data, and therefore experimental errors don't matter. Now I am playing another devil's advocate and I know how crazy this sounds to an unbiased experimental scientist (e.g. if they don't matter, why bother improving data reduction algorithms?). I guess maps produced in phenix do not use experimental errors in any way given that the maximum likelihood formalism implemented there does not. Although phenix is not immutable and my understanding may be outdated. But this is not the right forum for pondering this specific question. Cheers, Ed. PS. I fully realize that Francisco's question was more practical (and the answer to that is to run REFMAC without SIGFP record in LABIN), but isn't thread-hijacking fun? :) On Wed, 2012-05-23 at 10:05 +0300, Nicholas M Glykos wrote: Hi Francisco, I'll play devil's advocate, but a measurement without an estimate of its error is closer to theology than to science. The fact that the standard deviations are not used for calculating an electron density map via FFT is only due to the hidden assumption that you have 100% complete, error-free data set, extending to sufficient high (infinite) resolution. When these assumptions do not apply (as is usually the case with physical reality), then the simple-minded FFT is not the correct inversion procedure (and the data do not univocally define a single map). Under these conditions other inversion mathods are needed (such as maximum entropy) for which the standard deviations are actively being used for calculating the map. My twocents, Nicholas On Tue, 22 May 2012, Francisco Hernandez-Guzman wrote: Hello everyone, My apologies if this comes as basic, but I wanted to get the expert's take on whether or not the sigmaF values are required in the calculation of an electron density map. If I look at the standard ED equation, sigma's don't appear to be a requirement but all the scripts that I've looked at do require sigma values. I wanted to calculate the electron density for PDB id: 1HFS but the structure file only lists the Fo's, Fc's and Phases, but no sigmas. Would such structure factor file be considered incomplete? Thank you for your kind explanation. Francisco -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
Ed, I may be wrong here (and please by all means correct me), but I think it's not entirely true that experimental errors are not used in modern map calculation algorithm. At the very least, the 2mFo-DFc maps are calibrated to the model error (which can be ideologically seen as the error of experiment if you include model inaccuracies into that). And I supposed my statement may have more precise than helpful. Obviously model and experimental errors do factor into calculation of a 2mFo-DFc map - but is weight and structure factor calculation part of map calculation, or a distinct stage of data processing? I tend to think of it as separate from map calculation, but this may be up for debate (judging by the increasing number of statements along the lines of I looked at my mtz file in coot and saw X). [snip] Nevertheless, the perceived situation is that our models are not as good as our data, and therefore experimental errors don't matter. Now I am playing another devil's advocate and I know how crazy this sounds to an unbiased experimental scientist (e.g. if they don't matter, why bother improving data reduction algorithms?). The errors in our models are almost definitely more extensive than the errors in our measurements, but one try at answering this devil's advocate question would be to point out that the usual likelihood equations all require sigF (either as a component of sigma, or for bootstrapping sigma). I've only done limited testing related to this (it was actually for something else), but likelihood equations produce strange results if you try to get them to ignore sigF. Pete I guess maps produced in phenix do not use experimental errors in any way given that the maximum likelihood formalism implemented there does not. Although phenix is not immutable and my understanding may be outdated. But this is not the right forum for pondering this specific question. Cheers, Ed. PS. I fully realize that Francisco's question was more practical (and the answer to that is to run REFMAC without SIGFP record in LABIN), but isn't thread-hijacking fun? :) On Wed, 2012-05-23 at 10:05 +0300, Nicholas M Glykos wrote: Hi Francisco, I'll play devil's advocate, but a measurement without an estimate of its error is closer to theology than to science. The fact that the standard deviations are not used for calculating an electron density map via FFT is only due to the hidden assumption that you have 100% complete, error-free data set, extending to sufficient high (infinite) resolution. When these assumptions do not apply (as is usually the case with physical reality), then the simple-minded FFT is not the correct inversion procedure (and the data do not univocally define a single map). Under these conditions other inversion mathods are needed (such as maximum entropy) for which the standard deviations are actively being used for calculating the map. My twocents, Nicholas On Tue, 22 May 2012, Francisco Hernandez-Guzman wrote: Hello everyone, My apologies if this comes as basic, but I wanted to get the expert's take on whether or not the sigmaF values are required in the calculation of an electron density map. If I look at the standard ED equation, sigma's don't appear to be a requirement but all the scripts that I've looked at do require sigma values. I wanted to calculate the electron density for PDB id: 1HFS but the structure file only lists the Fo's, Fc's and Phases, but no sigmas. Would such structure factor file be considered incomplete? Thank you for your kind explanation. Francisco
