[ccp4bb] Question about stereo on LCD monitor
Dear CCP4BB users, Sorry, for non-CCP4 question. I'm looking for any freeware program for molecule model building, especially for perfect matching RNA duplex. Could you help me? I found some programs with options of de novo protein chain building, but without options of DNA/RNA chain building. With best wishes Rafal |--| |Rafal Dolot, Ph.D.| | | |Polish Academy of Sciences| |Centre of Molecular and Macromolecular Studies| |Department of Bioorganic Chemistry| |Sienkiewicza 112 | |90-363 Lodz, Poland | |Phone: +48(42)6803215 | |Cell: +48 502897781 | |--|
[ccp4bb] Software for RNA model building
Sorry, for mistake in the title of my last post. Please ingnore it. Dear CCP4BB users, Sorry, for non-CCP4 question. I'm looking for any freeware program for molecule model building, especially for perfect matching RNA duplex. Could you help me? I found some programs with options of de novo protein chain building, but without options of DNA/RNA chain building. With best wishes Rafal |--| |Rafal Dolot, Ph.D.| | | |Polish Academy of Sciences| |Centre of Molecular and Macromolecular Studies| |Department of Bioorganic Chemistry| |Sienkiewicza 112 | |90-363 Lodz, Poland | |Phone: +48(42)6803215 | |Cell: +48 502897781 | |--|
Re: [ccp4bb] Software for RNA model building
Dear Rafal, The program 'Coot' or (X)3DNA can easily do this for you. Regards Kristof On 12 May 2009, at 10:05, Rafal Dolot wrote: Sorry, for mistake in the title of my last post. Please ingnore it. Dear CCP4BB users, Sorry, for non-CCP4 question. I'm looking for any freeware program for molecule model building, especially for perfect matching RNA duplex. Could you help me? I found some programs with options of de novo protein chain building, but without options of DNA/RNA chain building. With best wishes Rafal |--| |Rafal Dolot, Ph.D.| | | |Polish Academy of Sciences| |Centre of Molecular and Macromolecular Studies| |Department of Bioorganic Chemistry| |Sienkiewicza 112 | |90-363 Lodz, Poland | |Phone: +48(42)6803215 | |Cell: +48 502897781 | |--| --- Kristof Van Hecke, PhD Biomolecular Architecture Celestijnenlaan 200F 3001 Heverlee (Leuven) Belgium ---
Re: [ccp4bb] Software for RNA model building
I dare say that make-na can do it even easier (no need to install anything): http://structure.usc.edu/make-na/server.html It is for *very* simple stuff though. James On May 12, 2009, at 1:11 AM, Kristof Van Hecke wrote: Dear Rafal, The program 'Coot' or (X)3DNA can easily do this for you. Regards Kristof On 12 May 2009, at 10:05, Rafal Dolot wrote: Sorry, for mistake in the title of my last post. Please ingnore it. Dear CCP4BB users, Sorry, for non-CCP4 question. I'm looking for any freeware program for molecule model building, especially for perfect matching RNA duplex. Could you help me? I found some programs with options of de novo protein chain building, but without options of DNA/RNA chain building. With best wishes Rafal |--| |Rafal Dolot, Ph.D.| | | |Polish Academy of Sciences| |Centre of Molecular and Macromolecular Studies| |Department of Bioorganic Chemistry| |Sienkiewicza 112 | |90-363 Lodz, Poland | |Phone: +48(42)6803215 | |Cell: +48 502897781 | |--| --- Kristof Van Hecke, PhD Biomolecular Architecture Celestijnenlaan 200F 3001 Heverlee (Leuven) Belgium ---
Re: [ccp4bb] phasing with se-met at low resolution
On Mon, May 11, 2009 at 05:22:25PM -0500, Pete Meyer wrote: P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one. Last time I emailed the RCSB about this (a few years back), it wasn't possible to search by phasing method. You can try using advanced search - keyword search - advanced and doing a full text search, but this is somewhat less than ideal. To be fair though, I suspect relatively few people searching the PDB are concerned about the phasing method used. Funny, I was looking at that just the other day. You basically need a local copy of the PDB and do some ugly grep/awk into the PDB files ... at least thats what I do (there might be better ways). Anyway, as of 28.04.2009 we have a total of 48969 entries with a line REMARK 200 METHOD USED TO DETERMINE THE STRUCTURE: which show (no guarantee!): Molecular replacement = 26436 Undefined (i.e. NULL, N/A etc) = 9349 MAD= 4125 SAD= 3028 Fourier methods= 2929 MIR= 1706 SIR= 512 Direct methods, ab initio = 327 Rigid-body refinement = 186 RIP= 3 UNKNOWN (everything else) = 504 This might add up to more than the total number of entries, since some have several methods listed. I tried to accomodate mis-spellings - lots of them available: MOLECULARE REPLACEMENT MOLECULAR REPLECEMENT MOLECULAR REPLCEMENT MOLECULAR REPLACEMET MOLECULAR REPL. MOLECULAR REPACEMENT MOLECULAR REEMPLACEMENT MOLECULAR PLACEMENT MOLECULAR EEPLACEMENT MOLECULAR PLACEMENT DIFFERENT FOURIER DIFFERECE FOURIER DFIFFERECE FOURIER etc. Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
