[ccp4bb] OT: VectorNTI alternatives - SUMMARY
Thanks for the 38 replies, both on and off the bboard. I have tested some of them and my favorites so far are ApE and Gentle which are free and quite good. But there may be others that are also good and I missed. Darren Summary: *Firstly*, good advice from Warren DeLano: 1. Be wary of relying upon free tools not based on open-source code. 2. Be extremely wary of free tools which come with a license manager. 3. Instead favor free software tools which strictly meet the established definitions of: Open Source: http://www.opensource.org/docs/osd, Free Software: http://www.gnu.org/philosophy/free-sw.html, or Public Domain: http://en.wikipedia.org/wiki/Public_domain since it is *only* those tools that can be safely taken for granted over the long haul. But be prepared to pay good money for good software! *Secondly*, if you are going to stop using VectorNTI, export valuable files in .gb format before the program locks. If this happens, contact Invitrogen and they (might) issue a short time extension as they did for me. *Recommended programs:* *Geneious *and *CLCbio *workbench are professional polished products competing with VectorNTI – but CLC free version is just a plasmid viewer really. Sebastiano and others - much much easier than VectorNTI is *ApE *( http://www.biology.utah.edu/jorgensen/wayned/ape/), which is multi-platform and very easy to use for simple tasks. I tried ApE and was really impressed, once I got past the very simple looking format. This would do most of the things required for designing vectors and works with .gb format files – Darren *Serial cloner *(http://serialbasics.free.fr/Serial_Cloner.html) suggested by James Stroud. It works only with fasta or .xdna files – so is really a DNA editor and seems to have limited Protein analysis functions, even displaying translated ORFs above DNA sequence. But splicing DNA sequences together seems efficient. Mark Brooks - recommended *BioEdit*: http://www.mbio.ncsu.edu/BioEdit/bioedit.html It has an old fashioned cluttered interface, but does do sequence editing, translation into proteins, ClustalW alignments and contig assemblies (a bit like ContigExpress in Vector NTI). It opens ABI files for sequencing data, to view the chromatograms. It uses the external programs such as clustalw alignments or cap3 to do the contig assemblies, and its licence doesn't expire! For storing everything, I put my primers, plasmid sequences, insert sequences in a MySQL database, with an HTML front end I wrote: http://plasmidb.sourceforge.net/ *Plasmi::db *also has a homespun feel to it, and only works with Firefox, for example (not other browsers). There is a primer designer page, for traditional cloning by restriction digestion etc.. I can't pretend it's in the same league as Vector NTI, though. The data is stored in a non-proprietary format; database tables which can be viewed with either the HTML pages, or MS Excel, for example. Andy Gulick recommends the *Workbench* suite at the *San Diego Supercomputer Center*. It allows you to maintain a database of protein and DNA sequence, has many tools, and allows you to create subprojects to help organize. http://workbench.sdsc.edu Yong-Fu Li suggested *Lasergene*, but not enthusiastically due to requirement to reformat files and not very good editing functions. Roger Dodd - *PlasmaDNA *which seems pretty good for the basics http://research.med.helsinki.fi/plasmadna/ . Christian Biertümpfel recommends another free tool: *pDRAW32 *( http://www.acaclone.com/ ). It runs natively under Windows and with the emulator wine on Linux. Francis Reyes - Not sure if it's been mentioned, but I personally use *EnzymeX *(http://mekentosj.com/enzymex/) .Also recommends PDF library organizer Papers (http://mekentosj.com/papers/) to be exceptional. Juan Sanchez Weatherby - GCK2 (*GeneConstructionKit*) and another * GeneInspector*. They where pretty amazing and with lots of features for plasmid design, keeping history, sharing, and lots more. I suppose they must have improved quite a lot over the years. I can't remember what the license was like (money wise) but I think you can download a free version (doesn't let you save or print things but shows what you can actually do with them). The link you need to find them is http://www.textco.com/products/index.html Bryan Lepore – Lots can be done just with with [1] *expasy tools *and [2] *sequence manipulation suite*, which is entirely downloadable for local use. http://www.bioinformatics.org/sms2/about.html (Darren says: I agree *SMS *is very useful indeed and can be run via their website – no installation) There is *GENtle *which has a whole slew of tools associated with it. There are versions for several platforms. http://gentle.magnusmanske.de/ it is pretty similar to Vector NTI (and open source for the ambitious). For Macs: Jovine Luca - *DNA Strider *(1.4) runs just fine on both Tiger and Leopard. For more info, you can contact the author directly:
[ccp4bb] comparison of maps, intensities and other basics
Dear CCP4BB List Members, first of all I am not a crystallographer, but I would like to get some things clear, things I did not find in Crystallography Made Crystal Clear and on the internet for now. I am trying to read electron density maps in the EZD format. These maps contain scaled values of electron density and size and shape of the unit cell. How can I convert the values of intensities (what is the unit of these values?) to the probabilities you can see in coot for example (1.03 electron / A^3), Once I have achieved this conversion, can I compare densities of different maps of different proteins? If not directly, is there a way to do so? Last, is there a way to know the experimental error made on intensity values of a map? Thanks in advance. -- Peter Schmidtke -- PhD Student - Molecular Modeling and Bioinformatics Group Dep. Physical Chemistry Faculty of Pharmacy University of Barcelona
[ccp4bb] Scientific Programmer Position - EMBL Hamburg
= Scientific Programmer EMBL Hamburg = A position for a scientific programmer is immediately available in the group of Thomas Schneider at EMBL Hamburg. The EMBL Outstation in Hamburg (htpp://www.embl-hamburg.de) has a wide research program in structural biology ranging from the development of synchrotron instrumentation via computational methods for structure determination and analysis all the way to structural and functional studies of molecules of biological interest. In the near future, the PETRA III synchrotron, one of the world's brightest sources for X-rays, will become operational. EMBL-Hamburg is constructing three beamlines on PETRA III and we expect many interesting structural biology projects to be carried out on this exceptional source. The group of Dr. Thomas R. Schneider is developing computational tools for mining ensembles of experimentally determined structures for information about the conformational flexibility of biological molecules and complexes. This information is then used to understand the function of the molecules or to provide a basis for the interpretation of low resolution structural data in terms of known fragments. For details, see Schneider TR (2004) Acta Cryst. D60:2269 and Mosca et al. (2008) BMC Bioinformatics 9:352 We are seeking a Scientific Programmer for the development and implementation of novel algorithms in structure determination and analysis. In addition to the development work, the successful candidate will be in charge of making our computational tools available to the scientific community by maintainance and distribution of the respective computer programs and by the provision of web-based services. There are also opportunities to engage into independent projects. The ideal candidate should have a strong background in structural biology. Experience in scientific programming, ideally using C++, is required. Previous experience in the construction of web-based services is a plus. The candidate should be interested in working in an international, multidisplinary research environment and in interacting with a broad user community. Excellent communication and interpersonal as well as English language skills are a necessity. For more information and to apply, please visit: http://www.embl-hamburg.de - Jobs For informal enquiries please contact me directly at: thomas.schnei...@embl-hamburg.de --- Dr. Thomas R. Schneider Project Coordinator e...@petra3 EMBL c/o DESY Notkestr. 85 fax: 0049-(0)40-89902-149 22603 Hamburg phone: 0049-(0)40-89902-190 Germany email:thomas.schnei...@embl-hamburg.de --- e...@petra3: http://www.embl-hamburg.de/services/petra/index.html ---
[ccp4bb] tutorial / pipeline for ligand fitting, refinement?
Hello, does anyone know of a tutorial which lays out some sort of pipeline, hopefully using CCP4 packages, to fit and refine a small molecule ligand please? cheers andy
Re: [ccp4bb] tutorial / pipeline for ligand fitting, refinement?