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
Hi Ed, I may be wrong here (and please by all means correct me), but I think it's not entirely true that experimental errors are not used in modern map calculation algorithm. At the very least, the 2mFo-DFc maps are calibrated to the model error (which can be ideologically seen as the error of experiment if you include model inaccuracies into that). This is an amplitude modification. It does not change the fact that the sigmas are not being used in the inversion procedure [and also does not change the (non) treatment of missing data]. A more direct and relevant example to discuss (with respect to Francisco's question) would be the calculation of a Patterson synthesis (where the phases are known and fixed). I have not done extensive (or any for that matter) testing, but my evidence-devoid gut feeling is that maps not using experimental errors (which in REFAMC can be done either via gui button or by excluding SIGFP from LABIN in a script) will for a practicing crystallographer be essentially indistinguishable. It seems that although you are not doubting the importance of maximum likelihood for refinement, you do seem to doubt the importance of closely related probabilistic methods (such as maximum entropy methods) for map calculation. I think you can't have it both ways ... :-) The reason for this is that model errors as estimated by various maximum likelihood algorithms tend to exceed experimental errors. It may be that these estimates are inflated (heretical thought but when you think about it uniform inflation of the SIGMA_wc may have only proportional impact on the log-likelihood or even less so when they correlate with experimental errors). Or it may be that the experimental errors are underestimated (another heretical thought). My experience from comparing conventional (FFT-based) and maximum-entropy- related maps is that the main source of differences between the two maps has more to do with missing data (especially low resolution overloaded reflections) and putative outliers (for difference Patterson maps), but in certain cases (with very accurate or inaccurate data) standard deviations do matter. All the best, Nicholas -- Nicholas M. Glykos, Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620, Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
Nicholas, My experience from comparing conventional (FFT-based) and maximum-entropy- related maps is that the main source of differences between the two maps has more to do with missing data (especially low resolution overloaded reflections) and putative outliers (for difference Patterson maps), but in certain cases (with very accurate or inaccurate data) standard deviations do matter. I'm curious - which programs have you used for maximum-entropy for map calculation? Pete
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
On Wed, 2012-05-23 at 10:02 -0500, Pete Meyer wrote: bviously model and experimental errors do factor into calculation of a 2mFo-DFc map - but is weight and structure factor calculation part of map calculation, or a distinct stage of data processing? Oh, I see. Sure, when the map coefficients are already available, the further calculation does need sigmas, or even Fo's/Fc's. I think the practical question Fransisco asked comes to this: If you have no SIGFP column in the input mtz-file, REFMAC will by default fail (phenix probably won't - at least I've seen datasets where all the sigmas were reset to 1.00 and it didn't change the output a bit). So one naturally things that sigmas are needed/used in map calculation. Again in practical terms, just remove the SIGFP from LABIN and it works, because refmac can do it both ways. Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
On Wed, 2012-05-23 at 18:06 +0300, Nicholas M Glykos wrote: This is an amplitude modification. It does not change the fact that the sigmas are not being used in the inversion procedure Nicholas, I am not sure I understand this - perhaps we are talking about different things. Even if by inversion procedure you mean simple calculation of (2fo-fc)*exp(i*phi), the fc is still technically a product of the refinement, which unless based on trivial least square target (i.e. no weights) does factor in experimental errors. The (2mFo-DFc) map is even more obviously dependent on the errors. Again, I believe that the differences will be minor, but if one calculates a map with refmac either with or without factoring in experimental errors, there will be *some difference*. Thus, the experimental errors will affect the resulting map. Could you please clarify? Cheers, Ed -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