[ccp4bb] Crystal Screen Optimization Server
Hi all, I’ve written a GUI to calculate 2D gradients for crystal screen optimization that people might find useful (much like a web 2.0 version of Hampton’s “Make Tray” utility). It’s intended to write scripts for a liquid handling robot our lab uses, but I’ve set up a public version up that gives a handy print out of recipes and records for people who make their optimization trays by hand. http://www.pageforaday.com/xtalwizard/table20.php I’d be happy to include an option to produce scripts for (other) robots… just e-mail me a template. P.S: It hasn’t been tested extensively, so please e-mail me if you notice any bugs. Thanks! _ Hotmail® goes with you. http://windowslive.com/Tutorial/Hotmail/Mobile?ocid=TXT_TAGLM_WL_HM_Tutorial_Mobile1_052009
Re: [ccp4bb] SUMMARY:vSystem virtual machine recommendation for crystallography?
Hello Everyone, Thank you for the replies to my questions regarding system virtual machine software. I have organized the replies into subheadings and summarized the comments below. The original question was: I would like to install a system virtual machine to run Ubuntu Linux as a guest OS on a 32-bit Vista laptop. The idea is to allow occasional use of crystallographic refinement programs while I'm away from lab. The laptop has an Intel Core 2 duo processor (2.0 GHz) and 3 GB RAM. There are popular software programs available (VMWare, Parallels, VirtualBox, etc.), but is anyone aware of any considerations that would make one better for the above purposes? For example, will one offer easy control over distributing hardware resources to prevent crippling Vista while running refinement within the guest Linux? The organized/summarized responses: Individual user experiences: - VirtualBox 2.2.2 to run Ubuntu 9.04 (guest) on WinXP 32-bit (native). Runs well with 512 Mbyte assigned memory with 64 Mbyte assigned graphics memory (dual core 2.4 GHz machine with 2 Gbyte total RAM). - VMWare used for various guest OSs. Found to work well. - Parallels useful for Linux as a guest; VMWare for Windows as a guest. - Qemu/Kvm and VirtualBox work well with Linux as a guest OS. Kvm found to be troublesome for Windows as a guest OS. - VMWare server 2 running CentOS (guest) on Linux (native) Software considerations: - VirtualBox is free - VirtualBox supports hardware virtualization, but it is off by default. Some others do as well. - Some distributions of VMWare are free - VMWare reported to be stable, has wide user-base, good documentation and can run dual-CPU General considerations: - No accelerated graphics performance (i.e. Coot/Pymol run slowly at ~15 frames/sec. with non-native graphics. - Previous threads recommend running graphics in native OS to take full advantage of hardware acceleration - Recommended to set up shared folders to transfer files between OSs to facilitate using native graphics software/hardware. - You have to reboot the machine to change the memory configuration, but people agreed the software is generally easy to configure for resource allocation. Thanks again to all who replied, -Andy Torelli -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry and Chemical Biology Cornell University =
Re: [ccp4bb] phasing with se-met at low resolution
measuring anomalous differences has nothing to do with resolution. measuring anomalous differences has nothing to do with Rmerge. measuring anomalous differences has EVERYTHING to do with signal and noise. (as does measuring anything else) If your average anomalous difference is going to be ~5%, then you need to be able to measure a 5% change in spot intensity, yes? So, if you take your native data, and compare the merged values of I+ and I- (known in Scala as Ranom), and they are already more than 5% different, then ... you are in trouble. But if Ranom for native data is less than 5%, then you stand a chance of measuring a 5% difference. That is, for native data, the true values of I+ and I- should be the same (within the Bijvoet ratio for the sulfurs, which is usually 0.5%), so comparing I+ and I- for native data is actually a very good way to get your expected anomalous error. You can improve this number by increasing redundancy, even if you reduce the exposure time to compensate. In fact, it is a VERY good idea to do this when trying to measure anomalous differences. Redundancy is good for anomalous, but bad for high-res data. Long exposures and fine slicing are good for high-res data, but bad for