[ccp4bb] question about DM
Hi all, Just a (naive?) question: how does one manage to introduce (and deal with) improper non-crystallographic symmetry in DM ? Or does one has to go to DMmulti for that (because, by definition, going from one crystal form to another crystal form is improper NCS) ? Ta, F. Vellieux
Re: [ccp4bb] Regarding refinement in Refmac5
Hi there, Not much information provided. How was the initial model refined ? Phenix ? It could be a problem with the Refmac refinement protocol (difficult to say with so little information) if you switched from Phenix to Refmac. How certain are you 1 - of the space group; 2 - that the crystal wasn't twinned ? You can have both and it can be annoying. Further, at this resolution I think you could use one of the SHELXes (forgot the terminology) for refinement, that could be more appropriate. F.V. Deepthi wrote: Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi
[ccp4bb] information received through the AFC: iycr2014
Dear colleagues, Information just received here through the AFC channels: the UN general assembly has declared that 2014 will be the international year of crystallography. Their statement (in French) as an attachment (sorry about including an attachment). F. Vellieux IYCr-FR.pdf Description: Adobe PDF document
Re: [ccp4bb] information received through the AFC: iycr2014
The press release in English: http://www.un.org/News/Press/docs/2012/ga11262.doc.htm Boaz: perhaps the crystallography congresses and events taking place in 2014 will be attended by UN peacekeeping force members as well (with their blue helmets) ? F.V. Boaz Shaanan wrote: Et alors, what will this mean for us? Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710
Re: [ccp4bb] information received through the AFC: iycr2014
Do you mean we should crush the helmets and solve the structure of the resulting powder blue coloured powder by crystallographic methods ? Sounds like a good proposal to be put forward to the UN to help them get rid of their used helmets ! David Schuller wrote: On 07/04/12 08:03, Vellieux Frederic wrote: The press release in English: http://www.un.org/News/Press/docs/2012/ga11262.doc.htm Boaz: perhaps the crystallography congresses and events taking place in 2014 will be attended by UN peacekeeping force members as well (with their blue helmets) ? Powder blue, one might say.
Re: [ccp4bb] how to get phase of huge complex
Hi Lisa, hi all, Please do not discard the alternative method(s) of conventional heavy atoms. Co-crystallisation or heavy-atom containing mother liquor soaks. You may remember that monster complexes have been solved in the past by such methods, and sometimes there are difficulties in crystallising the Se-Met version of a protein (the native protein gives crystals, the Se-Met version does not). And there is still some work going on regarding the development of novel (lanthanide-based) heavy-atom compounds that may provide both isomorphous and anomalous differences, the anomalous signal being extremely useful in the case of lanthanides and can be used on its own to solve 3D structures (one can then go back to the structure of the native macromolecule or complex if need be). See e.g. Talon, R. et al. (2011), J. Sync. Rad. 18, 74-78 (PMID: 21169697) and http://www.natx-ray.com/products/catalogue_consum_CSM002.html HTH, Fred. F.M.D. Vellieux (B.Sc., Ph.D., hdr) Institut de Biologie Structurale J.-P. Ebel CEA CNRS UJF LBM/ELMA 41 rue Jules Horowitz 38027 Grenoble Cedex 01 France Tel: +33 (0) 438789605 (direct line), +33 (0) 663482891 (mobile phone) Fax: +33 (0) 438785494 e-mail: frederic.velli...@ibs.fr LISA wrote: Hi all, My work is to solve huge complex containing 4 different proteins and total molecular weight is about 300 KD. I can purify the complex by co-expression them in E.coli. This complex contains 8 protein A, 2 protein B and 1 protein C and D. protein B and protein C have homology structures deposited in PDB database. No homology structure available for protein A and D, which contribute 60% of the whole molecular weight for the complex. Now I am trying to find a way to solve the phase of this complex. I am thinking of use sad or mad with se-Met. There total 111 Met residues in this complex. Is it possible to solve this complex by se-Met? Does someone have experience to solve huge complex structure with se-met? It is also very welcome for all the suggestion. Thank you. All the best, Lisa
Re: [ccp4bb] P321 space group reindex problem
Hi there, I am not certain that the thread is P321 space group reindex problem any more. But: trigonal (and hexagonal) space groups are (usually?) polar. The cell axis c can go up or can go down, and in order to get a consistent indexing you need to check both indexing systems when you scale additional data to your native (the indexing chosen by your first crystals defines the standard indexing - I must say that I haven't checked in the drawings of the international tables if having c going up or going down leads to a difference in that particular space group, P321, I'd need to draw both possibilities and check but I'm sorry I do not have the time right now - in fact it's too bad that the International Tables do not indicate Polar or Non-polar). For practical purposes, a derivative is considered non isomorphous when the differences in unit cell parameters exceed ca. 1% (this is because if you take 2 crystals from the same crystallisation drop and collect and process diffraction crystals from these 2 crystals, you will never get exactly the very same values for the unit cell parameters; non-isomorphism effects start at ca. 1% change and you'll never get 2 perfectly isomorphous crystals - even if you collect diffraction data twice from the same crystals you will not get perfect isomorphism). From the values mentioned, 1% of the cell parameters of the native for a and b is 1.81 Angstroem and for c 1.1 Angstroem (the angles do not matter for a trigonal space group). Had you obtained a value for a, b larger than ca. 183 Angstroem, or below ca. 109.2 Angstroem (only in the direction indicated by the changes mentioned in your mail - I ignored changes in the opposite direction) then you would have been able to say that the crystals were non-isomorphous to each other. For me they are isomorphous to each other and I ignore these small differences in unit cell parameters. The difference of 3% in Rfactor (I suppose this is |FP - FPH| / |FP|, no Sigma character on my keyboard to indicate the summations over h k l) is I think due to 2 different chemicals (heavy-atom compounds) in derivative 1 and derivative 2. Differences in R-factors at low resolutions are often associated with solvent effects, and I think you will have 2 different mother liquors and hence 2 different solvents in derivative 1 and in derivative 2. That is assuming that derivative 1 and derivative 2 were prepared using 2 different chemicals. And typically low-resolution data (below 15 Angstroem resolution or so) is kept out during phasing by the MIR method. To locate the heavy atom constellations in the 2 derivatives, you could compute and interpret difference Patterson maps - including automated interpretation, vector search and the likes -, you could use direct methods (the heavy atom constellation is similar to a small molecule because there are far fewer atoms there than in the full macromolecule, and direct methods work extremely well for small molecules - you would need to use the isomorphous differences in order to use direct methods; no mention is made of any anomalous signal so I do not know if you could this as well). HTH, Fred. Qixu Cai wrote: Why the 29% Rfactor indicate the derivatives are not isomorphous to native dataset? Native dataset cell constant: 181.39 181.39 110.217 90 90 120 derivative1 cell constant: 181.909 181.909 109.62 90 90 120Rfactor to native: 26% derivative2 cell constant: 181.527 181.527 109.32 90 90 120Rfactor to native: 29% The Rfactor at low resolution is larger than in high resolution. Could you please to help me figure out where the heavy atoms had been soaked into the crystal? Thank you very much. Best wishe, Qixu Cai 2012/5/30 Laurent Maveyraud laurent.maveyr...@ipbs.fr mailto:laurent.maveyr...@ipbs.fr Hi, it is therefore likely that your spacegroup is really P321... hopefully, your data set is not twinned, did you check that ? You are left with 2 possible indexing schemes, as already mentionned. Chek scaling derivative / native scaling for each indexation of the derivative : the lowest Rfactor will likely indicate the right one. It appears that you end up with Rfactor of about 29 %, which suggest that your derivatives are not isomorphous to your native dataset. How do cell parameters compare for each data set ? Check also how the Rfactor varies with resolution, you might still have usefull phasing info at low resolution. hope this helps laurent Le 30/05/2012 07:50, Qixu Cai a écrit : At first, I processed the data at P3 space group. But after phenix.xtriage analysis, the Xtriage told me the space group must be P321, so I used P321 to process my data, and got an acceptable Rmerge. Qixu Cai 2012/5/29 Phil Evans p...@mrc-lmb.cam.ac.uk mailto:p...@mrc-lmb.cam.ac.uk
Re: [ccp4bb] P321 space group reindex problem
Hi Ian, You're right the information is there... but not where I was expecting it (on the page corresponding to an individual space group). It had never occurred to me that it could be somewhere else. So thanks, and regards to Jasmine. Fred. Ian Tickle wrote: Hello Fred On 30 May 2012 07:55, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi there, But: trigonal (and hexagonal) space groups are (usually?) polar. The cell axis c can go up or can go down, and in order to get a consistent indexing you need to check both indexing systems when you scale additional data to your native (the indexing chosen by your first crystals defines the standard indexing - I must say that I haven't checked in the drawings of the international tables if having c going up or going down leads to a difference in that particular space group, P321, I'd need to draw both possibilities and check but I'm sorry I do not have the time right now - in fact it's too bad that the International Tables do not indicate Polar or Non-polar). It does, at least my edition (Vol. A: 5th ed., 2002, Table 10.2.1.2, p.806) does - ITC has everything you need to know about space groups (and a lot more besides)! See also this table that I made where all polar non-polar SGs are listed individually: http://www.ccp4.ac.uk/dist/html/alternate_origins.html Counting only the non-enantiomorphic ones, PG3 (4 SGs) and PG6 (6 SGs) are polar, whereas PG321 (3 SGs), PG312 (3 SGs), PG32 (1 SG) and PG622 (6 SGs) are non-polar. So in all 10 are polar and 13 are non-polar. A 2-fold axis perp to another axis always implies that there's no preferred direction along the other axis, so it's non-polar. Cheers -- Ian
Re: [ccp4bb] phaser: high z score but no sol
Hi, If you have the correct solution, clashes may be due to loops. It may be an idea to clip these off for the molecular replacement calculations (loops might be shorter in the structure-to-be-solved than in the search model, they may have different conformations in the structure-to-be-solved than in the search model, a common property of loops is that they sometimes have different conformations). You didn't provide much information about what was precisely done wrt molecular replacement calculations, I notice... So what I write is just guesswork. If you have little experience with molecular replacement, it may be a good idea to get advice from a colleague who has plenty and can sit next to you and guide you through the process (again, this is guesswork). HTH, Fred. LISA wrote: Hi all, I am trying to solve one structure by molecular replacement with phaser in CCP4. This a complex of a multi-domain domains with small ligand. I have structues of this protein in apo state and with other similar ligand. The space group of this crystal is P21. This crystal should have 4 molecules in ASU. I used the full protein as model but did get any sol and LLG is below zero. Then each domain were used as the search models in phaser with rotation and tranlsation. I can the get high z score (20), and LLG is raising. It looks like I get the right sol, but it have more 50 clashes. Why phaser give wrong sol with so high z socre? Can anyone give me some suggestion to solve my strucutes? Thank you. Best Lisa
Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
Hi, I think this practice (requesting the data) is getting more and more common these days with some scientists having published fake structures. You are far more protected from scientific misconduct when you provide the data to referees (this takes place through an editorial system - you should keep a copy of all correspondence and requests) than you are in your own lab where your next door neighbor may decide to use your results for his/her own benefit without giving you any credit. Should your paper be rejected and if you see your structure published by others soon afterwards, you could argue with an editor that your data may have been used by others provided that you have kept a copy of everything as mentioned before in this mail. Fred. Marc Kvansakul wrote: Dear CCP4BBlers, I was wondering how common it is that reviewers request to have a copy of the PDB coordinate file for the review purpose. I have just been asked to supply this by an editor after several weeks of review, after one of the reviewers requested a copy. Not having ever been asked to do this before I feel just a tad uncomfortable about handing this over… Your opinions would be greatly appreciated. Best wishes Marc Dr. Marc Kvansakul Laboratory Head, NHMRC CDA Fellow Dept. of Biochemistry| La Trobe University | Bundoora Rm 218, Phys Sci Bld 4, Kingsbury Drive, Melbourne, 3086, Australia T: 03 9479 2263 | F: 03 9479 2467 | E: m.kvansa...@latrobe.edu.au |
Re: [ccp4bb] Temp Fact Variance Analysis
Hi, You could try to lower the threshold used to contour the maps for these side-chains (middle mouse button if you have a 3 button mouse with a wheel in the middle). Quite often such side chains have lowish electron density that does not appear at (say) 1.0 or 1.5 sigma (in the 2mFo-DFc maps), but that appears at (say) 0.7 sigma. If you delete such side-chain atoms you'll probably end up with negative difference density appearing in the difference Fourier after the next round of refinement. So these residues must be checked individually, much work I know... To delete or not to delete, that is the question. Fred. Uma Ratu wrote: Hello, I run my model in Coot to do Temp Fact Variance Analysis. There are red bars from the B-factor Variance graphy. I click each red bar to exam the residues in Coot. Many of these residues do not have the electronic density on their side chains, especially Lys residues. Here is my questions: 1. The lack of electronic density is the cause of these red-bars? 2. How do I fix them? delete the side chains? The diffraction data is 2A, and data completness is 98.8%. Thank you for comments Ros
Re: [ccp4bb] Temp Fact Variance Analysis
Typo (my fingers type faster than my brain...) Original Message Subject:RE: [ccp4bb] Temp Fact Variance Analysis Date: Thu, 1 Mar 2012 14:56:06 + From: Oganesyan, Vaheh oganesy...@medimmune.com To: Vellieux Frederic frederic.velli...@ibs.fr References: caf6easvd8vw1hwfv4dl5sicttwqgcbzvraaexummnpv2qxb...@mail.gmail.com 4f4f8cb1.1070...@ibs.fr Fred, After deleting the difference density will be positive, IMHO. Vaheh
Re: [ccp4bb] MTZ file
Hi, The refinement program generates an mtz file with map coefficients (including difference Fourier coefficients) so you should use that one for model rebuilding in coot; for the next refinement rounds, at the beginning of each round you should provide the initial mtz file coming from data processing and post processing (1 in your terminology I suppose). This is because several refinement programs scale the Fobs (and sigmaFobs). Refmac is one of them I think; for data deposition, you deposit the same mtz file (1), and if you also wish to deposit map coefficients you can also extract the map coefficients from the final refinement and map calculation round mtz (using, say, sftools) and deposit those. Phaser does not carry out model refinement per se (adjusting atom positions and temperature factors). But the map coefficients present in the mtz file are generated in the same way (Sigmaa coefficients) with Phaser and with Refmac. There might be small difference in terms of the program code used internally but that shouldn't make much of a difference. Since a model that has seen refmac is (normally) improved, the electron density maps generated using the refmac mtz should be improved wrt the maps coming directly from Phaser (molecular replacement). Normally, the model you refine will have had the differences in the sequence between molecular replacement search model and 'your' structure corrected (gradually?), the solvent model is introduced and improved, ligands, ions etc are being introduced... Thus the maps improve seen that your model should reflect more and more what is present in the crystal as you build and refine. HTH, Fred. Uma Ratu wrote: Hello, I have a question about .mtz files used in model building. Here is how I did: Diffraction data - HKL 2000: .sca CCp4i: scalepack2mtz: .mtz (1) Phaser: In: template pdb .mtz(1) Out: model .pdb(1) .mtz(2) Refmac5: model .pdb(2) .mtz(3) Here is the question: 1. Coot check and refinment: which mtz file shoudl I use? 2. With further refinemnt by refmac5, which mtz file should I use? 3. When I deposit data, which mtz file to use? 4. What is the difference between .mtz(1) and the .mtz files generated from phaser and refmac? Thank you for advice Ros
Re: [ccp4bb] off-topic:schematic representations for secondary structure
I believe Procheck generates drawings such as those. It generates PostScript files, and if you need to have (eg) jpg files, a PostScript interpreter, screen capture and there you are (Gimp to select only the areas you're interested in) HTH, Fred. WENHE ZHONG wrote: Dear memebers, Thank you all firsit for the helps on my previous post about sequence alignment. They are really useful! Apologize for the off-topic question again. I usually use DSSP method to calculate the secondary structures on my sovled structures. I saw a nice schematic representation for secondary structure from PDB database website (please see the attached figure). Does anyone know whether there is a program to draw these schematics? Thank you. King regards, Wenhe