Andy, We do a lot of liganding fitting with CCP4. This is the general order of steps we take (post initial solution of the protein itself): 1) Build the potential ligand in CCP4 Sketcher a) Rename all the Hydrogens to H#, CCP4 Refmac has some issues with Hydrogens marked OH1, NH1, etc. To simplify things I normally just renumber all the Hydrogens starting from 1. Also makes for less hassle when using the definition file, as the labels in the definition file has to match the pdb of the ligand (this will be more important below). b) Use the regularize function with Refmac 2) Using Coot, load the protein and maps 3) Load the ligand and definition file (_mon_lib.cif) 4) Use the find ligand function in Coot (find it under other modeling tools) a) select the protein, map you want to search 5) If you find results you desire, merge those ligands with the main pdb 6) Run Refmac on the merged PDB with the library for the ligand in the library input space. Hope this helps, Scott On Thu, Feb 5, 2009 at 9:27 AM, ANDY DODDS andy.dod...@googlemail.comwrote: Hello, does anyone know of a tutorial which lays out some sort of pipeline, hopefully using CCP4 packages, to fit and refine a small molecule ligand please? cheers andy -- Scott D. Pegan, Ph.D. Senior Research Specialist Center for Pharmaceutical Biotechnology University of Illinois at Chicago
[ccp4bb] Choosing MR solutions in the case of perfect twinning with P41212?
It seems like this space group will be the death of me. I'm working on a structure in SG P41212 one molecule per asu that was solved with experimental SAD phases. The resolution is to 2.5 and the refinement is stuck at an R/Rfree of 30 and 33 with bonds rmsd of 0.011 and angles of 1.597 . The unit cell is 73.604 73.604 114.279 90.00 90.00 90.00. I'm considering the case of perfect twinning where the real s.g. is P41 masked under the higher symmetry in P41212. It seems to be the case in perfect twinning that the approach is to molecular replace the refined model into the lower space group. I reindexed my data to the lower space group P41 and molecular replaced into the reindexed data with Phaser. A single was solution was found with 2 mol per asu (39.6% solvent content) related by NCS. I've refined the now two fold ncs related structure in P41 to a much more respectable R/Rfree of 25.2 and 28.6 with rmsd bonds at 0.004 and angles at 0.865 refining with a twin law and NCS as implemented by phenix.refine. However I'm not happy: [1] a simmulated anneal omit map one of the monomers in P41 where 5 residues in a non crystal contact region of the molecule (I wanted to challenge the omit map) shows nearly no density. (the SA OMIT map was generated with phenix.autobuild using the same refine parameters as the final round of refinement) [2] the NCS selection is a little bit troubling. (maybe the phenix developers can chime in on this) reference = chain 'B' and (resseq 243:293 or resseq 310:370 ) selection = chain 'A' and (resseq 243:293 or resseq 310:370 ) seems as if resseq 243:293 is behaving differently than 310:370? [3] the densities of the side chains of a helix (not an xtal contact) are poorly defined, with geometry for the backbone not so good. There's talk about choosing the correct MR solution (see On the molecular-replacement problem in the presence of merohedral twinning: structure of the N-terminal half-molecule of human lactoferrinW. A. Breyer, R. L. Kingston, B. F. Anderson and E. N. Baker ) . I use phaser to pick my MR solution for P41. Could phaser possibly have chosen poorly? Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Choosing MR solutions in the case of perfect twinning with P41212?