anomalous. Resolution comes into play because the anomalous error will approach infinity as your spot intensity approaches zero, so you will never be able to measure anomalous differences for your highest resolution bin. The resolution to which you CAN measure anomalous differences (with a signal-to-noise ratio greater than one) will be the resolution where the cumulative Ranom rises to the Bijvoet ratio (5% in your case). That is, look for the resolution limit where the overall native Ranom is 5%, and that is the resolution to which you will probably get experimental phases. If there is no such resolution limit (Ranom 5% in all bins), then MAD/SAD will not work with your current data collection method. Higher redundancy is called for. However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. Programs and crystallographers alike can get confused by this. I know that there are still many structural biologists out there who just want to get the structure, but I remind you that you can already get the structure to ~50 A resolution with other techniques. Such as gel filtration. The success of phase extension does depend on resolution. Although I do not have a quantitative argument for it, the success of SAD structure determination at worse than 4 A does seem to drop precipitously. This could simply be correlated with the crappiness of the crystals, but it is important to remember that SAD relies heavily on density modification technology, such as solvent flattening and histogram matching, etc, and these methods loose a great deal of power as the resolution of the map decreases (and the protein-solvent contrast becomes less clear). IMHO it is ALWAYS better to collect MAD data, because then the dichotomous phase ambiguity is resolved experimentally. Two wavelengths are twice as good as one, even with the exposure time cut in half. -James Holton MAD Scientist Engin Ozkan wrote: Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, where I/sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick-Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other partial phases, and no data cut off at 3.6 A with an I/s of 15 in the last resolution bin. Are there any
Re: [ccp4bb] phasing with se-met at low resolution
Dear James, On Tue, May 12, 2009 at 11:26:55AM -0700, James Holton wrote: However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. Programs and crystallographers alike can get confused by this. I know that there are still many structural biologists out there who just want to get the structure, Completely agree. The big misconception is that the result of the X-ray experiment is anything else than 'just' such an electron density map. What we usually see as 'the structure' is only a model: a PDB file to help us measuring distances, looking at on the display and making nice pictures ... just a useful interpretation of the electron density. So at lower resolution one needs to think more like an EM structural scientist and not an X-Ray one I guess. The success of phase extension does depend on resolution. Although I do not have a quantitative argument for it, the success of SAD structure determination at worse than 4 A does seem to drop precipitously. Not just SAD, but also MAD, MIR, SIR et al (in my experience). Somewhere below 3.5-4A it becomes VERY hard to extend the phases to the full resolution of the dataset. Unless you have NCS (the more the better) - which is just great in those cases. So if one gets crappy crystals at least get them with a huge asymmetric unit ;-) This could simply be correlated with the crappiness of the crystals, but it is important to remember that SAD relies heavily on density modification technology, such as solvent flattening and histogram matching, etc, and these methods loose a great deal of power as the resolution of the map decreases (and the protein-solvent contrast becomes less clear). I always thought it had more to do with the look-and-feel of lower resolution maps (helices are big sausages, sheets blend into flat patches and side-chains are not visible): the methods modifying the density in real-space have probably different assumptions and default parameters (radii for masking, histograms becoming messy, absolute scaling nearly impossible etc). Also: the typical low resolution (20A and below) that is often neglected (beamstop size and masking? Overloads?) becomes more important. In the end 'resolution' comes into play in some way after all I guess - at least when we see 'resolution' as what it is mostly used in that context: a simple concept to describe several actual difficulties (poor crystals which only diffract to low resolution, weak experimental phases, anisotropy, radiation damage etc). Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] phasing with se-met at low resolution