Re: [ccp4bb] MAD
For those of you interested, the reply to Tassos' question can be found here: http://www.iucr.org/resources/commissions/crystallographic-computing/schools/school96/ccp4-program-system (on-line) as well as here, http://www.*ccp4*.ac.uk/manual.ps (a ps file). McLaughlin, Terry and Zelinka. And yes, I'm over 40 ! I have also dealt with LCF files... Fred. Anastassis Perrakis wrote: A, yes, inventor's names. Anyone reading who is less than 40 and knows what MTZ stands for? ;-) My favorite technique remains SADDAM - a side product of Gerard's War On Error, that never did catch-up with the masses - experimentally or as an acronym. A. On 19 Jan 2012, at 21:51, Petr Leiman wrote: It would be so much more convenient to call these techniques (MAD, SAD, etc.) by their inventor's name. This would simplify things immensely simultaneously eliminating CCP4BB MADisagreements. Although in our days of copyrights wars, the journals and perhaps conferences where these methods were presented for the first time would insist on using their names as part of the method's name... Petr On Jan 19, 2012, at 7:42 PM, Ethan Merritt wrote: On Thursday, 19 January 2012, Ian Tickle wrote: So what does this have to do with the MAD acronym? I think it stemmed from a visit by Wayne Hendrickson to Birkbeck in London some time around 1990: he was invited by Tom Blundell to give a lecture on his MAD experiments. At that time Wayne called it multi-wavelength anomalous dispersion. Tom pointed out that this was really a misnomer for the reasons I've elucidated above. Wayne liked the MAD acronym and wanted to keep it so he needed a replacement term starting with D and diffraction was the obvious choice, and if you look at the literature from then on Wayne at least consistently called it multi-wavelength anomalous diffraction. Ian: The change-over from dispersion to diffraction in MAD protein crystallography happened a couple of years earlier, at least with regard to work being done at SSRL. I think the last paper using the term dispersion was the 1988 Lamprey hemoglobin paper. The next two papers, one a collaboration with Wayne's group and the other a collaboration with Hans Freeman's group, used the term diffraction. WA Hendrickson, JL Smith, RP Phizackerley, EA Merritt. Crystallographic structure-analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation. PROTEINS-STRUCTURE FUNCTION AND GENETICS, 4(2):77–88, 1988. JM Guss, EA Merritt, RP Phizackerley, B Hedman, M Murata, KO Hodgson, HC Freeman. Phase determination by multiple-wavelength X-ray-diffraction - crystal-structure of a basic blue copper protein from cucumbers. SCIENCE, 241(4867):806–811, AUG 12 1988. WA Hendrickson, A Pahler, JL Smith, Y Satow, EA Merritt, RP Phizackerley. Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 86(7):2190–2194, APR 1989. On the other hand, David and Lilo Templeton continued to use the term anomalous dispersion for at least another decade, describing their diffraction experiments exploring polarization effects and other characteristics of near-edge X-ray scattering by elements all over the periodic table. Ethan Cheers -- Ian On 18 January 2012 18:23, Phil Jeffrey pjeff...@princeton.edu wrote: Can I be dogmatic about this ? Multiwavelength anomalous diffraction from Hendrickson (1991) Science Vol. 254 no. 5028 pp. 51-58 Multiwavelength anomalous diffraction (MAD) from the CCP4 proceedings http://www.ccp4.ac.uk/courses/proceedings/1997/j_smith/main.html Multi-wavelength anomalous-diffraction (MAD) from Terwilliger Acta Cryst. (1994). D50, 11-16 etc. I don't see where the problem lies: a SAD experiment is a single wavelength experiment where you are using the anomalous/dispersive signals for phasing a MAD experiment is a multiple wavelength version of SAD. Hopefully one picks an appropriate range of wavelengths for whatever complex case one has. One can have SAD and MAD datasets that exploit anomalous/dispersive signals from multiple difference sources. This after all is one of the things that SHARP is particularly good at accommodating. If you're not using the anomalous/dispersive signals for phasing, you're collecting native data. After all C,N,O,S etc all have a small anomalous signal at all wavelengths, and metalloproteins usually have even larger signals so the mere presence of a theoretical d difference does not make it a SAD dataset. ALL datasets contain some anomalous/dispersive signals, most of the time way down in the noise. Phil Jeffrey Princeton On 1/18/12 12:48 PM, Francis E Reyes wrote: Using the terms 'MAD' and 'SAD' have always been confusing to me when considering more complex phasing cases. What happens if you have
Re: [ccp4bb] MERGING THREE DATASETS
There are several ways to skin a cat. If you have processed your data with XDS, XSCALE can also do the job (you have to think of the unit cell parameters of the merged data set though - the default may not necessarily what you wish to have), and I am certain other data frame processing suites will have this offered as an option somewhere... (I personally like all the statistics provided by the combat / scala route though). Fred. Eleanor Dodson wrote: Do you still have the unmerged files? In that case just feed them to pointless and redo scala/etc.. If you only 3 merged data sets, you will have to use combat to convert the mtz files into an fake unmerged format then put them through pointless/scala to get relative scales and merge them.. Eleanor On 12/21/2011 11:15 AM, arka chakraborty wrote: Hi all, I have three datasets with same space-group and identical cell dimensions which I want to merge together. I remember that this was discussed in the blog some time back. Nevertheless, I can use some help! Thanks in advance, ARKO
Re: [ccp4bb] Movements of domains
A mixture between mathematical significance and biological significance as a part of the reply: you should also take into account the thermal vibrations of the atoms present in that domain, i.e. the thermal ellipsoids when you have one of the representations of anisotropic temperature factors (when these can be obtained, high enough resolution), together with the associated density smearing. Especially if you observe correlated thermal ellipsoids. If you have a small motion but that this motion can be (at least in good part) explained by the inherent thermal flexibility of all atoms in that domain then perhaps you can question the significance of this domain motion (at least in the publication). Fred. Filip Van Petegem wrote: Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com mailto:filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] raw data deposition
D Bonsor wrote: and allow someone else to have ago at solving the structure. I'd be careful there if there was a motion to try to implement a policy at SR sources (for academic research projects) to make it compulsory to publically release all data frames after a period (1 year ? 2 years ? 4 years) during which you are supposed to solve the structures you have collected the data for, so that others can have a go at it (and solve the structures for you): you may find yourself for example in between grants and need to spend all of your time looking for funding for a couple of years, with little or no staff working with you. With the trend we see of ever diminishing resources, this would mean that the very large and well funded labs and groups would solve their own structures, and solve those of smaller groups as well (and publish the latter). This would then mean (after a while) the concentration of macromolecular crystallography to only the lucky few who have managed to secure large grants and will therefore go-on securing such grants. You could call that evolution I suppose. We are already in a situation where the crystallographers who solved the structures are not necessarily authors on the publications reporting the structures... so is it time to go back to home sources (X-ray generators) for data collection ? Fred.
Re: [ccp4bb] raw data deposition
I must say that there were some emails exchanged between me and Gerard later, in which I pointed out that I wasn't against deposition of images (data frames). In fact, if SR sources kept user's data there would be one more structure from here in the PDB: HDD failure here, the data on a mirror HDD but the company in charge of maintenance erased the data frames and data processing statistics by accident. For a trypanosomal enzyme there is no chance that I can ever get funding now to replicate the work (protein production and purification, crystallisation, data collection) so that Table 1 could be produced for a manuscript. However, my email to the bb was provocative - I admit I was doing this willingly - to write that in such harsh funding times someone could start a career, get some small grant, enough to clone produce purify crystallize and collect a first data set. And then find him or herself without funding for X years (success rate = less than 10% these days). If this person then gets scooped by whoever, end of a promising career. Unfortunately, such a prospect doesn't seem to be science fiction any more nowadays. I hope this clears things. I wanted to be provocative and point out the difficulties we are all facing wrt funding so that we shouldn't set up a system that may result in killing careers. Our politicians do not need any help from us on that I think. Fred. Gerard Bricogne wrote: Dear Remy, You are right, and I was about to send a message confessing that I had been rash in my response to Fred's. Another person e-mailed me off-list to point out that sometimes a structure can be quickly solved, but that doing all the rest of the work involved in wrapping that structure into a good biological story for publication can take a very long time, and that it would be wrong for a SR source's forced disclosure policy to start imposing deadlines on that process. I entirely agree with both of you and admit that I reacted too quickly and with insufficient thought to Fred's message. However, as you point out yourself, this issue is related to a different question (SR sources' disclosure policy towards all data collected on their beamlines) from the original one that started this thread (deposition of raw images with the pdb entries they led to). The two topics became entangled through the idea of prototyping an approach to the latter by tweaking the storage and access features involved in the former. Many thanks to you and to the other correspondent for picking up and correcting my error. This however leaves the main topic of this thread untouched. With best wishes, Gerard. -- On Fri, Oct 28, 2011 at 01:38:29PM +0200, Remy Loris wrote: Dear Gerard, I cannot agree. Last year my group published a paper in Cell which contained a structure for which the native data were collected at a synchrotron around 1997. Various reasons contributed to the long lag period for solving this structure, but basically it all came down to money needed to do the work. Equally I am sure there are other cases for which a first good native data set is a breakthrough you wish to protect rather than hand it out to anyone who might potentially scoop you after you have put lots of money and effort into the project. Therefore: Images corresponding to structures I deposit in the PDB: No problem. That is what we do with processed data as well. But images of unsolved structures, I don't see why that should be enforced or done automatically by synchrotrons. Nobody deposits processed data without an accompanying structure either. I do agree that one could be given the option to deposit interesting data with which he/se will not continue for whatever reason. But this should be optional, and a clear consensus should emerge within the community as how the original producers of the data have to be acknowledged if these data are used and the results published by another team, especially if the use of that particular dataset is crucial for the publication. Remy Loris Vrije Universiteit Brussel and VIB
Re: [ccp4bb] Biological assembly
Hi, If, in your case, no possible asymmetric unit can contain A1-B2, then you deposit A1-B1 (or I suppose A2-B2...) and indicate to the PDB (like placing cards in the header cards) the operator to be used (and the subunit it applies to) in order to generate the most likely biological dimer. Normally the PDB can take care of that. Fred. Kayashree M wrote: Respected Sir, The space group is H3. if I generate the symmetry, it appears to be a dimer of trimers stacked one above the other with a rotation of 60 deg wrt each other, like this - A1, A2, A3 (in one trimer) stacked upon B1, B2, B3 (second trimer). So structure that is in the ASU is with A1-B1 while PISA predicts A1-B2. Thank you With Reagrds Kavya -CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote: - To: CCP4BB@JISCMAIL.AC.UK From: James Stroud Sent by: CCP4 bulletin board Date: 10/19/2011 10:41PM Subject: Re: [ccp4bb] Biological assembly On Oct 19, 2011, at 4:36 AM, Kayashree M wrote: We have a structure which is a homodimer in the asymmetric unit. PISA predicts most probable assembly as a dimer but this dimeric assembly is different from what is solved (offcourse we can generate the symmetry equivalent molecule and get that). This last sentence is a bit vague. Can you take the just dimer that PISA predicts, fit this dimer to the lattice (i.e. each monomer sitting correctly in density but retaining the dimeric relationship predicted by PISA), and then generate the complete lattice using just this fitted dimer and crystallographic symmetries? If so, that means that the PISA dimer is equivalent to the ASU you can deposit the PISA dimer as the ASU. James -- This message has been scanned for viruses and dangerous content by *MailScanner* http://www.mailscanner.info/, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by *MailScanner* http://www.mailscanner.info/, and is believed to be clean.
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Just to add to what has been said (written) before: coiled coiled or simply helices can be very problematic for M.R.. Human sacrifice has never given any positive result (as reported in the literature as far as I know), but heavy atom sacrifice could be attempted (heavy atom includes in my view atoms that are used for SAD, MAD - such as S, Se... - in addition to Au, Pt...) in parallel to your M.R. searches which may never provide you with an acceptable solution. Fred. Napoleão Valadares wrote: Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo
Re: [ccp4bb] Phenix building residues not found in input sequence file
Could you have an unexpected small subunit (or some other situation) giving additional density that Phenix is building into ? In other words, how sure are you of what is present in the crystal ? This is because I remember the enzyme methanol dehydrogenase where there was a small subunit which had not been evidenced for years... Fred. Allan Pang wrote: Hi everyone, Apparently my Phenix (1.6.3-473) is building in extra residues in the C-terminal region, which is not found in my input sequence file. It's trying to build a model which is longer than I expected. Phenix building 87 extra amino acids. Any clue what's wrong? I made sure that these amino acids are not residues found in the N-terminal region or anywhere else in the protein. Allan
[ccp4bb] important information for scientists with samples travelling via Lyon or Grenoble airports
Could be of importance to those who have to use the Lyon or Grenoble airports (to ship in or out), check out the update from Sept. 27, 2011. http://www.ill.eu/users/user-guide/safety/important-instructions-for-biological-samples/ Fred. http://www.ill.eu/
Re: [ccp4bb] Structure problem
Hi there, In crystallography there are so many places where you can have problems (and need to solve these problems) that I cannot list them all. There is no twinning in the data - you probably mean the data does not seem to indicate the presence of twinning but there might be twinning; what about the space group ? What about your crystal appearing to have one space group for one component (example the protein) but the space group for the other component (e.g. DNA, which could be partially disordered, that happens) being different - and you have processed the data in the apparent space group for the protein ? The crystal could contain the protein:DNA complex plus one of the components needed for the whole thing to pack (and form the crystals); the model might be sufficiently different from your structure that all the loops plus a good part of the core is improperly positioned - or you have domain and subdomain motions etc etc. There is one symptom: R-free seems to be stuck. What the reason is for this is unknown. There are cases in the literature where molecular replacement leads to this behaviour and where the crystallographers have to use experimental phasing (and understand what the problem was with molecular replacement afterwards, when the structure is solved). Fred. #HEW KAI LI KELLY# wrote: Hi, I am facing some problems in solving my structure now, so I am wondering if anyone is able to give me any tips and tricks on this matter. My protein-DNA complex structure diffracted to 1.5A. There are 4 missing residues, 2 on each terminal. There is no twinning in the data. The angles, the bonds, the rotamers and the Ramachandran plot are okay too. I am using molecular replacement for the phasing and the sequence homology between my protein and my homologous model is 33%. The electron density map for the protein looks very nice and there is also nice density for the DNA. Rfree converged from the initial 39%. However, Rfree refused to go down any further and it's still around 30-31%. Does anyone have any suggestions for me? Thank you in advance! Warmest Regards, Kelly Hew * * * * * *
Re: [ccp4bb] Crystypos [WAS: [ccp4bb] Mac OSX 10.7 Lion]
Well in fact, it all depends on the type of detector these small angels end up on and on the speed of this godly radiation. Only once you have considered both these elements can you say poor little things. My 2p worth. Fred. Ed Pozharski wrote: The best X-ray related typo I ever seen was the Small angel scattering - poor little things! On Fri, 2011-09-09 at 18:23 -0400, Patrick Loll wrote: Still doesn't beat my all-time favorite, an early Microsoft spell-checker that changed diffract to defrocked. I forgot to mention how delightful the spelling auto-correction feature can be. (It should have read nothing unusual in and of itself). That, at least, can be turned off.