Hi Francis, I would consider the possibility of a orthorhombic sg (a~b with twinning). If unlucky maybe even monoclinic. Cheers Roberto On 5 Feb 2009, at 18:10, Francis E Reyes francis.re...@colorado.edu wrote: It seems like this space group will be the death of me. I'm working on a structure in SG P41212 one molecule per asu that was solved with experimental SAD phases. The resolution is to 2.5 and the refinement is stuck at an R/Rfree of 30 and 33 with bonds rmsd of 0.011 and angles of 1.597 . The unit cell is 73.604 73.604 114.279 90.00 90.00 90.00. I'm considering the case of perfect twinning where the real s.g. is P41 masked under the higher symmetry in P41212. It seems to be the case in perfect twinning that the approach is to molecular replace the refined model into the lower space group. I reindexed my data to the lower space group P41 and molecular replaced into the reindexed data with Phaser. A single was solution was found with 2 mol per asu (39.6% solvent content) related by NCS. I've refined the now two fold ncs related structure in P41 to a much more respectable R/Rfree of 25.2 and 28.6 with rmsd bonds at 0.004 and angles at 0.865 refining with a twin law and NCS as implemented by phenix.refine. However I'm not happy: [1] a simmulated anneal omit map one of the monomers in P41 where 5 residues in a non crystal contact region of the molecule (I wanted to challenge the omit map) shows nearly no density. (the SA OMIT map was generated with phenix.autobuild using the same refine parameters as the final round of refinement) [2] the NCS selection is a little bit troubling. (maybe the phenix developers can chime in on this) reference = chain 'B' and (resseq 243:293 or resseq 310:370 ) selection = chain 'A' and (resseq 243:293 or resseq 310:370 ) seems as if resseq 243:293 is behaving differently than 310:370? [3] the densities of the side chains of a helix (not an xtal contact) are poorly defined, with geometry for the backbone not so good. There's talk about choosing the correct MR solution (see On the molecular-replacement problem in the presence of merohedral twinning: structure of the N-terminal half-molecule of human lactoferrinW. A. Breyer, R. L. Kingston, B. F. Anderson and E. N. Baker ) . I use phaser to pick my MR solution for P41. Could phaser possibly have chosen poorly? Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Off topic: Crystal degredation with age
/lurk_mode_off /dumb_question_on Dear All, I was recently trying to find references on how age may degrade a crystal, i.e. grow them and use them or preserve them as fresh as possible. I seem to remember seeing a couple of papers on this but my memory is fading and I have been unable to locate them. Can anyone jog my memory or tell me if I'm imagining things? I've found plenty on the protein prep etc. but nothing on the crystal. Thanks, 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: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here!Crystallization, how quaint! /dumb_question_off /lurk_mode_on
Re: [ccp4bb] Off topic: Crystal degredation with age
Some things improve with age. Here is one of my favorite stories: http://tinyurl.com/oldtrna The crystal structure of yeast phenylalanine tRNA at 2.0 Å resolution: cleavage by Mg2+ in 15-year old crystals Luca Jovine, Snezana Djordjevica and Daniela Rhodes We have re-determined the crystal structure of yeast tRNAPhe to 2.0 Å resolution using 15 year old crystals. The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details. In particular, the water structure around the tightly bound Mg2+ is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium- binding sites and the role of water molecules in coordinating the metal ions to the tRNA. We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg2+ binding. In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TΨC arm and also contacting a symmetry-related tRNA molecule. Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved. The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg2+. On Feb 5, 2009, at 11:11 AM, Edward Snell wrote: /lurk_mode_off /dumb_question_on Dear All, I was recently trying to find references on how age may degrade a crystal, i.e. grow them and use them or preserve them as fresh as possible. I seem to remember seeing a couple of papers on this but my memory is fading and I have been unable to locate them. Can anyone jog my memory or tell me if I'm imagining things? I've found plenty on the protein prep etc. but nothing on the crystal. Thanks, 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: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here!Crystallization, how quaint! /dumb_question_off /lurk_mode_on
Re: [ccp4bb] Off topic: Crystal degredation with age