Thanks, I do understand all of that. I gave some Rmerge and resolution values to give some idea about errors and noise expected in the data, and an idea for up to what resolution phases would be good. And if such low resolution phases ever yield a meaningful model. Both measures are flawed indicators, even though they are the most common measures of data among us. I will definitely check Ranom (which means I should try scala). What I was curious about is practical aspects: especially in cases in which it really worked. And I/we have gotten quite a few responses in MAD vs SAD, inverse beam strategies, radiation damage control, etc. The take home message for me was that noone agrees on the best data collection strategy, although I still have to read upon some of the case references that were sent. Another point is the success rate of software - be it direct methods based or Patterson based - with such data (where anomalous signal would die at even lower resolution) at solving the substructure. I have seen a reference where if the correct substructure could be provided in a test case, SAD was actually successful. In another case, they confirmed the correct selenium sites with a platinum derivative data to further proceed with phasing. To be honest, I can't ever get shelx to find my platinums with 6 A data :) I would also like to hear about phase extension at low resolution (which you have mentioned). Overall, it appears that with such data, there are too many places for failure. Thanks for everyone's interest. Later, I shall come up with a nice summary. Engin On 5/12/09 11:26 AM, James Holton wrote: measuring anomalous differences has nothing to do with resolution. measuring anomalous differences has nothing to do with Rmerge. measuring anomalous differences has EVERYTHING to do with signal and noise. (as does measuring anything else) If your average anomalous difference is going to be ~5%, then you need to be able to measure a 5% change in spot intensity, yes? So, if you take your native data, and compare the merged values of I+ and I- (known in Scala as Ranom), and they are already more than 5% different, then ... you are in trouble. But if Ranom for native data is less than 5%, then you stand a chance of measuring a 5% difference. That is, for native data, the true values of I+ and I- should be the same (within the Bijvoet ratio for the sulfurs, which is usually 0.5%), so comparing I+ and I- for native data is actually a very good way to get your expected anomalous error. You can improve this number by increasing redundancy, even if you reduce the exposure time to compensate. In fact, it is a VERY good idea to do this when trying to measure anomalous differences. Redundancy is good for anomalous, but bad for high-res data. Long exposures and fine slicing are good for high-res data, but bad for anomalous. Resolution comes into play because the anomalous error will approach infinity as your spot intensity approaches zero, so you will never be able to measure anomalous differences for your highest resolution bin. The resolution to which you CAN measure anomalous differences (with a signal-to-noise ratio greater than one) will be the resolution where the cumulative Ranom rises to the Bijvoet ratio (5% in your case). That is, look for the resolution limit where the overall native Ranom is 5%, and that is the resolution to which you will probably get experimental phases. If there is no such resolution limit (Ranom 5% in all bins), then MAD/SAD will not work with your current data collection method. Higher redundancy is called for. However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. Programs and crystallographers alike can get confused by this. I know that there are still many structural biologists out there who just want to get the structure, but I remind you that you can already get the structure to ~50 A resolution with other techniques. Such as gel filtration. The success of phase extension does depend on resolution. Although I do not have a quantitative argument for it, the success of SAD structure determination at worse than 4 A does seem to drop precipitously. This could simply be correlated with the crappiness of the crystals, but it is important to remember that SAD relies heavily on density modification technology, such as solvent flattening and histogram matching, etc, and these methods loose a great deal of power as the resolution of the map decreases (and the protein-solvent contrast becomes less clear). IMHO it is ALWAYS better to collect MAD data, because then the dichotomous phase ambiguity is resolved experimentally. Two wavelengths are twice as good as one, even with the
