[ccp4bb] I222 - P22121 space-group ambiguity
Hi everybody, I collected a number of X-ray data sets from crystals originating from the same cryst. drop. I solved the initial structure in P22121 space group by MR with Phaser locating two molecules (data to ~ 2.1 Angstr.); refined R/Rfree: 0.213/0.244. Processing of some of the other data sets with XDS/Aimless is consistent with I222 space group (resolution ~ 2.6 Ang.). I can locate one molecule. The unit-cell dimensions for I222 and the initial P22121 space groups for two of the data sets are: I222: a=87.8 b=101.18 c=123.63; P22121: a=93.34 b=105.47 c=122.98; I superposed the molecule in I222 onto one of the two located for the initially solved P22121; the orientation of the NCS-related molecule in P22121 differs from the crystallographic-symmetry related one in I222. Trying to solve this P22121 data set in I222 with MR, does not result in high Z scores, and maps do not look good. Some of the data sets that process in I222 to ~ 3 Angstr., I can also solve in P22121, locating two molecules (differences may not be that clear in this case, since the resolution is lower). Some other data sets process in P22121 with Aimless; with a substantial off-origin Patterson peak, indicating translational NCS. For these, Phaser positions two molecules that are related by crystallographic translational NCS. These two molecules are crystallographic-symmetry related in the original P22121 data set. I can also solve these data sets in I222 space group, with the overall Z score higher than for the P22121 data. I am uncertain, what the ‘true’ space group for some of my data sets is. Could it be that for data that process in P22121, but can be solved in I222, reflections that would indicate I222 space group were not collected? Alternatively, perhaps what I am seeing is that there is a (gradual) transition of the crystal lattice (between P22121 and I222 or vice versa), caused by variation in crystal handling/cooling or exposure to X-rays. It’s relevant to me, because in P22121 space group, a region of the molecule that is of biological interest makes NCS-related crystal contacts that are crystallographic-symmetry related in I222. Has anybody observed similar cases? I would appreciate comments. Cheers, Florian
Re: [ccp4bb] project and literature organization software (laboratory information management software)
Hi Tobias, There is Quartzy, which is free. https://www.quartzy.com I am not sure it covers all of your desired functionalities. Best regards, Florian On Apr 29, 2014, at 7:21 AM, Tobias Beck tobiasb...@gmail.com wrote: Dear all, I am looking for a software solution to organize many pieces of information 1.) Results from (bio)chemical experiments, such as spectral data, pictures. 2.) Project ideas, milestones, etc. 3.) Literature, including tags, short comments, etc. For example, for a certain project I would like to collect information about experiments conducted, then link this to literature/literature experiments and to project outlines. All this should be accessible for multiple users on different OS. I have briefly looked into PiMS (too much crystallography oriented), Contor ELN (only on Safari on Mac?), Labguru (nice, but not too flexible and mostly for biosciences) and Confluence (nice wiki, but so far no real literature plugin). I know that this sounds maybe a little bit like something called in German a 'eierlegende Wollmilchsau' http://en.wiktionary.org/wiki/eierlegende_Wollmilchsau But I would be happy to hear about what software people (and labs) have tried, liked/disliked and ideally the reasons. (I am aware that there was a similar query https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg24657.html, but this was more than 2 years ago) Thanks a lot! Best wishes, Tobias. -- ___ Dr. Tobias Beck ETH Zurich Laboratory of Organic Chemistry Vladimir-Prelog-Weg 3, HCI F 322 8093 Zurich, Switzerland phone: +41 44 632 68 65 fax:+41 44 632 14 86 web: http://www.protein.ethz.ch/people/tobias ___ --- Florian Schmitzberger, PhD Biological Chemistry and Molecular Pharmacology Harvard Medical School 250 Longwood Avenue, Seeley G. Mudd 127 Boston, MA 02115, USA Tel: 001 617 432 5601
[ccp4bb] Off-topic: 96-well plate PCR/plasmid purification
Dear All, I am looking for a bit of advice, and am interested to hear about experiences people have had with various commercially-available 96- well plate-based PCR, plasmid, and protein purification appliances and plates. Forgive me for comparing commercial vendors. We have a 96-well plate-compatible vacuum manifold (Qiavac). However, the plasmid-purification kits from this vendor do seem comparatively pricey to me. So, I am looking for a more cost-efficient system. How compatible are 96-well PCR purification plates/plasmid-binding and filter plates from different vendors with this type of vacuum manifold? From what I can see, it becomes difficult to avoid cross- contamination between wells, when using e.g. Whatman-type 96-well microplates with Qiavac, because the distance between the drip director of the DNA-binding/filter plate and a receiver plate (e.g. PCR plate) in the manifold is relatively long, and the alignment isn't great (drops end up on the rim). The alternative would be centrifugation directly on top of a receiver plate, but I am concerned about cross-contamination. Which type of membranes/filter plates were people most pleased with for (bacterial) lysate-clearing, avoiding clogging with lysates from ~ 5 ml bacterial cultures; and which DNA/plasmid-binding plates have most binding capacity and result in highest purity? Again cost- efficient vendors would be preferred. Regards, Florian
Re: [ccp4bb] The effect of His-tag location on crystallization