Here's another very similar case: http://www.ncbi.nlm.nih.gov/pubmed/12270703 On Thu, Feb 5, 2009 at 11:48 AM, William G. Scott wgsc...@chemistry.ucsc.edu wrote: Some things improve with age. Here is one of my favorite stories: http://tinyurl.com/oldtrna The crystal structure of yeast phenylalanine tRNA at 2.0 Å resolution: cleavage by Mg2+ in 15-year old crystals Luca Jovine, Snezana Djordjevica and Daniela Rhodes We have re-determined the crystal structure of yeast tRNAPhe to 2.0 Å resolution using 15 year old crystals. The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details. In particular, the water structure around the tightly bound Mg2+ is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium-binding sites and the role of water molecules in coordinating the metal ions to the tRNA. We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg2+ binding. In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TΨC arm and also contacting a symmetry-related tRNA molecule. Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved. The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg2+. On Feb 5, 2009, at 11:11 AM, Edward Snell wrote: /lurk_mode_off /dumb_question_on Dear All, I was recently trying to find references on how age may degrade a crystal, i.e. grow them and use them or preserve them as fresh as possible. I seem to remember seeing a couple of papers on this but my memory is fading and I have been unable to locate them. Can anyone jog my memory or tell me if I'm imagining things? I've found plenty on the protein prep etc. but nothing on the crystal. Thanks, 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: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here!Crystallization, how quaint! /dumb_question_off /lurk_mode_on
Re: [ccp4bb] Off topic: Crystal degredation with age
I had a structure that was done with crystals that were about a year old. Initial crystals appeared in a less then a day and diffracted very poorly. In trying to make room for more trays I reexamined the old trays before throwing them out and low and behold nice well diffracting crystals. Of course these are probably more the exception then the rule. Len Leonard Thomas Ph. D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry 620 Parrington Oval Norman, OK 73032 lmtho...@ou.edu Office: 405-325-1126 Lab: 405-325-7571 On Feb 5, 2009, at 2:05 PM, Nathaniel Echols wrote: Here's another very similar case: http://www.ncbi.nlm.nih.gov/pubmed/12270703 On Thu, Feb 5, 2009 at 11:48 AM, William G. Scott wgsc...@chemistry.ucsc.edu wrote: Some things improve with age. Here is one of my favorite stories: http://tinyurl.com/oldtrna The crystal structure of yeast phenylalanine tRNA at 2.0 Å resolution: cleavage by Mg2+ in 15-year old crystals Luca Jovine, Snezana Djordjevica and Daniela Rhodes We have re-determined the crystal structure of yeast tRNAPhe to 2.0 Å resolution using 15 year old crystals. The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details. In particular, the water structure around the tightly bound Mg2+ is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium-binding sites and the role of water molecules in coordinating the metal ions to the tRNA. We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg2+ binding. In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TΨC arm and also contacting a symmetry-related tRNA molecule. Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved. The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg2+. On Feb 5, 2009, at 11:11 AM, Edward Snell wrote: /lurk_mode_off /dumb_question_on Dear All, I was recently trying to find references on how age may degrade a crystal, i.e. grow them and use them or preserve them as fresh as possible. I seem to remember seeing a couple of papers on this but my memory is fading and I have been unable to locate them. Can anyone jog my memory or tell me if I'm imagining things? I've found plenty on the protein prep etc. but nothing on the crystal. Thanks, 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: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here!Crystallization, how quaint! /dumb_question_off /lurk_mode_on
Re: [ccp4bb] temperature after 30 minutes using microscopes ?
Here's the summary and some own experimental data. First off all thank you all for your replies. trick 1: put a glass bottle filled with water between fiberoptics and the mirrors of your base (Isolde Le Trong) trick 2: suspend plates to allow air circulation, with bulb under plate scopes (Jose Antonio Cuesta_Seijo) Halogen driven fiberoptics tested ∆1.2 F ~ 0.6 ˚C (Matthew Franklin) Zeiss LED has too much diffuse light and warms up (Clemens Grimm) Zeiss LED used at Diamond beamlines are very good (Sandy James) (I'm wondering if both of you had different bases, mine will have a mirror to focus the LED light where I want it to be) And here my own tests: I have a Zeiss Stemi2000 right now mainly to test the LED light, the LEDs are in the base and can't be moved around. In this case the contrast light is sufficient to visualize crystals and mount them. However the glass above the LED's really gets warm starting temperature was 20.9 ˚C and after 1 hour it stayed at 26.3 ˚C. Since the model I will most likely purchase will have a mirror, I assume heating should not be as dramatic, and keep in mind this will be only the mounting microscope with 50x magnification. The highend model I decided to go for fiberoptics, mainly because of brightness to take pictures etc (100x). I tried one of our neighbouring labs fiberoptics microscope and there was an increase over an hour of 0.3˚C compared to the surrounding environment. I'm expecting the smaller scope to arrive within the next 2-3 weeks and I'll send an update on the thermal aspects to the board then. Hope this information is useful to the community, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Biochemistry and Molecular Biology, W8708 615 North Wolfe Street Baltimore, MD 21205 Phone: +1-410-614-4742 Fax: +1-410-955-3655 Web: http://faculty.jhsph.edu/default.cfm?faculty_id=2101