Re: [ccp4bb] phasing with se-met at low resolution
Dear Engin, On Tue, May 12, 2009 at 12:20:31PM -0700, Engin Ozkan wrote: The take home message for me was that noone agrees on the best data collection strategy No - since you have to factor in at least half a dozen parameters: unfortunately there is no silver bullet :-( Another point is the success rate of software - be it direct methods based or Patterson based - with such data (where anomalous signal would die at even lower resolution) at solving the substructure. In general you need better data for finding the HA substructure this way then to solve it (where 'solve' can mean a lot of things, e.g. breaking the phase ambiguity and getting some meaningful map). In another case, they confirmed the correct selenium sites with a platinum derivative data to further proceed with phasing. To be honest, I can't ever get shelx to find my platinums with 6 A data :) How do you know SHELXD hasn't found them? In my experience at this kind of resolution you have to be careful to trust the usual criteria for a good solution (CCall 40% etc). Maybe looking at good old fashioned anomalous Patterson Harker sections? Also: I've never had good experiences with Pt derivatives ... low occupancy, VERY high B-values and generally a pain. I've seen people using them very successfully though. As Jim said: it's all about signal and noise. If you have lousy crystals (large noise) you need to go for a large signal: one of those Ta/W clusters maybe - and bootstrapping your way to the other derivatives from there? Depending on the quality of your crystals (and data collection) a weak Pt derivative might not be enough. Overall, it appears that with such data, there are too many places for failure. Yes, such data isn't forgiving - but that makes success even sweater. Remember that what we now see as a straightforward and nearly trivial project was one of those 'really difficult structures' only a few years back (and people did solve those structures). Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] phasing with se-met at low resolution
However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. If the map is a 6 Angstrom SeMet map you may well be right, since if the signal goes to 6 Angstrom the data at 7 Angstrom isn't that hot either. However if this was a Ta6Br12 6 Angstrom map then it can look quite pretty for the resolution because the 7 Angstrom SAD data in that case can be pretty good. Case in point it the one we collected for PP2a ABC holoenzyme cleared up all sorts of things about the partial molecular replacement solution, including some reassurance that the desperation WD40 ensemble MR solution was actually correct. At 6A, the WD40 looked somewhat like a Bagel (or a Bundt Cake if one is familiar) but the helices in one of the other subunits (A) were actually nicely resolved. Excitement may be warranted, even at 6 Angstrom. Phil Jeffrey Princeton
Re: [ccp4bb] phasing with se-met at low resolution
Dear James, I don't understand why measuring anomalous differences has nothing to do with resolution. Heavy atoms scatter anomalously because the inner shell electrons of the heavy atom cannot be considered to be free anymore as was assumed for normal Thomson scattering. As a result the atomic scattering factor of the heavy atom becomes complex and this compex contribution to the structure factor leads to non-equality of Friedel pairs in non-centro symmetric systems(excluding centric zone). This feature is taken advantage in phase determination. Since the inner shell electrons being relatively more strongly bound in heavy atoms contribute to anomalous scattering and its effect is more discernable for high angle reflections . Here the anomalous component of the scattering do not decrease much because of the effectively small atomic radii (only inner shell being effective). FOR HIGH ANGLE REFLECTIONS ANOMALOUS DATABECOMES IMPORTANT. Raja From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, 12 May, 2009 11:26:55 AM Subject: Re: [ccp4bb] phasing with se-met at low resolution measuring anomalous differences has nothing to do with resolution. measuring anomalous differences has nothing to do with Rmerge. measuring anomalous differences has EVERYTHING to do with signal and noise. (as does measuring anything else) If your average anomalous difference is going to be ~5%, then you need to be able to measure a 5% change in spot intensity, yes? So, if you take your native data, and compare the merged values of I+ and I- (known in Scala as Ranom), and they are already more than 5% different, then ... you are in trouble. But if Ranom for native data is less than 5%, then you stand a chance of measuring a 5% difference. That is, for native data, the true values of I+ and I- should be the same (within the Bijvoet ratio for the sulfurs, which is usually 0.5%), so comparing I+ and I- for native data is actually a very good way to get your expected anomalous error. You can improve this number by increasing redundancy, even if you reduce the exposure time to compensate. In fact, it is a VERY good idea to do this when trying to measure anomalous differences. Redundancy is good for anomalous, but bad for high-res data. Long exposures and fine slicing are good for high-res data, but bad for anomalous. Resolution comes into play because the anomalous error will approach infinity as your spot intensity approaches zero, so you will never be able to measure anomalous differences for your highest resolution bin. The resolution to which you CAN measure anomalous differences (with a signal-to-noise ratio greater than one) will be the resolution where the cumulative Ranom rises to the Bijvoet ratio (5% in your case). That is, look for the resolution limit where the overall native Ranom is 5%, and that is the resolution to which you will probably get experimental phases. If there is no such resolution limit (Ranom 5% in all bins), then MAD/SAD will not work with your current data collection method. Higher redundancy is called for. However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. Programs and crystallographers alike can get confused by this. I know that there are still many structural biologists out there who just want to get the structure, but I remind you that you can already get the structure to ~50 A resolution with other techniques. Such as gel filtration. The success of phase extension does depend on resolution. Although I do not have a quantitative argument for it, the success of SAD structure determination at worse than 4 A does seem to drop precipitously. This could simply be correlated with the crappiness of the crystals, but it is important to remember that SAD relies heavily on density modification technology, such as solvent flattening and histogram matching, etc, and these methods loose a great deal of power as the resolution of the map decreases (and the protein-solvent contrast becomes less clear). IMHO it is ALWAYS better to collect MAD data, because then the dichotomous phase ambiguity is resolved experimentally. Two wavelengths are twice as good as one, even with the exposure time cut in half. -James Holton MAD Scientist Engin Ozkan wrote: Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution
Re: [ccp4bb] phasing with se-met at low resolution
Dear Raja, FOR HIGH ANGLE REFLECTIONS ANOMALOUS DATA BECOMES IMPORTANT. Raja this is the theoretical point of view. As James pointed out, in real life the intensities of reflections at high resolution becomes comparable to the noise level so that the accuracy of which the reflections are measured increases significantly, rendering the anomalous difference useless - unfortunately. Tim
[ccp4bb] Mercaptoethanol
Hi. We are trying to prepare heavy atom derivatives of a protein in which a surface residue was mutated to cysteine. Mass spectrometry of the purified cysteine mutant showed an additional peak with a molecular mass that correspond to a mercaptoethanol-bound cysteine mutant. I am wondering whether the bound mercaptoethanol prevents the heavy atom from binding to the cysteine residue. Any information would be greatly appreciated. Wataru Kagawa
[ccp4bb] question on Akta Prime
Dear colleagues, I have an Akta Prime. I have always had problem with the reproducibility of the peak heights of the various peaks. Recently the pump is breaking. Does anyone have an idea what can cause the pump to break? It is more than once. Jackie Vitali
Re: [ccp4bb] Mercaptoethanol
Hi Bill: Thanks for the prompt reply. I will try purifying the protein without mercaptoethanol. Wataru On 2009/05/13, at 11:42, William G. Scott wrote: HI Wataru: I think it makes an R-S-S-R' bond, so the disulfide will prevent the heavy atom from binding, unless exchange is rapid. I hope all is well. Bill On May 12, 2009, at 7:17 PM, Wataru Kagawa wrote: Hi. We are trying to prepare heavy atom derivatives of a protein in which a surface residue was mutated to cysteine. Mass spectrometry of the purified cysteine mutant showed an additional peak with a molecular mass that correspond to a mercaptoethanol-bound cysteine mutant. I am wondering whether the bound mercaptoethanol prevents the heavy atom from binding to the cysteine residue. Any information would be greatly appreciated. Wataru Kagawa