On Wed, 2012-05-23 at 18:06 +0300, Nicholas M Glykos wrote: It seems that although you are not doubting the importance of maximum likelihood for refinement, you do seem to doubt the importance of closely related probabilistic methods (such as maximum entropy methods) for map calculation. I think you can't have it both ways ... :-) Nicholas, I think that we are not comparing ML to no-ML (or maximum entropy), but rather ML inflated by experimental errors vs pure ML that ignores them. I may be crazy or stupid (or both), but certainly not crazy/stupid enough to doubt the importance of maximum likelihood for refinement. (On the other hand, one who promises to never doubt maximum likelihood shall never use SHELX :) Cheers, Ed. -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs
[ccp4bb] crystallization analysis software
Does anyone know of a (non-commercial) software that can analyze results of a crystallization screen? What I am looking for is some way to tell what components/factors favor protein solubility/precipitation based on binary input (clear drop/precipitate). I did some googling, but please feel free to use lmgtfy on me :) Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
I just wanted to take a moment to thank all of the respondents to the post. Indeed, my question was more practical in nature since I wanted to see the density around the ligand in question. From the first suggestions, I quickly did manage to generate the maps and accomplish my goal (special thanks to Robbie for actually sending me the converted mtz file from the PDB cif entry). The additional comments have also been highly educational and helpful to further my understanding of some more in-depth crystallography concepts. Thank you, Francisco PS The PDB_REDO (http://www.cmbi.ru.nl/pdb_redo/hf/1hfs/index.html) was indeed a great resource and I'm certain to use it again. Thanks! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Francisco Hernandez-Guzman Sent: Tuesday, May 22, 2012 9:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Calculating ED Maps from structure factor files with no sigma Hello everyone, My apologies if this comes as basic, but I wanted to get the expert's take on whether or not the sigmaF values are required in the calculation of an electron density map. If I look at the standard ED equation, sigma's don't appear to be a requirement but all the scripts that I've looked at do require sigma values. I wanted to calculate the electron density for PDB id: 1HFS but the structure file only lists the Fo's, Fc's and Phases, but no sigmas. Would such structure factor file be considered incomplete? Thank you for your kind explanation. Francisco
Re: [ccp4bb] DNA/RNA modeling
Hi Jens, You can try the free DS Visualizer: http://accelrys.com/products/discovery-studio/visualization-download.php It comes with a free molecular builder for proteins, dna/rna and ligands. Please note that if you do need to make anything more interesting (energy calculations, minimizations, md, etc) will then need a license. I hope this helps. Francisco Full Disclaimer: I do work for Accelrys. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of jens j birktoft Sent: Wednesday, May 23, 2012 9:32 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DNA/RNA modeling Hi everybody, Does anyone know of a (non-commercial) software that are suitable for modeling DNA/RNA structures. Coot is great, however I am looking something that allows more flexibility Thanks -- Jens J. Birktoft e-mail: jens.birkt...@nyu.edumailto:jens.kn...@gmail.com slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025 Phone: 212-749-5057
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
On 05/23/12 08:06, Nicholas M Glykos wrote: Hi Ed, I may be wrong here (and please by all means correct me), but I think it's not entirely true that experimental errors are not used in modern map calculation algorithm. At the very least, the 2mFo-DFc maps are calibrated to the model error (which can be ideologically seen as the error of experiment if you include model inaccuracies into that). This is an amplitude modification. It does not change the fact that the sigmas are not being used in the inversion procedure [and also does not change the (non) treatment of missing data]. A more direct and relevant example to discuss (with respect to Francisco's question) would be the calculation of a Patterson synthesis (where the phases are known and fixed). I have not done extensive (or any for that matter) testing, but my evidence-devoid gut feeling is that maps not using experimental errors (which in REFAMC can be done either via gui button or by excluding SIGFP from LABIN in a script) will for a practicing crystallographer be essentially indistinguishable. It seems that although you are not doubting the importance of maximum likelihood for refinement, you do seem to doubt the importance of closely related probabilistic methods (such as maximum entropy methods) for map calculation. I think you can't have it both ways ... :-) The reason for this is that model errors as estimated by various maximum likelihood algorithms tend to exceed experimental errors. It may be that these estimates are inflated (heretical thought but when you think about it uniform inflation of the SIGMA_wc may have only proportional impact on the log-likelihood or even less so when they correlate with experimental errors). Or it may be that the experimental errors are underestimated (another heretical thought). My experience from comparing conventional (FFT-based) and maximum-entropy- related maps is that the main source of differences between