Re: [ccp4bb] comparison of maps, intensities and other basics
A map file stores a density value for each point on a grid. The units and nature of that item is not defined in the format of the map. A map can store any number of things. The actual values are defined by the process that created the map file. For electron density maps you will find that some contain values measured in e/A^3, others contain values that are normalized Z scores (The standard deviation of the variation about the mean is set to 1.0), or just a bunch of numbers with arbitrary and mysterious units. One tends to use e/A^3 when trying to relate the map to expected electron density or to compare one map to another. A normalized map is useful if you are interested in the frequency that a density value of that magnitude appears in the map. (Is this value common or rare?) One uses arbitrary values if one has an attachment to honesty. Calculating an electron density map in units of e/A^3 is not an easy task. The diffracted intensities are not measured, themselves, in real units. Their magnitude only has meaning as intensities relative to the other intensities in the same dataset. For the map to be expressed in units of e/A^3 the diffraction intensities must be expressed in units of e/Unit Cell (at least that is the convention). This is a hard problem and many papers have been written on the topic. If you have a well refined and complete model for the contents of the crystal you can use the calculated diffraction pattern as a template to scale the observed intensities and calculate maps in e/A^3, but this is an approximation as no model is complete or completely correct. The other big issue is that we cannot measure the one reflection that defines the average of the electron density in the crystal. It happens to always hit the beamstop. Because of this problem our maps usually have an average value of zero, which is of course wrong. Even when the density values are expressed in e/A^3 the intention is that each value in the map must have a number added to it to achieve the true value at that point. At least it's the same number everywhere in the map, although we don't know its value. Because of these issues and uncertainties, when maps are compared they are usually compared using a correlation coefficient. The correlation coefficient is relatively unaffected by these scaling problems and will usually give the same answer when given any of the kinds of maps I described. If you want a more detailed comparison of electron density values you really have to get into the details of each of the datasets and scaling that was applied to ensure that your results are meaningful. Estimating the error bars of an electron density map is another enormous problem. As you would expect, it depends critically on the origin of the map. The error analysis of a map calculated from MAD phasing is quite different than that of a map calculated using a refined model as a reference. One complication is that the error level is not necessarily the same everywhere in the map. In addition the errors at different regions of the map are not independent. The correlation of deviations at different regions of the map are likely more important to any analysis then any simple overall error bar. However, if you insist on an error level, my best guess would be to identify the regions of bulk solvent and calculate the rms deviation from the mean there. Since these regions should be flat, and deviations from the mean must be due to something that does not represent election density. We might as well call it error. Dale Tronrud Peter Schmidtke wrote: Dear CCP4BB List Members, first of all I am not a crystallographer, but I would like to get some things clear, things I did not find in Crystallography Made Crystal Clear and on the internet for now. I am trying to read electron density maps in the EZD format. These maps contain scaled values of electron density and size and shape of the unit cell. How can I convert the values of intensities (what is the unit of these values?) to the probabilities you can see in coot for example (1.03 electron / A^3), Once I have achieved this conversion, can I compare densities of different maps of different proteins? If not directly, is there a way to do so? Last, is there a way to know the experimental error made on intensity values of a map? Thanks in advance.