Re: [ccp4bb] omit map calculation in CCP4
sadaf iqbal wrote: Hello everyone, Do anybody know the correct procedure for calculating omit map in CCP4, if one need to reduce the bias from the Molecular Replacement structure. I am confused about the two options, one is fft map calculation while the other is OMIT map calculation in Map Mask Utilities window. Which one can be used for omit map? Regards Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Hi, As far as I know there are 2 ways of creating Bhat's OMIT maps within ccp4: - the program OMIT (not in ccp4i); - the program sfcheck, with option NOMIT. Otherwise people sometimes refer to OMIT maps as maps in which a specific part of the model is removed for the calculation of the phases used in the computation of the map; the map is then examined in that area to see if the density for the removed part reappears. There are many ways of computing such maps (including using a text editor to remove part of the model, or reset occupancies to 0.0 for the part that is going to be removed - and including computer programs not in ccp4). Fred.
Re: [ccp4bb] No reflections in resolution bin???
james09 pruza wrote: Dear CCP4BBers, Refmac is giving the error No reflections in resolution bin??? It seems there is no SigFP column. I wonder how to fix the problem. Thanks in advance. James If the error is indeed due to a missing SigFP column, there are 2 ways to go about it I think: reprocess the data and do not discard the SIGFP's; if you cannot (the data are from, say, 25 years ago - at a time when data processing programs did not provide estimated SD values), you could use sftools to generate a fake SIGFP column where the values are set to (say) 0.1 * FP (or .075, or .125...). Fred.
Re: [ccp4bb] Newbie Installation Question
Quoting you: I type ccp4 and I get no error, what happens if you type ccp4i (followed by carriage return) ? Yuri wrote: I downloaded the package for RHEL5. My default shell is sh. But I can change environments. I have progrmas that I must run in tcsh. I tried sourcing sh and csh setups. With the csh setup, after I changed the PATH in the ccp4.setup file (originally set to usr/local/bin), I type ccp4 and I get no error. I am a bit at loss with the sh setup I tried sourcing and I get a command not found message, which leads me to think I am not succeding in sourcing it. Someone mentioned something about some files are not readable... I tried running the executables in the /bin directory but nothing happens... Thank you On Fri, 29 Jul 2011 13:53:49 -0700, Iain Kerr wrote: What is the output when you try to launch ccp4i ? Are there any error messages ? Problems with Tcl/Tk will usually return an error about not being able to locate a specific library. Can you launch the programs independent of the GUI ? eg. if you go to where the executables are (CCP4/ccp4-6.2.0/bin) ./mtzdump You should see some output giving the program version etc. and a list of keyword options. If so, the installation is probably OK and as Ed suggested it could be an environment problem. Why don't you outline the specific steps you've taken so far during installation and environment set up - what shell are you using ? did you download the source code or binaries ? what Tcl/Tk version are you using and where did you get it from ? I can help with this, off the board and then if we find a solution you can post it on the BB. Iain On 7/29/2011 12:37 PM, Yuri wrote: I tried that too ... no success On Fri, 29 Jul 2011 15:28:58 -0400, Ed Pozharski wrote: On Fri, 2011-07-29 at 20:08 +0100, Yuri wrote: Dear all, I have just downloaded and installed the ccp4-6.2.0. It says all I should do next is source the /setup-scripts/csh/ccp4.setup file... I have done that, but I cannot launch the program... Any help is welcome...(it is probably something really stupid on my part...) Best, It is most likely that your default shell is bash and you are trying to source tsch script, which naturally fails. Try /setup-scripts/sh/ccp4.setup instead. -- Yuri Pompeu
Re: [ccp4bb] Phaser and Molrep gave different solutions
Hi, It's not a bad idea to read the Phaser manual for molecular replacement; see http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement Soon after the start, in a table on the right hand side, there is: TFZ score 5, have I solved it ? No. Hence with a TFZ score of 3.8 you do not have a solution using Phaser. Fred. Hubing Lou wrote: Dear all, I am stuck in a molecular replacement case and looking for advices. I have been working on a protein-DNA complex structure. Data was processed by HKL2000 to 2.6Ang and some of the data statistics are shown below: Space group: P21, Unit Cell: 54.73, 104.91, 78.40, 90, 100.2, 90 Redundancy: 2.8 (2.7) Completeness: 94.8 (93.1) Linear R-fac: 0.051 (0.442) Data quality was checked by Phenix.xtriage and there's no problem. I then prepared a model by Chainsaw. Our protein shares only 30% of sequence similarity with the model, but structurally they are in the same group and almost identical in apo form. Matthrews Coeff indaced two monomers in AU. I then ran Phaser in automated search mode and there's a solution with RFZ score 4.8, TFZ score 3.8. The electron density map was not bad with DNA double helix clearly seen. However Refmac5 couldn't get Rfree lower than 50%. I then changed to MolRep, ran self rotation function first then used the first 10 peaks for translation search. Again there's a solution but it is different from that from Phaser. I attached a picture here. Checking in coot, the packing is the same. But, the refinement couldn't get Rfree lower than 50%. I have tried to include NCS, TLS refinement in Refmac, both not working. Hope someone out there can help. Thanks very much for your time. Hubing
Re: [ccp4bb] Data from old tapes
Plenty of old drives at ESRF, for that purpose. If you don't find any other way, I could always read them there (the ESRF is in my backyard) and burn the data to DVD for shipping (or upload to megaupload - but our gov't is hunting down people who use such services, they think megaupload and other such services are only for making illegal use of the internet such as transferring ripped commercial DVD's or CD's). Fred. VAN RAAIJ , MARK JOHAN wrote: did you try looking if there is a company offering data recovery services from these kind of tapes? if there is, there may not be a need to buy a tape drive yourself. Mark Quoting Min, Xiaoshan: Dear CCP4 community, We have a few datasets that reside in tapes (Sony QG-112M 8 mm tape and Maxell DDS-2 4 mm tape). I have been searching the internet for tape drives ( and cable) but haven't found anything. Does anyone know where we can purchase compatible tape drives for these lovely tapes? Or if you have a spare working set sitting in your graphic room and would like to sell them, that will be wonderful. Thanks. Xiaoshan Min. Ph.D. Molecular Structure Amgen San Francisco 1120 Veterans Blvd. South San Francisco, CA 94080 Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Strange density in arginine
The distance to your modelled water seems to be in agreement with an ionic interaction. What are the components of all buffers in the crystallisation components ? Fred. ruheng wrote: Dear all, Recently we are working on an archaebacteria protein which was expressed and purified from /E.coli /by conventional procedures. After we solved the structure, we found that there is an extra density in one of the argninine as shown in the attached picture. It seems that the density is larger than a methyl group and the atoms in the density are not on one plane. So we are curious about whether the density is a kind of posttranslational modification of arginine residues in the protein, if it is, what kind of modification could it be? And most important, what is the biological significance of this kind modification? Any suggestions and dicussions are appreciated! Best, Heng http://skydrive.live.com/redir.aspx?cid=2f5a8d98562e91e4page=playresid=2F5A8D98562E91E4%21199type=1Bpub=SDX.PhotosBsrc=Photomailparid=2F5A8D98562E91E4%21198authkey=S1J8JneduIs%24 Strange density in arginine http://skydrive.live.com/redir.aspx?cid=2f5a8d98562e91e4page=browseresid=2F5A8D98562E91E4%21198type=5Bpub=SDX.PhotosBsrc=Photomailauthkey=S1J8JneduIs%24 查看幻灯片 http://skydrive.live.com/redir.aspx?cid=2f5a8d98562e91e4page=playresid=2F5A8D98562E91E4%21198type=5Bpub=SDX.PhotosBsrc=Photomailauthkey=S1J8JneduIs%24 下载全部 http://skydrive.live.com/redir.aspx?cid=2f5a8d98562e91e4page=downloadaszipresid=2F5A8D98562E91E4%21198type=6Bpub=SDX.PhotosBsrc=Photomailauthkey=S1J8JneduIs%24 添加更多照片 http://skydrive.live.com/redir.aspx?cid=2f5a8d98562e91e4page=uploadresid=2F5A8D98562E91E4%21198type=5Bpub=SDX.PhotosBsrc=Photomailauthkey=S1J8JneduIs%24 此相册包含 1 张照片，并且将在 2011/10/02 之前在 SkyDrive 上提供。 通过 Hotmail 共享您自己的幻灯片 http://g.live.com/9wc9zh-cn/f_photos
Re: [ccp4bb] hello
Hi, The R-factor you mention, is it an R-factor before any refinement of the model ? Like an R-factor at the very beginning of the entire modeling procedure, right after molecular replacement ? If this is so: you should always compare such initial R-factors to the R-factors for the atoms placed randomly in the asymmetric unit (in the crystal thus, see X-ray Structure Determination by Stout and Jensen, pp 246 in the edition I have here - 1968, 1st edition?). R for centric reflections = 0.83, R for acentric reflections = 0.59. In space group F4132 there are many centric reflections. The random R-factor may be around 0.65 or higher... (I didn't count), and if the value you quote (0.5) is indeed a starting R-factor it is well below that for a random distribution of atoms in the asymmetric unit. Matthews coefficient only gives a probability of the number of molecules in the asymmetric unit, but it does not provide you with that figure. You may well have plenty of solvent in your asymmetric unit. The most important thing are the electron density maps: do these provide indications of where the search model has to be modified in order to fit the target molecule ? Also, if you have a component missing from your molecular replacement calculations (as in the case you locate only one molecule instead of two), very often there is some additional proteinacious density appearing in the crystal contact regions between your correctly placed molecule and the ghost molecule (which you haven't positioned yet). So if R=0.5 is the starting R-factor just after molecular replacement, I think it is an appropriate R-factor for molecular replacement calculations. HTH, Fred. Afshan Begum wrote: Dear all, Could any one help me regarding my serious problem actually i have collected data at 3.0 and cut off 3.1 where the data statics showed the good values for the further processing. According to the methew coefficient there would be two molecule in the asymmetric unit but after running the molrep its provide only one monomer instead of two, for this reason R values is very high 50%. Actually homologous model having P6322 space group where as my one is F4132. I had tried to run phaser as well phenix but both were failed to process further. i really do not know how can i get the second chain in my structure. Please if you have some ideas i will appreciate and would be many thankful to you. Hope to hearing you soon Best Regards AFSHAN **
Re: [ccp4bb] XDS question
the space group is I422 do you have any other suggestion? Yes, how certain are you of the space group? For myself, I'm never entirely certain of the space group until I have solved the structure... I always keep in mind the other possibilities for space group assignment, if need be. And sometimes the obvious space group is not the space group of the final structure. The computer programs we use only give hints of the solution, but these are only hints. Remember that crystals behave as they want, the fact for example that I(equiv.1) is approximately equal to I(equiv.2) is approximately equal to I(equiv.3) etc does not mean that the relationship between intensities is in fact an equality, it can be just an approximation... With crystals I have learned, everything is possible. It might be an idea to enclose parts of relevant XDS output files for our perusal. Using standard input parameters (for spot selection) as well as your spot selection input parameters. Fred. Marco Lolicato wrote: Thank you to all! @Frederic I have a problem with the following sentence: if I collect all spots I get good map, but it is impossible to solve the structure by molecular replacement - if you have a good map (I assume electron density map) then the structure is solved... for me a good map is a map I can interpret. You're right, I said good map instead of good output values. @Konstantin It is possible to process diffraction spots from both crystals using XDS. The procedure is described here (under 'Index and integrate multiple-crystal diffraction'): http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Tips_and_Tricks I tried but with no success! :( @Kay and all the others The following links are the images: http://www.facebook.com/photo.php?fbid=2127189780277set=o.136323896385679type=1theater http://www.facebook.com/photo.php?fbid=2127189180262set=o.136323896385679type=1theater http://www.facebook.com/photo.php?fbid=2127188820253set=o.136323896385679type=1theater ...then a little bit more details... so, if I process only the strong spots I have those cell parameters: a=b=96.66 c=112.26alpha=beta=gamma= 90 f I process all the spots I have those cell parameters: a=b=216.4 c=112.4 alpha=beta=gamma= 90 In both cases the space group is I422. Thank you again to all, do you have any other suggestion? Marco
Re: [ccp4bb] XDS question
Hi, I have a problem with the following sentence: if I collect all spots I get good map, but it is impossible to solve the structure by molecular replacement - if you have a good map (I assume electron density map) then the structure is solved... for me a good map is a map I can interpret. There have been several reports in the literature of structures where you have layers of normal intensities interspersed with layers of very weak spots in the diffraction patterns. Sometimes with a change in space group assignment when you go from integrating the strong spots only to integrating the entire pattern including the very weak intensities. I don't have a reference off my head right now, some other people on the bb may have such references available. But differences in spot intensities (including reassignment of the space group) is described in Morales et al. (2000), Acta Cryst D56, 1408-1412. So the reply I can give is the following: if the diffraction pattern with these strong diffraction intensities interspersed with weak diffraction intensities only comes from one crystal, then processing the strong spots only does not explain the diffraction pattern and is wrong. Similarly with processing the weak diffraction spots only (which would be difficult to do in practice... see below). The model produced should explain the diffraction pattern seen (in terms of space group, layers of molecules arranged in the crystal...). If the diffraction pattern originates from 2 crystals (in different orientations, a case I've had with one large crystal plus a satellite crystal attached to it in the same loop), it is in principle possible to integrate only the diffraction spots from only one of the crystals. No problems for the larger crystal that diffracts more strongly (which is what I did with my data set - the second lattice was ignored). To process the diffraction from the smaller crystal would be tricky: you should have some version of the data frame processing software that processes first the spots coming from the large crystal and would produce a copy of the input frames where the optical density corresponding to the processed reflections is set (on the images, on the frames) to say 0, for the entire range of frames processed. Then you would repeat the autoindexing and frame processing (integration) to take care of the diffraction from the satellite crystal. I don't think such a modification of the data processing programs is available. But could you explain more clearly the problem? Fred. Marco Lolicato wrote: Dear all, I have a particular problem... so, I have a beautiful crystal with nice diffraction pattern at 2.7A. The diffraction images are composed by very strong spots and weak spots. With XDS, if I collect all spots I get good map, but it is impossible to solve the structure by molecular replacement. If I collect only the strongest spots (STRONG_PIXEL= 99), I'm able to solve a very good structure... My problem is: I was trying to get the apo-structure of my protein. I obtained nice crystals of the apo-protein, but using the method above, in the structure I have found also the ligand!! (probably incorporated during the overexpression). My protein is a multimer and, biochemically, I found that the endogenus ligand bond to the protein is in the ratio 1:6. ...and I got a crystal in this way. So, is there a way to analyze all spots in the diffraction pattern to have a structure of the apo-protein? Is a good idea discard the strongest spots and try to analyze only the weak spots? If yes, how I can do it? All the best, Marco
Re: [ccp4bb] unsubscription request
Hi, What about the ccp4 web page, from which you can follow the link to http://www.ccp4.ac.uk/ccp4bb.php ? HTH, Fred. Angela (Shaoxu) Li wrote: Hi there, I wish to unsubscribe to the mailing list. But I'm unsure as to how I can do that. Your help will be greatly appreciated. Best wishes, Angela
Re: [ccp4bb] Change cell parameter
Zhiyi Wei wrote: Dear all, I have a P2 derivative dataset with beta=89.6. I try to change the beta to 90.4 to be consistent with the native dataset. Should I do sth with the HKL, like applying a matrix? Thanks a million! Best, Zhiyi Hi, Personally I would use sftools (no ccp4i GUI), to be run in a terminal sftools read mymtz.mtz set cell [then you specify the new cell] write mynewmtz.mtz stop However, before changing cell parameters I would think twice... Further, each data set in an mtz file can have its own cell dimensions. Differences in unit cell parameters of less that 1% (I think this is the consensus) are still isomorphous, over this you have non-isomorphism. There is a paper on this (Crick ?). Fred.