the two maps has more to do with missing data (especially low resolution overloaded reflections) and putative outliers (for difference Patterson maps), but in certain cases (with very accurate or inaccurate data) standard deviations do matter. In a continuation of this torturous diversion from the original question... Since your concern is not how the sigma(Fo) plays out in refinement but how uncertainties are dealt with in the map calculation itself (where an FFT calculates the most probable density values and maximum entropy would calculate the best, or centroid, density values) I believe the most relevant measure of the uncertainty of the Fourier coefficients would be sigma(2mFo-DFc). This would be estimated from a complex calculation of sigma(sigmaA), sigma(Fo), sigma(Fc) and sigma(Phic). I expect that the contribution of sigma(Fo) would be one of the smallest contributors to this calculation, as long as Fo is observed. I wouldn't expect the loss of sigma(Fo) to be catastrophic. Wouldn't sigma(sigmaA) be the largest component since sigmaA is a function of resolution and based only on the test set? Dale Tronrud All the best, Nicholas
Re: [ccp4bb] DNA/RNA modeling
Hi Jens, You can try NAMOT (Nucleic Acid MOdelling Tool), from the links below. http://namot.sourceforge.net/ http://sourceforge.net/projects/namot/ Best, Tuhin. Tuhin Bhowmick Prof. S. Ramakumar’s Lab Biocrystallography and Computation Department of Physics Indian Institute of Science Bangalore, 560012 India On Wed, May 23, 2012 at 10:18 PM, Francisco Hernandez-Guzman francisco.hernan...@accelrys.com wrote: Hi Jens, ** ** You can try the free DS Visualizer: http://accelrys.com/products/discovery-studio/visualization-download.php** ** ** ** It comes with a free molecular builder for proteins, dna/rna and ligands. Please note that if you do need to make anything more interesting (energy calculations, minimizations, md, etc) will then need a license. ** ** I hope this helps. ** ** Francisco ** ** Full Disclaimer: I do work for Accelrys. ** ** *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *jens j birktoft *Sent:* Wednesday, May 23, 2012 9:32 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] DNA/RNA modeling ** ** Hi everybody, ** ** Does anyone know of a (non-commercial) software that are suitable for modeling DNA/RNA structures. Coot is great, however I am looking something that allows more flexibility ** ** Thanks ** ** ** ** -- Jens J. Birktoft e-mail: jens.birkt...@nyu.edu jens.kn...@gmail.com slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025 Phone: 212-749-5057
[ccp4bb] Off-topic Thrombin cleavage
Dear community, I am trying to cleave a hexaHis tag from my protein prior to crystallization. As I was setting up my digestion, my protein started to precipitate as soon as I added the recommended thrombin buffer. My question is, if anyone has encountered this, how well does it cleave without thrombin buffer? or even, without the CaCl2? Any other buffer/conditions known to work? thanks in advance
Re: [ccp4bb] DNA/RNA modeling
For the RNA I recommend Eric Westhof's Assemble. F On May 23, 2012, at 9:32 AM, jens j birktoft wrote: Hi everybody, Does anyone know of a (non-commercial) software that are suitable for modeling DNA/RNA structures. Coot is great, however I am looking something that allows more flexibility Thanks -- Jens J. Birktoft e-mail: jens.birkt...@nyu.edu slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025 Phone: 212-749-5057
Re: [ccp4bb] crystallization analysis software
Ed, I looked into this a number of years ago, and remember the following papers. I did not actually use any of the databases described due to IT issues at the time. I hope this helps. Kind regards, Bryan LISA: an intranet-based flexible database for protein crystallography project management http://scripts.iucr.org/cgi-bin/paper?S0907444901009295 HalX: an open-source LIMS (Laboratory Information Management System) for small- to large-scale laboratories http://scripts.iucr.org/cgi-bin/paper?S0907444905001290 Xtrack - a web-based crystallographic notebook http://scripts.iucr.org/cgi-bin/paper?S0907444902012696 CLIMS: Crystallography Laboratory Information Management System http://scripts.iucr.org/cgi-bin/paper?za5055 These I found while looking up the others. MOLE: A data management application based on a protein production data model http://onlinelibrary.wiley.com/doi/10.1002/prot.20291/full Design of a data model for developing laboratory information management and analysis systems for protein production http://onlinelibrary.wiley.com/doi/10.1002/prot.20303/full The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory http://scripts.iucr.org/cgi-bin/paper?S0907444911007943 -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Wednesday, May 23, 2012 12:24 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization analysis software Does anyone know of a (non-commercial) software that can analyze results of a crystallization screen? What I am looking for is some way to tell what components/factors favor protein solubility/precipitation based on binary input (clear drop/precipitate). I did some googling, but please feel free to use lmgtfy on me :) Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