[ccp4bb] Postdoctoral Fellow, Membrane Protein Structural Biology
We are seeking talented and highly motivated Postdoctoral Fellows to work within the Structural Biology Department of GNF on the structure and function of eukaryotic membrane proteins associated with human disease. Projects will utilize GNF's state of the art robotic eukaryotic protein expression, bio-analytic, crystallization and X-ray data collection systems to investigate and determine the structures of novel membrane proteins. The position provides a unique opportunity to conduct research in an exceptional multi-disciplinary environment studying many aspects of the biological sciences from pure biology to translational drug discovery research. The successful candidate should ideally have experience in membrane protein expression, purification and analysis, a desire to learn structural biology techniques and a recent Ph.D. in biochemistry, biophysics or related discipline. The Genomics Institute of the Novartis Research Foundation (GNF) located in the Torrey Pines area of San Diego, CA focuses on paradigm-shifting technologies, making possible new approaches to complex biomedical problems. Pioneering technology platforms in genomics, proteomics, chemistry, structural biology, computation biology and engineering have been established as part of an integrated, multidisciplinary approach to biomedical research and drug discovery. The mission is to develop and apply innovative technologies to the discovery of new biological processes and a detailed understanding of their underlying molecular mechanisms ultimately leading to new or improved human therapeutics. Currently the institute consists of approximately 500 scientist dedicated to various areas such as Neurobiology, Cancer Biology, Immunology, Metabolic/Cardiovascular and Infectious Disease. Applications or informal enquiries should be directed to G. Spraggon gsprag...@gnf.org and Andreas Kreusch akreu...@gnf.org : Job Code GS08-008 10675 John Jay Hopkins Drive San Diego, CA 92121
Re: [ccp4bb] Choosing MR solutions in the case of perfect twinning with P41212?
This sounds like a case I had last year. It looked like P41212 (in fact I solved it by MR in P43212 and refined the data in shelxl reasonably well. Two superimposed molecules at 50% occupancy began to emerge from the density - this was at very high resolution). It took me a while to get there, but in reality it turned out to be P212121 with TWIN 0 1 0 1 0 0 0 0 -1 I generated the new asymmetric unit (2 molecules) from the old (one molecule) and refined from there. The improvement in R-factor was immediately obvious with both shelxl and phenix.refine. Hope that helps, Cheers, Charlie Francis E Reyes wrote: It seems like this space group will be the death of me. I'm working on a structure in SG P41212 one molecule per asu that was solved with experimental SAD phases. The resolution is to 2.5 and the refinement is stuck at an R/Rfree of 30 and 33 with bonds rmsd of 0.011 and angles of 1.597 . The unit cell is 73.604 73.604 114.279 90.00 90.00 90.00. I'm considering the case of perfect twinning where the real s.g. is P41 masked under the higher symmetry in P41212. It seems to be the case in perfect twinning that the approach is to molecular replace the refined model into the lower space group. I reindexed my data to the lower space group P41 and molecular replaced into the reindexed data with Phaser. A single was solution was found with 2 mol per asu (39.6% solvent content) related by NCS. I've refined the now two fold ncs related structure in P41 to a much more respectable R/Rfree of 25.2 and 28.6 with rmsd bonds at 0.004 and angles at 0.865 refining with a twin law and NCS as implemented by phenix.refine. However I'm not happy: [1] a simmulated anneal omit map one of the monomers in P41 where 5 residues in a non crystal contact region of the molecule (I wanted to challenge the omit map) shows nearly no density. (the SA OMIT map was generated with phenix.autobuild using the same refine parameters as the final round of refinement) [2] the NCS selection is a little bit troubling. (maybe the phenix developers can chime in on this) reference = chain 'B' and (resseq 243:293 or resseq 310:370 ) selection = chain 'A' and (resseq 243:293 or resseq 310:370 ) seems as if resseq 243:293 is behaving differently than 310:370? [3] the densities of the side chains of a helix (not an xtal contact) are poorly defined, with geometry for the backbone not so good. There's talk about choosing the correct MR solution (see On the molecular-replacement problem in the presence of merohedral twinning: structure of the N-terminal half-molecule of human lactoferrinW. A. Breyer, R. L. Kingston, B. F. Anderson and E. N. Baker ) . I use phaser to pick my MR solution for P41. Could phaser possibly have chosen poorly? Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310 35 Stirling Highway Crawley WA 6009 Australia charles.b...@uwa.edu.au +61 8 6488 4406
[ccp4bb] PHIDM AND FOMDM from DM
Dear all, I am using the density modification tool in ccp4 to generate improved phases for/from my model. I find that the electron density map I generate using Fobs, and density modified phases (PHIDM) are not the same as that generated using Fobs, phicalc (original calculated phases) and FOMDM (new improved figure of merit post dm). Can someone please explain to me why this is so? Thanks in advance, Xie