Re: [ccp4bb] need proper suggestion http://scripts.iucr.org/cgi-bin/paper?ms0234
Afshan Begum wrote: Dear All, I have a severe prob lam to performed my ligand binding study with corresponding protein. I have taking the native diffraction data at 1.75 A and after that i have performed soaking as well co-crystallization experiment with my inhibitors. Problem is that at the active site phosphate ion always bind instead of inhibitors. I have used 1.6 M ammonium phosphate conc at the crystallization recipe which is a very weak inhibitor of my protein whereas the ligand is already clinically applicable but due to the very high conc. of phosphate i have not achieved my target. If some one can suggest me what else i can replace with ammonium phosphate or any other suggestions would be appreciated. I have tried to grown crystals some other condition but the crystal was not diffracted beyond 3.5 A. Best Regards AFSHAN I think you could get some hints from the following publication, J. Appl. Cryst. (1988) 21, 426. The transfer of protein crystals from their original mother liquor to a solution with a completely different precipitant, Schreuder H et al. http://scripts.iucr.org/cgi-bin/paper?ms0234 The aim would then be to (try to) transfer your crystals to another mother liquor that does not contain any phosphate. HTH, Fred.
Re: [ccp4bb] determining the domain for overexpression and crystallization
Hi, There's a whole bunch of programs that can help you out there. The 2 methods I think of right now are DISPROT (there's a server I believe, http://www.ist.temple.edu/disprot/ ) - Must admit I haven't been to that one for quite a while; DISPROT provides areas of your sequence with high probability of disorder hydrophobic cluster analysis. There are many others as well, can't think of them right now. Also, common sense (like trying to crystallise with and without tags) can be helpful. You may have to try to crystallise several constructs. And there's more than just compact and stable to crystallisation. Monodispersity is quite important too. Fred. Xianhui Wu wrote: Dear all, Before we try to study the crystal structure of an unknown protein, we need to determine the sequence that can fold into a compact and stable 3D domain. What kinds of methods can we choose? -- Best regards, XH Wu
Re: [ccp4bb] I/sigmaI of 3.0 rule
For myself, I decide on the high resolution cutoff by looking at the Rsym vs resolution curve. The curve rises, and for all data sets I have processed (so far) there is a break in the curve and the curve shoots up. To near vertical. This inflexion point is where I decide to place the high resolution cutoff, I never look at the I/sigma(I) values nor at the Rsym in the high resolution shell. As a reviewer, when I have to evaluate a manuscript where very high Rsym values are quoted, I have no way of knowing how the high resolution cutoff was set. So I simply suggest to the authors to double check this cutoff, in order to ensure that the high resolution limit really corresponds to high resolution data and not to noise. But I certainly do not make statements such as this one. I have seen cases where, using this rule to decide on the high resolution limit, the Rsym in the high resolution bin is well below 50% and cases where it is much higher. Like 65%, 70% (0.65, 0.7 if you prefer). So, in my opinion, there is no fixed rule as to what the acceptable Rsym value in the highest resolution shell should be. Fred. Van Den Berg, Bert wrote: There seem to be quite a few “rule” followers out there regarding resolution cutoffs. One that I have encountered several times is reviewers objecting to high Rsym values (say 60-80% in the last shell), which may be even worse than using some fixed value of I/sigI. On 3/3/11 9:55 AM, Ed Pozharski epozh...@umaryland.edu wrote: On Thu, 2011-03-03 at 12:29 +0100, Roberto Battistutta wrote: Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? There is none. Did editor ask you to follow this suggestion? I wonder if there is anyone among the subscribers of this bb who would come forward and support this I/sigmaI 3.0 claim. What was your I/sigma, by the way? I almost always collect data to I/sigma=1, which has the downside of generating somewhat higher R-values. Shall I, according to this reviewer, retract/amend every single one of them? What a mess. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
Hi, I don't think XDS generates an Rpim value, does it? The XDS CORRECT strep provides the old fashioned Rsym (R-FACTOR) plus R-meas and Rmrgd-F. The curves look all the same though Fred. Ed Pozharski wrote: On Thu, 2011-03-03 at 16:02 +0100, Vellieux Frederic wrote: For myself, I decide on the high resolution cutoff by looking at the Rsym vs resolution curve. The curve rises, and for all data sets I have processed (so far) there is a break in the curve and the curve shoots up. To near vertical. This inflexion point is where I decide to place the high resolution cutoff, I never look at the I/sigma(I) values nor at the Rsym in the high resolution shell. Fred, while your procedure is definitely more sophisticated than what I do, let me point out that the Rsym is genuinely a bad measure for this, as it depends strongly on redundancy. Does more robust measures (e.g. Rpim) show similar inflexion? I suspect it will at least shift towards higher resolution. Cheers, Ed.
Re: [ccp4bb] Processing Laue data
Hi, A Laue diffraction pattern is a diffraction pattern recorded using polychromatic (white) radiation. Hence the beam line optics is for focusing the radiation onto the sample (the crystal) but not to select a single wavelength (a monochromator). Just to make it simple to understand. Contrary to monochromatic data collection, the sample (the crystal) is not moving, bringing the reciprocal lattice into diffraction condition is carried out by having this range of wavelengths instead. In order to visualise this, you can make a simple drawing of the reflection conditions, with 2 limiting spheres (that for the minimal wavelength and that for the maximum wavelength of the incoming radiation). All reciprocal lattice points that are in between these 2 limiting spheres are in reflection condition using the polychromatic radiation. Now of course, between diffraction frames, you can rotate your crystal (and also translate it to expose a fresh part of the crystal to the radiation - for X-rays). So the crystal during exposure is stationary. Also note that at ILL (Grenoble) there is Laue (polychromatic) neutron diffraction. So it's not limited to X-rays. Fred. Pius Padayatti wrote: To all Laue experts up here How does a Laue data is collected? Thanks in advance to all PSP On Fri, Jan 28, 2011 at 3:43 AM, REX PALMER rex.pal...@btinternet.com mailto:rex.pal...@btinternet.com wrote: What programs are available for processing Laue data to produce an intensity data set? Are explanatory notes or publications available? Rex Palmer Birkbeck College -- Pius S Padayatti
Re: [ccp4bb] Problem with refinement and positive electron density
Judith Reeks wrote: Dear All, I am currently refining a structure using the latest experimental version of Refmac (5.6) and there seems to be a problem with my Fo-Fc map. There is a region where I have fitted residues to the electron density but after refinement there is still positive electron density assigned to the region despite the fact that residues fit the electron density (see link below for a screenshot). Multiple rounds of refinement have yet to get rid of this problem. I have checked the PDB file and there does not appear to be any problems with this region. Has anybody seen something like this before? http://i1083.photobucket.com/albums/j382/jreeks/Screenshot2011-03-01at1613402.png Regards, Judith Reeks ja...@st-andrews.ac.uk School of Chemistry University of St Andrews Yes, I have encountered such a situation before, where the occupancy was not set to 1.0. For some reason the occupancy had been set well below one. This is the first thing I would check. I can't remember right now why this had happened. Fred.
Re: [ccp4bb] .pir file
Hi Careina, Just an example of a pir file which I just generated (using Bart Hazes program mcfman): P1;MALDH_ Just a title TKVSVVGAAGTVGAAAGYNIALDIADEVVFVDIPDKEDDTVGQAADTNHGIAYDSNTRVR QGGYEDTAGSDVVVITAGIPRQPGQTRIDLAGDNAPIMEDIQSSLDEHNDDYISLTTSNP VDLLNRHLYEAGDRSREQVIGFGGRLDSARFRYVLSEEFDAPVQNVEGTILGEHGDAQVP VFSKVSVDGTDPEFSGDEKEQLLGDLQESAMDVIERKGATEWGPARGVAHMVEAILHDTG EVLPASVKLEGEFGHEDTAFGVPVSLGSNGVEEIVEWDLDDYEQDLMADAAEKLSDQYDK IS* I don't think the pir format has changed much since the subroutine that extracts the sequence from a coordinate file was written. As you will see the (ASCII) format is pretty simple, one start record, one title record followed by records containing the sequence in the one-character abbreviation, 60 characters per line, with * to end the sequence. The start record is P1; followed by 6 characters. I never had any problems with the pir files generated by mcfman, and I always have used an editor (vi is the one I use) to convert other formats to this format. Fred. Careina Edgooms wrote: Dear CCP4 mailing list I have a relatively simple question. How do I get sequence file in .pir format which is required for many programs? I normally use fasta format but some programs eg arpwarp do not allow me to use that Thanks for your help Careina
Re: [ccp4bb] Molrep with two models
The molecular replacement program does not know about your molecule being a single polypeptide chain. The problem is fit two bodies therefore the program fits two bodies. The centre of mass of whatever you wish to position is placed the standard asymmetric unit used by the program. If it happens that the center of mass of the second domain - linked to the (already positioned) first domain by the polypeptide linkages - does not fall within that asymmetric unit, then it won't be this one that will be provided in the resulting pdb file. An equivalent will be provided. Checking the symmetry equivalents (including limited translations) and writing out the correct equivalent to replace that you do not wish to have is the way to go. Just as you did. In case of a search using the entire molecule, this problem does not occur. And if you go from a molecular replacement solution provided by program X to an automatic model rebuilding program called Y, these two programs will not necessarily use the same conventions. Hence you may end up with the model after automatic model rebuilding that does not superimpose with the input file coming from molecular replacement. Same reason there. Does that answer your question? Fred. Christian Roth wrote: Dear all, I tried MolRep with a two domain protein. I have cut the two domain as one domain rotates which prevent a search with the complete model. After I finished the first run. I put this solution as fixed input model in the second molrep run with the second domain. The resulting solution positioned the tow expected molecules, but not nearby the two already found domains. Interestingly a symmetry mate would be at the correct position. I could manually write out the coordinates of the symmetry molecule to put the two domains together, but I thought Molrep would position it close to the already found domains because they belong together and in fact they are one polypeptide chain. Why does this not happen? Do I have to incorporate additional information or constrains for the Molrep run? Thanks and Best Regards Christian
Re: [ccp4bb] CNS and protein structure refinement
Hi Rex, There will be small differences in particular due to the different ways of treating the solvent. How large of a difference are you talking about? Normally the difference should not be very large... And if this related to solvent effects, it will affect the low resolution reflections with a decrease in the effect when you go to higher and higher resolutions (but never completely disappear). Fred. REX PALMER wrote: Does anyone still use CNS ? Do we expect Rfree from CNS for example to be different from the value given by Refmac at the end of the refinement? Rex Palmer Birkbeck College
Re: [ccp4bb] MR problem in determining the number of identical molecules in ASU
Ting-Wei Jiang wrote: Dear experts, Sorry for a simple question but confusing me so much! Does it make bad effects on determining the number of identical molecules in ASU by choosing low symmetry space group. For example,If I choose lowest symmetry(p4) instead of higher one(p43212). Does it cause any trouble in determining structure while we try to find the solution by molrep. If molrep compare the patterson of observe and model in P1 space group, it doesn't matter in choosing a lower s.g.? Any suggestion will be appreciated. Thanks in advance. Ting wei For molecular replacement: it does not matter if you carry out the rotation search in P422 or P43212. To understand this, ask youself the following question: what is the symmetry of the Patterson compared to that of the crystal? The symmetry of the Patterson is obtained from the symmetry of the crystal by converting all translation operators to the corresponding non-translation operators and adding a centre of symmetry. Hence for the rotation function, the symmetry of the Patterson will be P4/mmm for space group P422 and P4/mmm for P43212. Hence no difference for the rotation function. Different for P4 though: the symmetry of the Patterson there is P4/m. Hence the rotation function can be computed once for all space groups that have Patterson symmetry P4/mmm. No need to repeat the rotation function calculations several times. The situation is different for the translation function however. This is the stage at which you distinguish between all these space groups (including the pair of enantiomorphs P41212 and p43212). Failure to assign the proper space group cannot give you a satisfactory model. HTH, Fred.
[ccp4bb] adding gaussian noise to an mtz data column
Before re-inventing the wheel... Is there anywhere some software (freely available software, I mean) that can add some Gaussian noise to data. The data is currently stored in a data column in an mtz (not phase data, but amplitudes, sigma values...) but can be exported to another format if required. Before writing a computer program to do this, does anyone know if this can be done without writing any code. If it can then obviously I won't write new code. Thanks, Fred.
Re: [ccp4bb] adding gaussian noise to an mtz data column
Thanks Ian, I think this is good enough for what I have in mind. So I did not have to reinvent the wheel after all... Fred. Ian Tickle wrote: Hi Fred Doesn't sftools do this - from 'man sftools': CALC F COL Fsimulated = COL Fcalc ran_g 10 * + Create a column with label Fsimulated which contains the value of column Fcalc plus 10 times a random number from a Gaussian distribution with average = 0 and variance = 1 Cheers -- Ian On Fri, Feb 4, 2011 at 12:40 PM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Before re-inventing the wheel... Is there anywhere some software (freely available software, I mean) that can add some Gaussian noise to data. The data is currently stored in a data column in an mtz (not phase data, but amplitudes, sigma values...) but can be exported to another format if required. Before writing a computer program to do this, does anyone know if this can be done without writing any code. If it can then obviously I won't write new code. Thanks, Fred.
Re: [ccp4bb] Mg2+ or water
James Stroud wrote: On Dec 20, 2010, at 1:53 PM, Jacob Keller wrote: what is the .odp file extension? http://tinyurl.com/mjokqs A .odp file is an open document presentation. It is the open version of a power point presentation. http://en.wikipedia.org/wiki/OpenDocument An .odp file is an ISO standard--like the country code you dial when you call your favorite Aunt. You can open a .odp file with the free office suit called OpenOffice. Just download it from http://www.openoffice.org/ and start double-clicking to open the file just like you would if you were using some other presentation software. Also, Jlliu Liu set a good example by sending the document in an open format so anyone can open it (even though some may not have heard of an odp file before). *By using an open format, Jlliu Liu has catered to convenience rather than catering to ignorance,* and has increased the range of people who can provide him with help. James But there was a double extension in the name of the file provided initially (.png.odp if I remember well). Programs that check viruses in incoming emails remove all files that carry double extensions because this is a way to hide the real nature of the file. They also remove .exe files and others (like having too many spaces in a file name). Fred.
Re: [ccp4bb] If it is a new structure?
Hi, Last couple of times I asked myself the same question (what does it look like?) I used ssm (or PDBeFold as seems to be called now). http://www.ebi.ac.uk/msd-srv/ssm/ HTH, Fred. Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help.
Re: [ccp4bb] Space group vs. gap between Rwork and Rfree?