Hi Ed, I am not sure I understand this - perhaps we are talking about different things. Even if by inversion procedure you mean simple calculation of (2fo-fc)*exp(i*phi), the fc is still technically a product of the refinement, which unless based on trivial least square target (i.e. no weights) does factor in experimental errors. The (2mFo-DFc) map is even more obviously dependent on the errors. Again, I believe that the differences will be minor, but if one calculates a map with refmac either with or without factoring in experimental errors, there will be *some difference*. Thus, the experimental errors will affect the resulting map. Could you please clarify? Yes, we are talking about different things. I refer to the case that we have an amplitude term with its uncertainty (no matter whether it is Fo or Fo^2 or Fo-Fc or 2mFo-DFc or ...) plus a phase with its uncertainty. In normal everyday applications we use FFT which ignores (i) the uncertainties of both terms, (ii) the missing data. By doing an FFT we produce a map which exactly reproduces the input data (even if they are missing data which are reproduced with an amplitude of zero). What I have been saying is that in the presence of uncertainties and missing information the data do not define a single map, but a whole set of maps which are statistically consistent with the data and the question then arises : 'which map should I be looking at ?'. I happen to mention the maximum entropy method as a possible solution to this problem. I think that we are not comparing ML to no-ML (or maximum entropy), but rather ML inflated by experimental errors vs pure ML that ignores them. I may be crazy or stupid (or both), but certainly not crazy/stupid enough to doubt the importance of maximum likelihood for refinement. (On the other hand, one who promises to never doubt maximum likelihood shall never use SHELX :) We definitely talk about different things. My arguments had nothing to do with treatment of errors in refinement. The question I was tackling was how you go from |F|,sig(|F|),phase to a map in the presence of errors and missing data. Nicholas -- Nicholas M. Glykos, Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620, Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
In SHELXL. a refinement program sometimes used by small molecule crystallographers, all Fourier map for at least the last 20 years were weighted by Ic^2/(Ic^2+sigma^2(I)), where Ic is the calculated squared structure factor and sigma(I) is the square root of 1/w. w is the weight assigned to a reflection in the refinement (e.g. w=1/(sig(I)^2+(gI)^2), where sig(I) is the esd of the measured intensity I and g is a small constant. This purely empirical scheme appears to result in a significant reduction in the noise level of the map, at least for typical small molecule structures. Such schemes have been called 'maximum likelihood by intuition', a proper maximum likelihood treatment taking the esds of the intensities into account would of course do much better. George On 05/23/2012 06:59 PM, Dale Tronrud wrote: On 05/23/12 08:06, Nicholas M Glykos wrote: Hi Ed, I may be wrong here (and please by all means correct me), but I think it's not entirely true that experimental errors are not used in modern map calculation algorithm. At the very least, the 2mFo-DFc maps are calibrated to the model error (which can be ideologically seen as the error of experiment if you include model inaccuracies into that). This is an amplitude modification. It does not change the fact that the sigmas are not being used in the inversion procedure [and also does not change the (non) treatment of missing data]. A more direct and relevant example to discuss (with respect to Francisco's question) would be the calculation of a Patterson synthesis (where the phases are known and fixed). I have not done extensive (or any for that matter) testing, but my evidence-devoid gut feeling is that maps not using experimental errors (which in REFAMC can be done either via gui button or by excluding SIGFP from LABIN in a script) will for a practicing crystallographer be essentially indistinguishable. It seems that although you are not doubting the importance of maximum likelihood for refinement, you do seem to doubt the importance of closely related probabilistic methods (such as maximum entropy methods) for map calculation. I think you can't have it both ways ... :-) The reason for this is that model errors as estimated by various maximum likelihood algorithms tend to exceed experimental errors. It may be that these estimates are inflated (heretical thought but when you think about it uniform inflation of the SIGMA_wc may have only proportional impact on the log-likelihood or even less so when they correlate with experimental errors). Or it may be that the experimental errors are underestimated (another heretical thought). My experience from comparing conventional (FFT-based) and maximum-entropy- related maps is that the main source of differences between the two maps has more to do with missing data (especially low resolution overloaded reflections) and putative outliers (for difference Patterson maps), but in certain cases (with very accurate or inaccurate data) standard deviations do matter. In a continuation of this torturous diversion from the original question... Since your concern is not how the sigma(Fo) plays out in refinement but how uncertainties are dealt with in the map calculation itself (where an FFT calculates the most probable density values and maximum entropy would calculate the best, or centroid, density values) I believe the most relevant measure of the uncertainty of the Fourier coefficients would be sigma(2mFo-DFc). This would be estimated from a complex calculation of sigma(sigmaA), sigma(Fo), sigma(Fc) and sigma(Phic). I expect that the contribution of sigma(Fo) would be one of the smallest contributors to this calculation, as long as Fo is observed. I wouldn't expect the loss of sigma(Fo) to be catastrophic. Wouldn't sigma(sigmaA) be the largest component since sigmaA is a function of resolution and based only on the test set? Dale Tronrud All the best, Nicholas -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