Hi Petr, Usually IDXREF suggests more than one Bravais lattice that is consistent with your diffraction images; hence it is (sometimes) worthwhile trying to INTEGRATE in all possible Bravais lattices and this allows you to eliminate a number of possibilities (poor profiles during integration, instability of refinement, poor Rmerge values at the level of CORRECT). This leaves you with a smaller number of choices for the Bravais lattive. And I personally (at the end) integrate in the correct Bravais lattice instead of integrating in P1 and then doing the space group assignment at the level of CORRECT. I get improved Rmerge values then. I've had a case where one of the students here was wrongly advising my student about an orthorhombic space group, and my advice to carry out integration in all Bravais lattices suggested by IDXREF, integration led in orthorhombic gave a molecular replacement solution that did not refine properly and a molecular replacement solution that refined in a hexagonal space group. The Rmerge values (at the level of CORRECT) vere very similar both for P222 and for P6 integration. HTH, Fred. Petr Kolenko wrote: Dear colleagues, I appreciate any help, or any suggestion with my difficult data. Many thanks at least for consideration. I work with datasets at 3.6AA of resolution. Integrated with XDS, scaled with SCALA. After integration and scaling in P1, POINTLESS suggested space group I432: Space group confidence: 0.95 Laue group confidence: 1.000 Total probability: 0.97 After any longer refinement (more than 20 cycles), there was always a big gap between Rwork and Rfree (0.22 and 0.33), although the structure looks quite good at this resolution. I tried also space group I23, here are my statistics: Data processing (I23 vs I432): Rmerge - 0.124 vs 0.134 high resolution Rmerge - 0.649 vs 0.732 low resolution Rmerge - 0.048 vs 0.037 The gap between Rwork and Rfree was stabilized in I23 using tight NCS restraints: Rwork vs Rfree: I23 withouth NCS: 0.228 vs 0.334 I23 with NCS: 0.249 vs 0.296 I43 : 0.228 vs 0.326 My question is, what would you recommend me to close the gap? Or is this Rfree pointing out a lower real symmetry (I23) than suggested by POINTLESS or PHENIX.XTRIAGE (I432)? I have tried different selection of free reflections in I432 dataset already, but with almost the same results. Many thanks for any response. Petr
Re: [ccp4bb] Structure based and motif based sequence alignment
Muhammed bashir Khan wrote: Dear All; I have structures of two protein one full-length while the other truncated at the c-terminus(one from prokaryote while the other from eukaryotes). Now I want to do the sequence alignment of these two proteins from all species in such a way that the structure based sequence remain constant while extending the sequence only at the c-terminus. Remember the structure are known only for the two proteins. Any suggestion will be highly appreciated! Regards and have a nice weekend. Bashir Hi there, Ages ago, for this type of work (fine-tuning sequence alignments), I was loading pre-aligned (or not pre-aligned) sequences and was editing the alignment by hand using a sequence alignment editor. This editor was working on VAX/VMS systems, which no-one uses anymore (I haven't touched VMS in many many years). So I had a look at what sequence alignment editors are available today using google, and I came across this: Jalview (http://www.jalview.org ). Unfortunately, it seems it does not wish to install on my Linux box so I don't know if the software does what you want it to do. And it is not clear to me exactly what you mean by extending the sequence only at the c-terminus. Fred.
Re: [ccp4bb] Problem with finding of spots
Hi, I agree with what has been mentioned about fuzzy spots. But what seems obvious as well is that the resolution for spot picking should be limited (to 3.5 or 4 A resolution). It is difficult to judge from an image of a diffraction pattern, but it seems to me from this image that the spots do not extend to the limits of the detector, whereas the spot picking algorithm wishes to find spots on the entire detector surface. Fred. Petr Kolenko wrote: Dear colleagues, I am working on one dataset that is hard to process. The data are about 3A of resolution. As we are not able to reproduce the experiment again, I have to use this one, collected in a dirty way. The problem starts immediately with finding of spots. I have tried HKL2000, XDS, D*trek, ipmosflm, imosflm, but none of them gave a good read-out of the images. All the programs find some spots in wrong positions and the real spots are not covered. Here is an example: http://kolda.webz.cz/image-predictions.jpg The data were collected in-house, Saturn 944++ CCD, and all the necessary information should be in the header properly. I checked the distance, other parameters, but the problem is with finding of correct or real spots on the image. This should be even header-independent, should not? All the programs fail (or even crash) in this routine. Does anyone have any suggestion, please? Btw, we have several structures in the PDB from this experimental setup. This is the first problem I have met. Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de mailto:petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] relationship between B factors and Koff
I can direct you to PDB entry 1EWY, where the average isotropic temperature factor for the major component of the complex is ca. 47 A**2 and that for the smaller component is ca. 69 A**2. Similar values than the ones you are reporting. I am assuming some sort of disorder, or if you prefer, wobbling of the smaller component at the lever of the binding site. Fred. Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
I do not know if that's really cynical: I've had the case of a referee recommending manuscript rejection because the title of the manuscript was not appropriate. The editor followed the advice of the referee. A proper refereeing job would have been to suggest that the authors change the title of the manuscript, not suggesting to the editor that the manuscript should be rejected! So I think we can have different opinions on this. Sometimes referees do a good job in evaluating manuscripts, sometimes they do not. Fred. Eleanor Dodson wrote: Oh cynic! Eleanor On 10/27/2010 09:01 PM, Simon Kolstoe wrote: Surely the best model is the one that the referees for your paper are happy with? I have found referees to impose seemingly random and arbitrary standards that sometime require a lot of effort to comply with but result in little to no impact on the biology being described. Mind you discussions on this email list can be a useful resource for telling referee's why you don't think you should comply with their rule of thumb. Simon
Re: [ccp4bb] what package provides libg2c.so.0 on Ubuntu ?
Hi, For questions relating to a specific package (if it is in rpm format, I don't know what the Uuntu installation software uses as packages), you can use the rpm search web site http://rpm.pbone.net/ (in addition to http://www.rpmfind.net/ , can't search in rpmfind.net at the moment, it says there are too many connections); rpm.pbone.net lists rpm files that contain libg2c, but none for Ubuntu (perhaps Ubuntu does not use rpm files); there is a package search utility on the Ubuntu site, but no package is found containing libg2c. I would try the following: download a .rpm file for your architecture (e.g. i386); use rpm2targz to convert the rpm file into a tar.gz file; examine the content of the tar file and if possible install all files relevant to libg2c on your system; another possibility would be to locate the source code for libg2c, download it, compile it and install (the latter sometimes works for me, sometimes not because it requires installing several additional libraries - sometimes they seem not to be available - and sometimes compilation fails because of errors found in the code by the compiler). Fred. Edward A. Berry wrote: I'm helping set up crystallography programs on a ubuntu system, and we're stuck because one program (scalepack) needs the library libg2c.so.0 . I understand this is absent from modern distributions because gcc discontinued support for g77 and f2c in recent releases. However on fedora there are compatibility packages like compat-libf2c which allow running old executables compiled with g77. Is something like this available for ubuntu? Or is there some other trick to get scalepack running? I understand ubuntu is used by many crystallographers, and while I'm sure most of them use mosflm or XDS, I'm sure someone has tried setting up denzo/scalepack. the system: Linux x 2.6.31-22-generic #65-Ubuntu SMP Thu Sep 16 15:48:58 UTC 2010 i686 GNU/Linux gcc (Ubuntu 4.4.1-4ubuntu9) 4.4.1 Thanks, eab
Re: [ccp4bb] Refinement
I second that. Incorrect space group assignment usually causes this behaviour of having R and R-free stuck at very high values. It is useful to go back to the data processing stage and carefully consider all Bravais lattices (and associated space groups) that the autoindexing routine finds consistent with your diffraction data frames. Fred. Jim Fairman wrote: Did you check your data for twinning and/or pseudo symmetry using phenix.xtriage or Pointless? If the space group is incorrect, these programs will also assist you in selecting the correct one. What were the Z-scores for the rotation and translation functions? On Mon, Oct 18, 2010 at 7:39 PM, Jyotica Batra batra.jyot...@mayo.edu mailto:batra.jyot...@mayo.edu wrote: Hi All I have a dataset at 1.9A, spacegroup-P212121 (unit cell: 37.7, 39.52, 231.72, 90, 90, 90), I used MR phaser and got a structure solution with LLG= 320 (1copy/a.u) . During refinement, the R-free (50%) and R-factors (42%) never go down. I have tried refining in phenix and refmac both, but still the high R-free problem persists. At this point I'm seeking help to know the possible reasons for this high R-free. Thanks in advance! Jyotica -- Jim Fairman, Ph D. Post-Doctoral Fellow National Institutes of Health - NIDDK Lab: 1-301-594-9229 E-mail: fairman@gmail.com mailto:fairman@gmail.com james.fair...@nih.gov mailto:james.fair...@nih.gov
Re: [ccp4bb] protein ligand energy
Rex Palmer wrote: Can anyone reccomend a free download program that will calculate the energy of a protein/ligand complex? The ligand has been modelled in. Thanks Rex Palmer Birkeck College Hi Rex, I think any refinement program such as CNS will do this - problem is, since these programs are aimed at refinement, you need a file containing observations (crystallographic data or NMR observations file). There is also within CNS the input file model_stats.inp, I have no idea of what this one does (I cannot remember if I have used it in the past), if it provides the energy terms that you may require. example (from a minimize run): | Etotal =0.14E+07 grad(E)=38.876 E(BOND)=5061.584 E(ANGL)=8964.251 | | E(DIHE)=8542.700 E(IMPR)=2466.346 E(VDW )=6787.298 E(PVDW)=27.910 | | E(XREF)=0.13E+07 Here there is an X-ray energy term, obviously (this is taken from a refinement run, at the beginning of energy minimization). Now of course perhaps this is not what you are looking for. Fred.
Re: [ccp4bb] which Linux workstation for crystallography to buy?
Hi, Frankly, any vendor or assembler of PC's will do. Things to make sure to have on your PC: an NVIDIA graphics board in order to get nice graphics (their Linux drivers are fine; I don't know their current range of boards, here we buy middle-range boards, not the cheapest ones that are specific for cheap PC's and gaming in mind - so you'll need to check this out on their web site). It also helps to have one or two Firewire ports in case. Our employer decides which brand of computers we must buy, until fairly recently it was DELL (I have one of those), now it's LENOVO (formerly IBM) and several colleagues have some of these and they are fine too. A long time ago I had a workstation that had been put together by a local shop from parts and that was fine too. The choice is mainly whether or not you want a laptop or desktop. With a laptop, you can have a second (large) monitor sitting on your desk, but take the PC with you in the evening so that you can work at home if the family allows you to (with a desktop and a suitable graphics board, you can also have 2 monitors on your desk, this is what they have on the beamlines at the ESRF). Linux flavour: I personally do not like Ubuntu very much because it seems you cannot have a root account, so that all admin has to be done using sudo commands. Over here, the team in charge of computers and networks decides for us which flavour of Linux has to be installed on our machines. Until recently, we had Fedora Core (I think that with Secure Linux that installed automatically these were very secure computers for them because at first, everything was forbidden as a security risk - like printing is not allowed; or accessing the internet is not allowed etc; I have Fedora Core 8 which was fine as soon as the Secure Linux protection was removed). 1 year ago, it was Scientific Linux that had to be installed, the reason being that the updates are not as frequent as those on Fedora Core (and also, the distro is targetted towards scientific applications). The ESRF has SUSE Linux on their computers, and they work fine. Fred. Benini Stefano (P) wrote: Dear All, I need to buy a Linux workstation to run crystallographic software and graphics like ccp4, mosflm, coot., etc., Could you please suggest me a good combination of hardware and which linux operating system to install (ubuntu?)? I can spend about 1500€ Technology evolves so fast that I really want to be up to date not to be already late! Thank you very much in advance Stefano Stefano Benini, Ph.D. Assistant Professor _http://pro.unibz.it/staff2/sbenini/_ Bio-organic Chemistry Laboratory Faculty of Science and Technology Free University of Bolzano Piazza Università, 5 39100 Bolzano, Italy Office (room K2.11): +39 0471 017128 Laboratory (room E.012): +39 0471 017901 Fax: +39 0471 017009 ***
Re: [ccp4bb] unknown density
Hi, I did a little bit of modeling in your density (starting from a nicotinamide ring, the positioned nicotinamide is enclosed). The middle part looks suspiciously like a 6 membered ring. Could it be a molecule in a half-chair conformation? There is only the blob that is perpendicular to the ring that would remain unexplained... If it is indeed a molecule in a half-chair conformation then you'd need to ask a chemist what it could be. Note that I did not try to distort the nicotinamide ring to a half-chair conformation Blobology is a difficult branch of our trade... I've had a case where I had to state in the publication that a sodium (I think it was) ion had been introduced in the model to explain a density feature but that it wasn't known what this spherical blob of density in fact corresponded to. Fred. Priscila Oliveira de Giuseppe wrote: Hi, everyone, I solved a structure at 1.7 A. After fitting all residues into the model, I found an extra density at the catalytic site. The components of the crystallization condition did not explain it (crystallization condition: 40% PEG200, 0.1 M Tris-HCl, protein buffer: PBS) Neither substrates nor products of this enzyme fitted this density. I modelled two PEG molecules with half occupancy. They fitted very well, but I am not convinced that this is the best explanation. Does anyone figure out what this density can be? Attached to this e-mail I send the maps and a pdb file with the residues surrounding the ligand density. Thanks Priscila nicotinamide.pdb Description: Protein Databank data
Re: [ccp4bb] changing spacegroup
Another possibility is with sftools, set spacegroup option Fred. PS not in ccp4i Graeme Winter wrote: Hi Tim, Is it as easy as reindex hklin a.mtz hklout b.mtz eof symm P43212 eof This will simply (and correctly) reassign the symmetry operations. Is this what you meant? Best wishes, Graeme On 30 September 2010 10:49, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Hello, we are currently preparing a tutorial for a practical from lysozyme data. pointless picked the spacegroup P41212 and phaser should show the students that P43212 is the correct spacegroup. How do I best change the scala-mtz-file to that spacegroup? Can I tell pointless (and eventually rerun scala), or is there another program to choose (I would normally use xprep, but I'd like to show the students the 'ccp4i-way'). Thanks a lot, Tim -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.9 (GNU/Linux) iD8DBQFMpF00UxlJ7aRr7hoRAlRUAKD4dRIpButVwaXId7Nk1TDGtLcDUACfdn2O K5uVTF3ZmG1kv+VNbQI05+4= =YSVm -END PGP SIGNATURE-
Re: [ccp4bb] .cv to .mtz conversion
ccp4i Reflection Data Utilities Convert to/modify/extend MTZ Input reflection file is in XPLOR/CNS format etc Fortran format (7X,3I5,6X,E12.4,7X,E12.4,6X,I2) Skip first 6 lines (if # is part of the .cv file) [I think, I have counted the space before the INDEx for example giving me 7X; basically it's a question of counting the characters in the INDEx records] HTH, Fred. Marni Williams wrote: Hey I have a problem with converting a CNS created file in .cv format to .mtz for use in CCP4. I suspect it is because the Fortran model and the column labels are incorrect but I have no idea how to fix this. Here are the first few lines of the .cv http://the.cv file: ### NREFlection= 98435 ANOMalous=FALSe DECLare NAME=FOBS DOMAin=RECIprocal TYPE=REAL END DECLare NAME=SIGMA DOMAin=RECIprocal TYPE=REAL END DECLare NAME=TEST DOMAin=RECIprocal TYPE=INTE END INDEx002 FOBS= 0.2276E+02 SIGMA= 0.1094E+02 TEST= 0 INDEx003 FOBS= 0.8401E+03 SIGMA= 0.9090E+01 TEST= 0 INDEx004 FOBS= 0.1032E+04 SIGMA= 0.1418E+02 TEST= 0 ## any advice would be greatly appreciated. Regards, Marni Williams -- Molecular Parasitology Laboratory Department of Biochemistry School of Biological Sciences Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria South Africa 0002 Tel: +27 12 420 2990 Fax: +27 12 362 5302 Email: ma...@tuks.co.za mailto:ma...@tuks.co.za
Re: [ccp4bb] pdf to text
What you could try to do is print out the pdf file, then locate a scanner with a suitable scanning software. Several scanning software have the possibility of generating word processing program output or ASCII format. Since the pdf file is text only (no figures etc) then it should be OK. You just have to go through the output generated and check for errors (such software is not perfect and produces errors here and there). HTH, Fred. Oganesyan, Vaheh wrote: Colleagues, I have coordinates of “small” molecule in pdf format, i.e. its an image of pdb file and it doesn’t let me select text. The molecule has 150 atoms, so doing it by hand is not an option. How would one go around it? The molecule is polymyxin B and doesn’t exist in databases like CSD, Hic-up or PDB. If some of you happen to have it please share. Thank you. PS The pdf is available from ACS website as a supplementary material and is also attached here. Thank you. /*/Vaheh Oganesyan, PhD/*/ /*/Antibody Discovery and Protein Engineering/*/ /*/MedImmune, LLC./*/ /*/1 MedImmune Way/*//*/, Gaithersburg, MD 20878/*/ /*/o/*//*/ganesy...@medimmune.com/*/ mailto:oganesy...@medimmune.com
[ccp4bb] interesting article about science funding in the Financial Times
Dear all, Giuseppe Zaccai showed me an interesting article about science funding (from F.T.). I thought it could be of interest to all of us since we have to apply for funding, and succeeding in securing funds is getting more difficult all the time. The scan of the article can be found on: http://www.facebook.com/photo.php?pid=1140070l=ded4b09cafid=1612253929 This paper deals with the shift of funding away from fundamental, basic research to applied research that can guarantee a return on investments in a very short time. The article is concerned with science funding in Britain. However, I know it applies equally well for science funding in France, in Europe and I fear the same trend might apply to the rest of the world as well. I think the Financial Times cannot be considered as an ultra-left wing revolutionary journal, they are only concerned with the progression and development of the economy (and analyses of the economies etc). Clearly, in this article, the FT is saying Careful - you're going down the wrong road there. Fred.