Hi Pete, I'm curious - which programs have you used for maximum-entropy for map calculation? Thanks, I thought no-one would ask :-) http://utopia.duth.gr/~glykos/graphent.html Don't download the program today. Or tomorrow. This coming weekend there will be a new release which will contain MacOSX executables. Nicholas -- Nicholas M. Glykos, Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620, Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/
Re: [ccp4bb] Off-topic Thrombin cleavage
Thrombin works pretty good without any added calcium. We routinely added thrombin to whatever buffer the protein is in provided it doesn't have a lot of DTT. Some beta-mercaptoethanol is alright. What is the source of your thrombin? -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yuri Pompeu Sent: Wednesday, May 23, 2012 12:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic Thrombin cleavage Dear community, I am trying to cleave a hexaHis tag from my protein prior to crystallization. As I was setting up my digestion, my protein started to precipitate as soon as I added the recommended thrombin buffer. My question is, if anyone has encountered this, how well does it cleave without thrombin buffer? or even, without the CaCl2? Any other buffer/conditions known to work? thanks in advance
[ccp4bb] Off topic: Complexes cloning
Dear all A off-topic question - is there a practical limit for overexpression of membrane protein complexes (about 200 kDa in total)? This includes chaperones and my target proteins. Can I amplify the gene cluster and simply clone into a plasmid? Thank you.
Re: [ccp4bb] DNA/RNA modeling
On Wed, May 23, 2012 at 12:54 PM, Mark J van Raaij mjvanra...@cnb.csic.es mailto:mjvanra...@cnb.csic.es wrote: even more flexible than COOT? - the only thing I can think of that I haven't worked out how to do in COOT is grabbing and moving one atom at a time. On 23/05/12 19:50, jens j birktoft wrote: Thanks Mark. As a matter of fact that is what I am really looking for. The ability to move one or a few selected atoms/fragments at a time. And you thought that Coot was not flexible enough to do that? :-( Coot FAQ 4.23 http://www.biop.ox.ac.uk/coot/doc/coot-faq.html#How-do-I-_0022grab_0022-just-one-atom-in-Real-Space-Refinement_002fRegularization_003f Also: If you don't want regularization/refinement, use the Rotate/Translate tool and Ctrl-click the atom(s) to move them one at a time. If you want on the fly regularization/refinement of fragments that include atoms that don't move when you drop them you need to fix them with the Fix Atom tool before regularization/refinement. HTH, Paul.
[ccp4bb] Off-topic: probing hydrophobic surface with ANS
Hi, Could someone refer me to some papers on using ANS to estimate amount of hydrophobic surface in a protein? I tried the classic way by STEP 1: titrating my protein into 1uM ANS till saturation to get the molar fluorescence activity of ANS, then STEP 2: titrate ANS into protein to get amount of fluorescene at plateau so that I can estimate how much ANS is bound per protein molecule. However, for this protein I am dealing with, I can never get the STEP 1 done because the fluorescence signal never saturates. It keeps going up. Are there other ways of getting around this? Thanks, Huiming Li
Re: [ccp4bb] DNA/RNA modeling
and if everything else fails including kicking the trash can one could indeed read the manual/FAQ - thanks Paul On Wed, May 23, 2012 at 4:27 PM, Paul Emsley paul.ems...@bioch.ox.ac.ukwrote: On Wed, May 23, 2012 at 12:54 PM, Mark J van Raaij mjvanra...@cnb.csic.es wrote: even more flexible than COOT? - the only thing I can think of that I haven't worked out how to do in COOT is grabbing and moving one atom at a time. On 23/05/12 19:50, jens j birktoft wrote: Thanks Mark. As a matter of fact that is what I am really looking for. The ability to move one or a few selected atoms/fragments at a time. And you thought that Coot was not flexible enough to do that? :-( Coot FAQ 4.23 http://www.biop.ox.ac.uk/coot/doc/coot-faq.html#How-do-I-_0022grab_0022-just-one-atom-in-Real-Space-Refinement_002fRegularization_003f Also: If you don't want regularization/refinement, use the Rotate/Translate tool and Ctrl-click the atom(s) to move them one at a time. If you want on the fly regularization/refinement of fragments that include atoms that don't move when you drop them you need to fix them with the Fix Atom tool before regularization/refinement. HTH, Paul. -- Jens J. Birktoft e-mail: jens.kn...@gmail.com slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025 Phone: 212-749-5057