[ccp4bb] Competition organised by the Scientist: Proteopedia voted first place
Dear ccp4bb subscribers, I have just learned that Proteopedia (the wikipedia-style encyclopedia of macromolecular structures) has been voted winner in this year's competition organized by the Scientist ( http://www.the-scientist.com/ , readers and judges choice). So thanks to all who voted, this only shows the importance of our beautiful structures. Some of the comments on the Scientist's web site: Very absorbing. Kept me looking (and learning) with it for a long time (Holmes) Very informative and a good resource (Kirby) So thanks to all who voted for our lovely structures, anything that can make our structures understood by as many people as possible and (possibly) bring back some funding to our Science is always welcome. Fred.
[ccp4bb] Post-doctoral position available at IBS - please do not reply to me !
POST-DOCTORAL POSITION DATE OF AVAILABILITY : starting as soon as possible, for 2 years (ANR contract). TITLE : purification and crystallization of ABC transporters POSITION: the position is opened at the Institute for Structural Biology (IBS) in Grenoble in the team of Jean-Michel Jault and will be carried out in the framework of a collaborative project between chemists and biologists, both in Lyon (IBCP and UCB) and in Grenoble (IBS and UJF) with funding from an ANR grant. JOB DESCRIPTION: Multi-Drug Resistance (MDR) is an increasingly challenging problem in the biomedical field and is widespread from tumour cells to microbes. In bacteria, resistance to multiple antibiotics is often associated with the presence of MDR pumps and several of these belong to the largest superfamily of membrane proteins, the ABC (“ATP-Binding Cassette”) transporters, which include some human members whose dysfunction is involved in severe pathologies (cancer, cystic fibrosis, adrenoleukodystrophy…). This project will focus on two bacterial ABC transporters that we routinely purify in high yields in their functional states, with the goal to crystallize them and solve their 3-D structure, using both classical detergents and newly synthesized ones. JOB REQUIREMENTS: the applicant should hold a PhD in Biochemistry and have a strong background in protein biochemistry (purification and biochemical/biophysical characterisation) and in protein crystallography. Additional knowledge in membrane proteins will be appreciated. SCIENTIFIC ENVIRONMENT: the Institut de Biologie Structurale (Institute for Structural Biology) is located in the Grenoble “Polygone Scientifique” area, in the close vicinity of the EMBL outstation, ESRF and ILL, i.e. it is one of the most renowned scientific area in France for biological research. At the IBS, the post-doc will benefit from a technical environment devoted to purification, characterization and crystallization of membrane proteins (MP3, PAOL and high throughput membrane protein crystallization platforms), as well as the scientific expertise associated with these. More information available from http://www.psb-grenoble.eu/ and http://www.ibs.fr/?lang=en TO APPLY: please send a CV and the names of two referees to Jean-Michel Jault : jean-michel.ja...@ibs.fr
Re: [ccp4bb] How to find out Rmerge from the refinements..?
Hussain Bhukyagps wrote: Dear all, i want to know that how can we find Rmerge from the refinements done in CNS..?? Hi, I think you have the terminology wrong: Rmerge (or Rsym nowadays when most diffraction data is recorded from a single crystal) is provided by the data frame diffraction software, not by a refinement program. Internal agreement of the data so to speak. Can you tell us more what you are looking for? R-factor and R-free perhaps? Fred.
Re: [ccp4bb] XSCALE
anna delprato wrote: Hello All; I've just started using XDS and have scaled three data sets from the same crystal - unmerged. It was a SAD experiment My question is concerning which R values to use as data processing statistics. I don't find any Rsymm or even Redundancy values in the LP files. I came across an exmple where Rr.i.m / Rmeas was reported but I don't understand the distinction between these values. Thank you in advance. Anna Dear Anna, The classical R-sym (on intensities) in the files XCALE.LP and CORRECT.LP is called R-FACTOR observed. There is another R-sym value called R-meas, plus something called Rmrgd-F defined in Diederichs Karplus (1997), Nature Struct. Biol. 4, 269-275 (this is all given above the tables in the .LP files). In case of anomalous diffraction, there is no R-ano as such but other values are provided. WRT the redundancy, I am afraid you have to recompute an approximate value yourself using the number of observations and number of unique reflections (this is what I do all the time). I suppose one could always write a jiffy program to compute the correct values using both files INTEGRATE.HKL and XDS_ASCII.HKL, but I haven't done it myself... Yet ? Fred.
Re: [ccp4bb] Accuracy of the position of coordinates
I would like to know the accuracy (error) of the position of the coordinates This is provided by the output of the refinement software. An example (CNS-refined structure, from the pdb header, there is an input file called xtal_pdbsubmission.inp REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM LUZZATI PLOT(A) : 0.36 REMARK 3 ESD FROM SIGMAA (A) : 0.45 REMARK 3 CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT(A) : 0.49 REMARK 3 ESD FROM C-V SIGMAA (A) : 0.60 Another example, from Phenix: REMARK 3 ERROR ESTIMATES. REMARK 3 COORDINATE ERROR (MAXIMUM-LIKELIHOOD BASED) : 0.41 A third example, from Refmac: REMARK 3 ESTIMATED OVERALL COORDINATE ERROR. REMARK 3 ESU BASED ON R VALUE(A): 0.356 REMARK 3 ESU BASED ON FREE R VALUE (A): 0.279 REMARK 3 ESU BASED ON MAXIMUM LIKELIHOOD (A): 0.282 REMARK 3 ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2): 12.096 Can't provide you with an example for Buster because I requested the software last year and I do not know what happened to that request. The coordinate errors are estimated using statistical methods. Relevant papers to read are those of Randy Read and of Vittorio Luzzatti (and probably of others), Luzzatti V. (1952), Acta Cryst. 5, 802-810; Read, R.J. (1986), Acta Cryst. A42, 140-149. There are probably papers by the Russian school (Lunin and others) but I can't locate them right now... Fred.
Re: [ccp4bb] Accuracy of the position of coordinates
Received from John Helliwell and thus forwarded to the bb - why should you miss important information? Original Message Subject:Re: [ccp4bb] Accuracy of the position of coordinates Date: Tue, 3 Aug 2010 09:08:30 +0100 From: John R Helliwell jrhelliw...@gmail.com To: Vellieux Frederic frederic.velli...@ibs.fr References: 4c57c841.2080...@ibs.fr Dear Fred, I think the Cruickshank Diffraction Precision Index and its specific reformulation by David Blow are a better estimate of overall coordinate errors. These two papers are in Acta D. Greetings, John On Tue, Aug 3, 2010 at 8:41 AM, Vellieux Frederic frederic.velli...@ibs.fr mailto:frederic.velli...@ibs.fr wrote: I would like to know the accuracy (error) of the position of the coordinates This is provided by the output of the refinement software. An example (CNS-refined structure, from the pdb header, there is an input file called xtal_pdbsubmission.inp REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM LUZZATI PLOT(A) : 0.36 REMARK 3 ESD FROM SIGMAA (A) : 0.45 REMARK 3 CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT(A) : 0.49 REMARK 3 ESD FROM C-V SIGMAA (A) : 0.60 Another example, from Phenix: REMARK 3 ERROR ESTIMATES. REMARK 3 COORDINATE ERROR (MAXIMUM-LIKELIHOOD BASED) : 0.41 A third example, from Refmac: REMARK 3 ESTIMATED OVERALL COORDINATE ERROR. REMARK 3 ESU BASED ON R VALUE(A): 0.356 REMARK 3 ESU BASED ON FREE R VALUE (A): 0.279 REMARK 3 ESU BASED ON MAXIMUM LIKELIHOOD (A): 0.282 REMARK 3 ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2): 12.096 Can't provide you with an example for Buster because I requested the software last year and I do not know what happened to that request. The coordinate errors are estimated using statistical methods. Relevant papers to read are those of Randy Read and of Vittorio Luzzatti (and probably of others), Luzzatti V. (1952), Acta Cryst. 5, 802-810; Read, R.J. (1986), Acta Cryst. A42, 140-149. There are probably papers by the Russian school (Lunin and others) but I can't locate them right now... Fred. -- Professor John R Helliwell DSc
Re: [ccp4bb] Will 3mFo-2DFc maps have less model bias than 2mFo-DFc maps?
Hi, You take the output mtz from the refinement program (let's assume it's called refine_1.mtz). Command line mode: sftools read refine_1.mtz col 1 2 3 4 # assuming the mtz contains H K L 2FOFCWT PHI2FOFCWT FOFCWT PHI2FOFCWT cal col 3FO2FCWT col 1 col 3 + set types F P F P R F write 3fo2fc.mtz col 5 2 3 4 quit (or stop, can't remember which) That's it... Fred Armando Albert de la Cruz wrote: Does anyone have got a script to compute 3fo2fc map with CCP4? Armando El 29/07/2010, a las 23:38, Ian Tickle escribió: On Thu, Jul 29, 2010 at 8:25 PM, Pavel Afonine pafon...@lbl.gov wrote: Speaking of 3fo2fc or 5fo3fc, ... etc maps (see classic works on this published 30+ years ago), I guess the main rationale for using them in those cases arises from the facts that 2Fo-Fc = Fo + (Fo-Fc), 3Fo-2Fc = Fo +2(Fo-Fc) To be precise, it is actually 2mFo-DFc for acentric reflections and mFo for centric reflections I prefer to think of it rather as 2mFo - DFc = DFc + 2(mFo-DFc) for acentrics and mFo = DFc + (mFo-DFc) for centrics. Then it also becomes clear that to be consistent the corresponding difference map coefficients should be 2(mFo-DFc) for acentrics and (mFo-DFc) for centrics. Cheers -- Ian
Re: [ccp4bb] Will 3mFo-2DFc maps have less model bias than 2mFo-DFc maps?
Hi Ian, If you read my post carefully, and know the naming conventions, you will see that the mtz naming convention and mtz labels are those that are provided by Phenix... I was aware that there will be differences (I was assuming minor ones - in terms of map appearance) to the proper coefficients when using other refinement software. So I believe you ! Fred. Ian Tickle wrote: Hi Fred You have to be careful here because believe it or not, not all programs output the same coefficients for 'minimal bias' maps, so depending on which program Hailiang is using for SF calculation/refinement he may or may not get the right answer! You are assuming the difference map coefficient is (mFo-DFc) for both acentrics centrics so you are expecting to calculate: 3mFo-2DFc = (2mFo-DFc) + (mFo-DFc) for acentrics 2mFo-DFc = Fo + (mFo-DFc) for centrics However as I have been at pains to point out on numerous occasions the correct difference map coefficient is 2(mFo-DFc) for acentrics (i.e. 2 times half the peak height from an acentric mFo-DFc map), and (mFo-DFc) for centrics (i.e. 1 times the full peak height from a centric mFo-DFc map). This fact tends to be obscured if you think of it as Fo+(Fo-Fc) instead of Fc+2(Fo-Fc), which was my real objection to thinking of it in the way Pavel suggested. In fact the last time I checked (recently) neither Refmac nor Buster got it right (details on request!) - not only that but they get it wrong in different ways: at least they are inconsistent with what in my view are the correct coefficients, which is based on my understanding of Randy Read's 1986 paper, and no-one has yet provided me with a rationale for the formulae used by Refmac Buster. The CCP4 version of Sigmaa now gets it right, but that's only because I recently fixed it myself. I can't speak for phenix.refine, I suspect it gets it completely correct, since Pavel is on the case! So I think the safest CCP4 approach is to use Sigmaa to recalculate the map coefficients, then use FFT to combine them. This will require something like the following input to FFT: LABIN F1=FWT F2=DELFWT PHI=PHIC SCALE F1 1 0 F2 0.5 0 (check the FFT doc!) in other words: 3mFo-2DFc = (2mFo-DFc) + 0.5*(2(mFo-DFc)) for acentrics 1.5mFo-0.5*DFc = Fo + 0.5*(mFo-DFc) for centrics Note that this gives the coefficient 1.5mFo-0.5DFc for centrics, not 2mFo-DFc as suggested in your paper (sorry I couldn't see the rationale for that choice). Again this becomes much clearer if you write 3mFo-2DFc as Fc + 3(mFo-DFc) i.e. 3 times half height (= 1.5 times true height), so to be consistent the centric coefficient should also be 1.5 times true, or Fc + 1.5(mFo-DFc). I think it's important to get the centric reflections right (particularly in tetragonal and cubic space groups!) because obviously the centric phases tend to be better determined than the acentric ones. Cheers -- Ian On Fri, Jul 30, 2010 at 9:24 AM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi, You take the output mtz from the refinement program (let's assume it's called refine_1.mtz). Command line mode: sftools read refine_1.mtz col 1 2 3 4 # assuming the mtz contains H K L 2FOFCWT PHI2FOFCWT FOFCWT PHI2FOFCWT cal col 3FO2FCWT col 1 col 3 + set types F P F P R F write 3fo2fc.mtz col 5 2 3 4 quit (or stop, can't remember which) That's it... Fred
Re: [ccp4bb] non-symmetric tetramer ?
Non-symmetric tetramers: you can check out Tete-Favier et al (1993), Acta Cryst. D49, 246: the quaternary structure was assumed to have local 222 symmetry. It turned out this was not exactly the case: the actual symmetry of the object (the molecule) was pseudo 2t2t2t. So in addition to 2-fold axes being common as an assembly mechanism (leading to the quite common 222 symmetry) you can have deviations from this symmetry. Fred. Fred wrote: Dear CCP4bb, Could someone please, point me to some references about non-symmetric tetramers? If I have a tetramer composed by 4 identical subunits, it'll always have a P4 point group symmetry? Thank in advance, Tomb
Re: [ccp4bb] non-symmetric tetramer ? 2nd round
Hi, To quote you: even my P222 experimental envelop does have a 4-fold axis - this is not suprising, a particle with 222 symmetry does not have 4-fold symmetry. There are 3 mutually perpendicular 2-fold axes that intersect at the origin (of the particle, of the molecule) [and for the nomenclature, these axes are named the P Q and R axes]. Fred. Fred wrote: Thanks all of you who promptly replied my question. I should have been more precise. I was referring to the symmetry of the tetrameric particle (point symmetry) at the molecular level not at the atomic level. This question has arisen because I have collected some SAXS data of my protein in solution and I don't have a molecular model to superpose to the experimental envelop. Others experimental data, gel filtration and NAT-PAGE, suggest a tetrameric particle. On the other side, P1, P2, P222 and P4 experimental envelops are quite different. So, I am not sure which symmetry to take. Considering the native state (no ligands at all), 4 identical subunits and that the interface of oligomarization have to be conserved, I would take P222 or P4. However, I can be able to imagine such spacial arrangement without a 4-fold axis at the molecular level. Indeed, even my P222 experimental envelop does have a 4-fold axis. I appreciate if you could add some more comments on this. Thanks in advance, Fred
[ccp4bb]
Vandana Kukshal wrote: hello sir , recently i have collected one data of 3.0 A of a protein having no sequence homology with any known PDB . but while fold prediction i got 100 % identical fold with some of the protein . space group of my protein is P622 and showing 6 molecule in a assymetric unit. the homologous fold proteins are trimer protein. can i run MR in this case . how i should proceed. i m trying for MIR also . regards vandana I suggest that you ccp4i, then phaser within ccp4i (or Alexeai Vaguin's program, Molrep i think it's called). The easiest and perhaps most clever approach would be to give as a search model the trimer, and look for 2 copies in the asymmetric unit. Otherwise (if your protein does not assemble as trimers, for example as a dimer of trimers) then you give as a search model a single monomer, and look for 6 copies. But this can be tricky. You may have to edit your search model before. In any case, you should always remove the waters present in the pdb of your search model. HTH, Fred. PS how do you know you have 6 molecules in the asymmetric unit?
Re: [ccp4bb] problem in annealing
Hi, I think the MAXCHN errors has been fixed in recent versions of CNS. You must be using CNS v1.0 or v1.1. So either you download and install a recent version (the latest is v1.3), or you do as the error message suggests: you edit your anneal.inp file, you locate MAXCHN= or MAXCHN = (or maxchn= or maxchn = - can't tell you which one since I have removed previous versions of CNS on my workstation) and you increase the figure after the = sign. I cannot remember if the topology error message is related to the problem with an insufficient number of chains set in the input file. HTH, Fred. Hussain Bhukyagps wrote: Hi, I'm facing problem in refinement. i have a pdb and it is showing the following error in the step of annealing. -- Torsion Topology -- - ERROR: A tree has too many chains. Please increase MAXCHN and re-run. %TORSION:TOPOLOGY error encountered: Fatal Topology Error (CNS is in mode: SET ABORT=NORMal END) * ABORT mode will terminate program execution. *
[ccp4bb] Fifth Max Perutz prize of the E.C.A. to Prof. Claude Lecomte
News just received from the French Crystallographic Association (and thus forwarded to ccp4bb): The fifth Max Perutz Prize of the European Crystallographic Association goes to to Professor Claude Lecomte from Nancy Université and CNRS, France. Claude Lecomte is recognised for his contributions to the measurement and analysis of high resolution X-ray crystallographic charge densities and the insight it has provided. He is acknowledged for pioneering work on high-resolution analyses and charge density modelling of polypeptide and other macromolecular structures based on the analysis of multiple electron density determinations using non-spherical scattering factors. He applied this formalism to hydrogen bonding, porphyrins and metastable states of light-induced spin-transition complexes and to the evaluation of the electrostatic potential from diffraction data on the above and on zeolites, zeolite-like materials and proteins. Prof Lecomte will present an acceptance Lecture at the Opening Ceremony of ECM26 in Darmstadt. http://www.ecanews.org/ecaprize.php My congratulations to him. Fred.
Re: [ccp4bb] Density peaks to build
Hi, Can't rotate the picture so that I can't see the distance between the nitrogen and the green blob. The green blob is elongated. Sometimes what happens is that you can have 2 waters (partial occupancies), in some unit cells in your crystals the water occupies site 1, in the other unit cells the water occupies site 2. Obviously in the model you build, care should be taken during refinement that these waters do not push each other away (VdW repulsion). Anyway blobology is a difficult part of our trade. Fred. Fatima Fonseca wrote: Dear CCP4BB users, I have 2 questions: I'm refining a very high-resolution structure (1.08 A) and I need help to solve a blob of positive density close to a modified Arg (N-omega-hydroxy-L-Arginine) - picture1. I tried to model a third conformation there but it didn't work - picture2. In another protein model (1.35 Aº) I have a lot of positive peaks in the difference map that I model as water molecules and after Refmac these waters have very high B factors (100 - 200) and appear as blobs of positive density. They make H bonds with 2-4 water molecules/protein residues within 2.66 - 3.0 A. Any suggestions would be precious. Fátima
Re: [ccp4bb] Need suggestion on classification of a protease
Hi, If you do not know anything about peptidase (protease) classification, I'd suggest you have a look first at the Merops peptidase data base: http://merops.sanger.ac.uk/ Will tell you what (type of) classes there are, for example (and based on what). Fred. Gowriishankar Raju wrote: Dear Friends, We are doing research on membrane protease. We need to know to how to classify the protease in invitro and insilico levels. Is there is any advanced techniques or methods available, to classify the protease in insilico level? Please give suggestions on this. Waiting for your valuable suggestions. Thank you. With Regards, Gowriishankar R
Re: [ccp4bb] Perfect crystal with weird diffraction pattern
Hi Hui, I think most of us can't do much with rar archive files. This is a Windows thing I believe and my Linux system tells me Archive type not supported... Fred. hui yang wrote: Hi all, I just collected a data set from a long-spindle-shaped crystal. The data has been scaled to P1 space group with HKL2000, and the final overall Rmerge is 0.208. What is strange is that, the diffraction pattern looks all the same at different rotation angles, and the diffraction point outside the 4A cycle are excluded automatically and thus lead to a final resolution of 4.4A. I’m wondering 1) Whether this crystal is twinned? 2) What is the actual space group of it? A picture of the crystal, three images of the diffraction pattern (1, 90 and 180), the sca file and log file have been attached to this mail for your information. Any suggestions and comments will be highly appreciated. Thank you very much in advance for your inputs. Sincerely, Hui Yang
Re: [ccp4bb] OFF: Intein-chitin purification method
Petr Kolenko wrote: Dear all, I am working on crystallization of a protein purified using intein-chitin binding domain. There is 86% identity with protein with already known high-resolution structure, but I am not able to get any crystals. I heard that reversed phase chromatography is not good for crystallization, e.g. Has anyone of you observed something similar for intein-chitin chromatography? Is there any serious structural damage to the target protein during the purification? -- Petr Kolenko kole...@imc.cas.cz mailto:kole...@imc.cas.cz http://kolda.webz.cz The failure in crystallisation can be due to so many reasons... including protein damage (ending up with a mixture of components is probably one of the worse cases) Sometimes you need to modify your protein in order to crystallise it, or even use an analog from another organism. Fred.
Re: [ccp4bb] Lodovico...
Dear all, A tribute to Lodovico (pictures + letters + a song) can be found on http://www.crystalerice.org/Erice2010/Lodovico/index.htm Fred.
Re: [ccp4bb] measure of anamolous signal
Vandu Murugan wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan When processing the data, ensure that Bijvoet pairs are not merged. The data processing software should provide you with an R-ano value and that is already a start. The values provided should tell you if you have an anomalous signal or not. You may also have to play with the number of frames to integrate in order to obtain an optimal anomalous signal in the resulting data set. There are several publications describing Sulphur SAD, but one of them which is freely available on the Web can be found here: http://www.stoe.com/pages/brochure/labnote_genix_cu.pdf , Schiltz M (pp 4-6). Good luck! Fred.
Re: [ccp4bb] Prof. Lodovico Riva di Sansevenino passed away last night
Dear all, An obituary for Prof. Lodovico Riva di Sanseverino (in English, written by Prof. Carlo Mealli) can be found on http://www.cristallografia.org/uploaded/181.pdf (www.cristallografia.org is the web site of the Italian Crystallographic Association, Associazione Italiana di Cristallografia) Fred.
[ccp4bb] voting on The Scientist web site for our macromolecular structures
Dear all, I think we all think that macromolecular structures are the best there is... And now we have a chance to prove it to the world. The Scientist is organizing a competition to elect the best website. One of the competitors is the Proteopedia web site (http://www.proteopedia.org). We now have our chance to prove to the world that our beloved structures are indeed beautiful and important, buy voting on this site: http://www.the-scientist.com/videoawards (of course, if you are going to vote for another web site, then I suggest you do not participate in the voting process ! ;-) ). Fred.
Re: [ccp4bb] Matthews coefficient
Hi, No such thing as a stupid question. If the resolution is sufficient, arp_warp does a good job. But it is a reconstruction and REFINEMENT program. But checking the proper packing (that there are indeed contacts in the 3 dimensions of space to form the crystal): takes only 10 seconds using a graphics program, so there is no need to automate that. And trying to do everything completely automatically (without any human intervention and checks) can lead to big problems (for example if the space group has been wrongly assigned). You may end up publishing a wrong structure if you rely entirely on totally automated crystallographic software without checking anything. Fred. Francois Berenger wrote: Hello, I am not a crystallographer, so I will ask a maybe stupid question. When you say After molecular replacement you should check the result with the model building program of your choice and correct as many errors as possible before running a refinement program. Can this step be automated in some way (without any human intervention)? Are there programs to do this? I am interested even if some program does only one part of the job. Thanks a lot, Francois.
Re: [ccp4bb] molecular replacement
Hi there, If there are a few clashes (acceptable - usually surface loops that enter the surface part of another molecule or subunit) then you can delete the parts of loops that usually have poor density and that clash, refine, compute a new map and see if the loop can be traced (usually it can be). If there are really severe clashes, then your solution is not a proper solution. Also, have you checked the packing of the molecules in the crystal? Do you have contacts (in the 3 directions of space) that explain the crystal itself, with solvent regions or channels? This is also indicative of a real solution or if your solution to the MR problem is garbage. Further, if you can have a search model (located somewhere in the PDB) that is already a homodimer, then the molecular replacement calculations should be repeated. Better results simply because there are more vectors involved. Fred. Xianhui Wu wrote: Dear All, I am trying to resolve a protein structure with the use of Molecular Replacement. However, some part of protein are overlap in the interface of homodimer. Would someone please give me suggestions? Thank you! -- Best regards, WuXH
Re: [ccp4bb] PEG 1000
R.Srinivasan wrote: Dear All, I have got initial crystals in a condition with PEG 1000. The PEG 1000 stock we had in our lab was rock solid and when i heated it to about 50 degrees for 15 to 20 minutes it became a solution. We thought the compound has got out dated or something like that and bought a brand new bottle from Sigma and this is rock solid too. Is this something characteristic of PEG1000? The hit condition says its 12.5% w/v PEG 1000 but it apparently seems i could never get a powder of it. So my question is, Can i go ahead using this melted solution form of PEG1000 for setting up optimizations? Thank you all in anticiaption, Vasan All higher molecular weight PEG's are solid. Some are stored as flakes, but as you mention some are rock solid. And it's very difficult to get them out of the container (breaking the bottle is sometimes necessary). What I would go for personally would be mechanical grinding because I do not know what happens to the PEG when it's heated to 50 deg or higher. But perhaps you could take your bottle, cut the content in half, make a powder out of the one half and use the other one with this heating method and see if there are any differences. Or else if you happen to have an analytical chemistry service at hand, provide them with a small sample of each of the 2 and ask them to check if there is any difference. Fred.
Re: [ccp4bb] creating new ligand cif file
There is an option in Phenix to generate the necessary cif file for Phenix refinement. But that's a Phenix file, not a REFMAC file (I think, I never tried taking the cif file generated by Phenix to use it in REFMAC). As an example, I am enclosing a cif file that was generated by Phenix for NAD. Fred. Message du 21/06/10 12:41 De : Paul Lindblom lindblom.p...@googlemail.com A : CCP4BB@JISCMAIL.AC.UK Copie à : Objet : [ccp4bb] creating new ligand cif file Hi all, I need to create a cif file for a new ligand that does not exist in the pdb, so far. Normally refmac created such a cif file when I merged the ligand to the structure, but this time it stops with a fatal error. I think the alternatives are sketcher and the prodrg server. Can anybody tell me if I have to define the geometric restraints by myself, or is it possible to take just the output files and put them into refmac.? And if I have to define the geometric restraints, how to do this? Thanks, Paul # data_comp_list loop_ _chem_comp.id _chem_comp.three_letter_code _chem_comp.name _chem_comp.group _chem_comp.number_atoms_all _chem_comp.number_atoms_nh _chem_comp.desc_level NAD NAD ' ' ligand 70 44 . # data_comp_NAD # loop_ _chem_comp_atom.comp_id _chem_comp_atom.atom_id _chem_comp_atom.type_symbol _chem_comp_atom.type_energy _chem_comp_atom.partial_charge _chem_comp_atom.x _chem_comp_atom.y _chem_comp_atom.z NADN7NN NH2. 2.6986 -6.1207 3.004 NADC7NC C . 1.537 -6.6555 2.3517 NADO7NO O . 1.3898 -7.8687 2.2732 NADC3NC CR6. 0.4091 -5.7431 1.9755 NADC4NC CR16 . -0.2797 -5.0126 2.9515 NADC5NC CR16 . -1.381 -4.2342 2.5783 NADC6NC CR16 . -1.7709 -4.2029 1.2337 NADN1NN N . -1.0959 -4.9106 0.3178 NADC2NC CR16 . -0.0379 -5.6633 0.6517 NADC1DC CH1. -1.5719 -4.8804 -1.1622 NADO4DO O2 . -1.3484 -3.6272 -1.9309 NADC4DC CH1. -2.1668 -3.7109 -3.0354 NADC5DC CH2. -2. -2.3363 -3.602 NADO5DO O2 . -1.347 -1.5451 -3.9358 NADPN P P . -1.5662 -0.0959 -4.4972 NADO1NO OP . -1.0974 -0.0374 -5.9333 NADO2NO O . -3.035 0.2582 -4.431 NADO3 O O2 . -0.744 0.9221 -3.6379 NADPA P P . -0.3546 2.3201 -4.2438 NADO1AO OP . -1.6084 3.2286 -4.2603 NADO2AO O . 0.1652 2.1131 -5.6917 NADO5BO O2 . 0.7742 2.9381 -3.3472 NADC5BC CH2. 0.4584 3.9355 -2.4289 NADC4BC CH1. 0.5056 3.5799 -0.9628 NADO4BO O2 . 1.6648 4.0546 -0.3156 NADC1BC CH1. 1.361 4.0835 1.0286 NADC2BC CH1. -0.0848 4.4799 1.1094 NADO2BO OH1. -0.7347 3.7827 2.1075 NADC3BC CH1. -0.6372 4.1573 -0.2 NADO3BO OH1. -1.6585 3.2415 -0.0848 NADN9AN N . 2.2055 4.9952 1.7726 NADC8AC CR15 . 2.2653 6.3255 1.6131 NADN7AN N . 3.1449 6.8137 2.4992 NADC5AC CR56 . 3.5563 5.8132 3.2885 NADC6AC CR6. 4.5276 5.6879 4.29 NADN6AN NC2. 5.249 6.8437 4.7336 NADN1AN N . 4.7981 4.4881 4.8196 NADC2AC CR16 . 4.2084 3.3883 4.3291 NADN3AN N . 3.3409 3.443 3.3085 NADC4AC CR56 . 2.991 4.6313 2.7967 NADC3DC CH1. -3.4086 -4.4391 -2.5803 NADO3DO OH1. -3.9339 -5.1909 -3.5949 NADC2DC CH1. -2.9817 -5.297 -1.4587 NADO2DO OH1. -3.0403 -6.6279 -1.7966 NADH1DH HCH1 . -2.1022 -4.2335 -0.6748 NADH4DH HCH1 . -1.7309 -4.2424 -3.7186 NADH4BH HCH1 . 0.4797 2.6144 -0.8821 NADH1BH HCH1 . 1.4694 3.1891 1.3961 NADH2BH HCH1 . -0.1583 5.4339 1.2721 NADH3BH HCH1 . -0.9583 4.9641 -0.6299 NADH3DH HCH1 . -4.0703 -3.8052 -2.2618 NADH2DH HCH1 . -3.5406 -5.1319 -0.6798 NADH71N H HNH2 . 2.8124 -5.2214 3.0642 NADH72N H HNH2 . 3.3238 -6.6844 3.3588 NADH4NH HCR6 . 0.0257 -5.0151 3.8693 NADH5NH HCR6 . -1.834 -3.6797 3.2336 NADH6NH HCR6 . -2.5483 -3.6886 0.9687 NADH2NH HCR6 . 0.4339 -6.1535 -0.0382 NADH51N H HCH2 . -2.9863 -2.4496 -4.4054 NADH52N H HCH2 . -2.9761 -1.8462 -2.9472 NADH51A H HCH2 . 1.0746 4.6774
[ccp4bb] Fwd: Re: [ccp4bb] creating new ligand cif file
Received from Peter Zwart: Original Message Subject:Re: [ccp4bb] creating new ligand cif file Date: Mon, 21 Jun 2010 07:08:18 -0700 From: Peter Zwart phzw...@lbl.gov To: Vellieux Frederic frederic.velli...@ibs.fr References: 4c1f4f26.2070...@ibs.fr it is the same format actually. 2010/6/21 Vellieux Frederic frederic.velli...@ibs.fr: There is an option in Phenix to generate the necessary cif file for Phenix refinement. But that's a Phenix file, not a REFMAC file (I think, I never tried taking the cif file generated by Phenix to use it in REFMAC). As an example, I am enclosing a cif file that was generated by Phenix for NAD. Fred.
Re: [ccp4bb] NOT Job Advertisement but model refinement
This has been told (written, in fact) on this bb many times. The purpose of refinement is not to bring the R-factor down, but to obtain a model that satisfies best all available data (including biochemical data). Anyway, as to what you can do: it's always possible to question the choice of TLS groups. I do not know how you select yours. What I personally do is to connect to the TLSMD server, lauch the computation, then have a look at the output curve. There is a steep descent at first, and (usually) a break in the curve. This break in the curve I use to tell me how many TLS groups to be used during refinement. One can always change the refinement program to see if an improved geometry together with more appropriate R and R-free values can be obtained (both at the same time). But you do not give much information on how you have built and refined your model so far... Also, sometimes automated model rebuilding programs give excellent results. Fred. atul kumar wrote: dear all I am trying to solve structure of data set at 1.9 A,r merge 9.3,it belongs to space group P3221 unit cell 160.78 157.32 135.62 90.0 90.0 90.0,i has no NCS ,i did tls as well but after water addition the r factor and r free is stuck at 22 and 25 respectively. kindly guide me how can i bring down the R factor. Thankin you. Regards Atul
Re: [ccp4bb] Ligand present in only one monomer in NCS
Hi Andy, I'd say fairly common, and there can be several reasons. One is that the entrance of the active site (in case of crystal soaks) is blocked in some subunits. Also, different affinities in the different active sites, plus allosteric effects, are possibilities to consider. The latest example here I can give you (unpublished yet) is a mutant of an NAD-binding tetrameric enzyme. The crystals were grown by co-crystallisation. Out of the 4 active sites, only 2 of them contain cofactor plus substrate analogue. It is likely that the scientists who do large scale analyses of the PDB using automated programs have done a systematic search in the PDB and could provide you with accurate statistics. I wouldn't know any reference to such work (I am not in that field myself). Fred. ANDY DODDS wrote: Hello, I am solving a structure of an enzyme, which crystallises as a dimer. We have pretty good evidence that this operates as a dimer in vitro, also. We have an inhibitor of this enzyme, which we are keen to visualise by X-ray methods. We seem to have very strong density in which we can model our inhibitor, with good stats and no negative density. However, there is only density in one of the monomers, nothing in the other. The SG is P212121, and although I can postulate why this may have happened (if this is indeed what HAS happened), different solvent channel accessibility etc., I would like to know how common this was and, if possible, some literature regarding this, if the board would be so kind? Regards, Andrew.
Re: [ccp4bb] Converting cif-files into pdb-files / reading cif -files into coot
Hi Christian, Had exactly the same problem (converting an mmCIF file into a PDB). I located and installed CIFTr . The version I have running here is ciftr-v2.053 . I am afraid I can't remember exactly where I downloaded it from. I think it one of the PDB associated files. Fred. Christian Engel wrote: Dear All, I am looking for a ccp4 program that reads in cif-files and converts them into pdb-files, including the CRYST1 card. Can anybody suggest a solution? I didn't find any in ccp4i, e.g. the coordinate utilities. I also tried COOT to read in cif-files (downloaded from the pdb server). For one example it crashed, for others it doesn't colour the bonds properly if Bonds (Colour by Atom) is chosen but shows all bonds in one colour. Is that a known feature? If I save these coordinates in pdb-format and try to read it back, I get an ERROR saying: Wrong ASCII format of an integer. Thanks for any suggestions Christian Engel */Mit freundlichen Grüßen / Best regards / Cordialement/* Dr. Christian Engel Sanofi-Aventis Deutschland GmbH RD CAS Structural Biology FFM Industriepark Hoechst Bldg. G877, Room 020 D-65926 Frankfurt am Main t: +49 69 305 12946 f: +49 69 305 80169 w:_www.sanofi-aventis.de_ 125 Jahre Arzneimittel aus Deutschland von sanofi-aventis * Sanofi-Aventis Deutschland GmbH · Sitz der Gesellschaft: Frankfurt am Main · Handelsregister: Frankfurt am Main, Abt. B Nr. 40661 Vorsitzender des Aufsichtsrats: Hanspeter Spek - Geschäftsführer: Dr. Martin Siewert (Vorsitzender), Ulf Bialojahn, Dr. Matthias Braun, Peter Guenter, Prof. Dr. Dr. Werner Kramer, Dr. Klaus Menken, Dr. Heinz Riederer *
Re: [ccp4bb] Far to good r-factors
Hi Paul, I've seen that type of behaviour before for low resolution structures. On such structures, either I have a very hard time getting at the same time a good geometry, good R-factors and satisfactory electron density, or things go very smoothly and all the statistics (model geometry, R-factors) plus electron density are fine. Too bad I have no way of predicting when things will be going well. Two examples where things went very smoothly: glyceraldehyde-phosphate dehydrogenase from Trypanosoma brucei brucei (PDB id: 2X0N re-refined fairly recently with Phenix); malate dehydrogenase from Archaeoglobus fulgidus (PDB id: 2X0I also re-refined with Phenix) The only thing you have to check is that the relative weighting of the X-ray term vs. the geometry term is appropriate, so that you do not lower the R-factors while the geometry is getting worse. HTH, Fred. Paul Lindblom wrote: Hi everybody, once more I need your help. I solved the structure of an enzyme at resolution of 1.9 A. Now I was trying to get a complex and soaked some ligand to my crystals. I could solve the structure (and see poor density for my ligand or something else) at 3.0 A by molecular replacement using my 1.9A structure as a starting model. But the problem is now, that I got an R-work of 16.34 and an r-free of 20.23 for the new 3.0 A structure - without adding any waters or solvent/ligand molecules. The r-factors are even better than the ones I got for the 1.9A structure. So I think something is wrong with the whole thing. I observed twinning for both data and used the twin refinement option in refmac, but the results stay more or less the same. Any suggestions what to do? Thanks a lot, Paul
Re: [ccp4bb] How to show bonds between ligands and metal which are around 1.8-2.0 Ao far away, in pymol..??
Hussain Bhukyagps wrote: Hi! their is no connection between the metal and ligand in my pdb.. how can i generate bonds between them using pymol..?? by the way the distance between ligands and metal is 1.8-2.0 Ao...?? Hussain Hi, What about specifying the bonds with CONECT records in the PDB file? From the PDB specs section: CONECT Overview The CONECT records specify connectivity between atoms for which coordinates are supplied. The connectivity is described using the atom serial number as found in the entry. CONECT records are mandatory for HET groups (excluding water) and for other bonds not specified in the standard residue connectivity table which involve atoms in standard residues (see Appendix 4 for the list of standard residues). These records are generated by the PDB. Record Format COLUMNS DATA TYPEFIELD DEFINITION - 1 - 6 Record name CONECT 7 - 11 Integer serial Atom serial number 12 - 16 Integer serial Serial number of bonded atom 17 - 21 Integer serial Serial number of bonded atom 22 - 26 Integer serial Serial number of bonded atom 27 - 31 Integer serial Serial number of bonded atom 32 - 36 Integer serial Serial number of hydrogen bonded atom 37 - 41 Integer serial Serial number of hydrogen bonded atom 42 - 46 Integer serial Serial number of salt bridged atom 47 - 51 Integer serial Serial number of hydrogen bonded atom 52 - 56 Integer serial Serial number of hydrogen bonded atom 57 - 61 Integer serial Serial number of salt bridged atom Details * Intra-residue connectivity within non-standard (HET) residues (excluding water) is presented on the CONECT records. * Inter-residue connectivity of HET groups to standard groups (including water) or to other HET groups are represented on the CONECT records. * Disulfide bridges specified in the SSBOND records have corresponding CONECT records. * Hydrogen bonds and salt bridges have CONECT records. * No differentiation is made between donor and acceptor for hydrogen bonds. * No differentiation is made between atoms with excess negative or positive charge. * Atoms specified in the connectivity are presented by their serial numbers as found in the entry. * All atoms connected to the atom with serial number in columns 7 - 11 are listed in the remaining fields of the record. * If more than four fields are required for non-hydrogen and nonsalt-bridge bonds, a second CONECT record with the same atom serial number in columns 7 - 11 will be used. * These CONECT records occur in increasing order of the atom serial numbers they carry in columns 7 - 11. The target-atom serial numbers carried on these records also occur in increasing order. * The connectivity list given here is redundant in that each bond indicated is given twice, once with each of the two atoms involved specified in columns 7 - 11. * For nucleic acids, Watson-Crick hydrogen bonds between bases may be listed, but this is optional. * For hydrogen bonds, when the hydrogen atom is present in the coordinates, PDB generates a CONECT record between the hydrogen atom and its acceptor atom. * For NMR entries, CONECT records for all models are generated describing heterogen connectivity and others for LINK records. Verification/Validation/Value Authority Control Connectivity is checked for unusual bond lengths. Relationships to Other Record Types CONECT records must be present in an entry that contains either non-standard groups or disulfide bonds. Example 1 2 3 4 5 6 7 1234567890123456789012345678901234567890123456789012345678901234567890 CONECT 1179 746 1184 1195 1203 CONECT 1179 1211 1222 CONECT 1021 544 1017 1020 1022 1211 1222 1311 Known Problems Only five digits are available for the atom serial number, but some structures have already been received with more that 99,999 atoms. Changing the field length would make earlier entries incorrect. CONECTs to atoms whose coordinates are not in the entry (e.g., symmetry-generated) are not given.
Re: [ccp4bb] Possible sulphate
Rex Palmer wrote: What seems to be a possible sulphate has been identified in our electron density. What steps could/should be taken to confirm or consolidate this assignment that would satisfy referees? Rex Palmer Birkbeck College Geometry of the interactions (and the shape of the electron density). Anomalous map. Even if you have only diffraction data collected at low wavelength you can always compute an anomalous map and see if the sulphur shows up. Fred.
[ccp4bb] asking for less red tape for European research funding
Dear fellow cristallographers, It has come to my attention that there is a petition at the moment, directed at the *European Council of Ministers and the Parliament, asking for less red tape and cumbersome financial regulations from the EU concerning European research funding.* http://www.trust-researchers.eu Hence this petition might be of interest to you if you think we are wasting too much time in order, for example, to write grant proposals for the Framework funding programs. Hope this information can be useful to you. And have a nice end of the day. Fred.
Re: [ccp4bb] pdb file deposition
Hi, What happens usually is that some solvent atoms are in fact closer to a symmetry-related molecule than the one you are working on. The PDB has programs to reposition these water molecules closer to the correct macromolecule or subunit, and they will provide you with the modified waters and ask you to approve (or not). So I personally ignore these warning messages until I receive news from the PDB. HTH, Fred. Azadeh Shahsavar wrote: Dear All, In depositing a pdb file, after validation step, an error comes up: *Solvent Atoms* The following solvent molecules lie farther than expected from the protein. Can any one give me some advice about it? deleting these water molecules results in a large increase of R factor, by the way. Thank you in advance, A
[ccp4bb] problem with residue numbering and Coot
Hi folks, I'm having a problem with Coot. Molecular Replacement solution to the phase problem. The MR model has one small stretch of residues missing, for which there is density in the maps (both normal and difference density maps). Hence I have built the model into this density. Except that there is a problem with the standard residue numbering scheme one has to use in this class of enzymes. The numbering jumps from residue number 103 to residue number 105. As a result, Coot won't accept that the residues be linked together and forces them apart. The initial refinement round has corrected this somewhat (but still not entirely, even though I had introduced a LINK record in the PDB file before refinement). So I don't seem to be able to use Coot to correct this. Hence the question: is there any way to force Coot to accept that these two residues be connected together even though the numbering scheme is not sequential? I haven't found the correct option anywhere. And I really do not want to use a sequential numbering scheme for refinement and model building only, since it would means plenty of residue number editing (by hand) to re-introduce all of this weird residue numbering scheme, everywhere in the subunit. Thanks, Fred.
Re: [ccp4bb] problem with residue numbering and Coot
Dear All, I have received the following suggestion from a crystallographer. The bottom part of the email was indicating that the mail was confidential, so I have removed the name of the author of that email (together with this section about confidentiality) and I am therefore forwarding the message below. What worked for me was to temporarily renumber residue 105 to 104 (this I did with a text editor, not with Coot), fix the bond by real-space refinement, and immediately change residue 104 back to 105. I did not need to have a LINK card in the PDB file. That did the trick, so I thought I should inform the bb even though it is confidential. Fred. Dear Fred, Here’s what you should do: 1. Briefly renumber your protein to sequential numbering (103, 104 etc.) in Coot. 2. Use real space refine zone in coot to tidy up the peptide bond between residue 103 and 104. 3. When it looks good renumber the residues back to the standard numbers (103, 105 etc.) in Coot 4. Add this line to your PDB header: LINKR C TRP A 103 N ASN A 105 TRANS 5. Refine in Refmac. There will be no problem with it breaking the bond any more because the TRANS word tells it to link the two amino acids together as normal. 6. Bob’s your uncle! Hope this helps.