Human leukotriene C4 synthase (PDB accession code: 2UUI) is another example, illustrating how an N-terminal polyhistidine-tag, in conjunction with metals, presumably facilitated crystallization. On Jun 27, 2012, at 12:04 PM, Brad Bennett wrote: I think it was an N-terminal RGS-type His tag in 3O8Y (human lipoxygenase) that mediated crystal contacts with a symmetry related molecule. As I recall, this tag composed a B-strand that formed a nice interface with a native B-strand of the symmetry related molecule. Pretty cool... -Brad On Wed, Jun 27, 2012 at 11:00 AM, Phoebe Rice pr...@uchicago.edu wrote: With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 27 Jun 2012 10:14:58 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of R. M. Garavito rmgarav...@gmail.com) Subject: Re: [ccp4bb] The effect of His-tag location on crystallization To: CCP4BB@JISCMAIL.AC.UK Most of the comments you will get will be anecdotal in that people will report the successful results and do not take the time or effort to characterize the less successful results. This often occurs because the tagged portion of the protein is most often disordered, even in the best crystals. Thus, other than saying tagging on this end works, but tagging on that end doesn't, there is little more you can say. Each case will be different, and it is almost impossible to arrive at any generalized conclusion. We prefer C-terminal tagged proteins for a number of reasons, but if an N-terminally tagged protein crystallizes well, so be it. Of the dozens of N- and C-tagged protein structures we have solved in my lab and with collaborators, I have only seen one case of an ordered His-tag: the His residues had coordinated Cd ions, which proved essential for getting good crystals. However, beyond that there was not much more to say. For your protein and the resulting crystals, an N-terminally tagged protein crystallized well. Whether you can draw any more conclusions from these results depends on characterizing crystals of both N- and C-tagged proteins. Just assuming that the C-tagged protein is trying to crystallize in the same or related crystal form as the N-tagged protein is an unwarranted assumption without experimental evidence to back it up. That is why most groups just run with the winner. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com On Jun 26, 2012, at 9:06 PM, weliu wrote: Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
[ccp4bb] Off-topic: PDB deposition of multiple structure factor files
Dear All, With my most recent PDBe deposition, in addition to the native data, I had intended to deposit the anomalous data, used for structure determination, and make it available for download. This turned out to be less straightforward than I had anticipated, because the current PDB convention is to only allow a single structure factor file for experimental data (usually the native dataset), available for download from the PDB. In my case, the anomalous data were concatenated with the native data into a single cif file (this worked and made sense, because both for both datasets the unit cell dimensions are virtually identical). I imagine it would be beneficial to be able to make available more than a single structure factor file, including the ones derived from experimental phasing, in the PDB, along with the final coordinates, without concatenating the data into a single file (which may lead to confusion to users when downloaded). Is this anything the PDB is already working to implement in the near future (perhaps via the coming PDBx format)? Best regards, Florian
Re: [ccp4bb] Off-topic: PDB deposition of multiple structure factor files
Dear Mark, This is interesting. I also had submitted my data via the PDBe (European portal). While they allow deposition of multiple datasets, only a single file can apparently be made available for download from the site. In contrast to your case, for my deposition the second deposited dataset is not explicitly listed though. Cheers, Florian On Apr 27, 2012, at 2:35 PM, Mark J van Raaij wrote: again, it looks like this is particular to the US portal. We submit via the European www.pdbe.org and can submit multiple datasets. See 2XGF for an example. Note: I think from www.rcsb.org only one file can be downloaded, but www.pdbe.org clearly shows both. Although you are in the US, you can use the pdbe deposition tool AUTODEP - or the Japanese one, if you like. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 27 Apr 2012, at 20:23, Florian Schmitzberger wrote: Dear All, With my most recent PDBe deposition, in addition to the native data, I had intended to deposit the anomalous data, used for structure determination, and make it available for download. This turned out to be less straightforward than I had anticipated, because the current PDB convention is to only allow a single structure factor file for experimental data (usually the native dataset), available for download from the PDB. In my case, the anomalous data were concatenated with the native data into a single cif file (this worked and made sense, because both for both datasets the unit cell dimensions are virtually identical). I imagine it would be beneficial to be able to make available more than a single structure factor file, including the ones derived from experimental phasing, in the PDB, along with the final coordinates, without concatenating the data into a single file (which may lead to confusion to users when downloaded). Is this anything the PDB is already working to implement in the near future (perhaps via the coming PDBx format)? Best regards, Florian --- Florian Schmitzberger, PhD Biological Chemistry and Molecular Pharmacology Harvard Medical School 250 Longwood Avenue, Seeley G. Mudd 123 Boston, MA 02115, US Tel: 001 617 432 5603
Re: [ccp4bb] Substitution to glycerol during crystallogenesis
Dear Toby, I don't think there is a basic problem using glycerol in crystallization. Glycerol will affect the vapour pressure (if it is not present in the well/precipitant solution) and 10 % glycerol is ~ 1.3 molar concentration. During equilibration the drops may increase in volume, decreasing the protein concentration. Thus, when using glycerol I think it is generally beneficial to start with a high protein concentration. Perhaps, you can concentrate your protein sample further. I have on several occasions observed immediate precipitation upon mixing protein solution (containing glycerol) and precipitant solution; drops then cleared up after a short period of time (and crystals eventually formed). In this case, the crystallization experiment starts in the supersaturated zone, and moves towards an undersaturated concentration, traversing the (metastable) zone where nucleation and crystallization can happen (rather than the other way around, which seems the more traditional approach with crystallization by vapour diffusion). Enrico Stura published a recent article, describing an effect of glycerol on crystallization. Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth Des. 11 :2755–2762. You could replace glycerol with ethylenglycol or a small molecular weight PEG (e.g. 400), which may also have a stabilizing effect on your complex. Regards, Florian On Apr 3, 2012, at 7:49 AM, Toby Longwood wrote: Dear all, My question is related to a sample preparation. I’m working with a complex that can be stabilized with glycerol (at least 10%) during purification. The use of detergents does not help. After purification, the sample is homogeneous (EM) and can be concentrated (3-4mg.mL-1) . I already set up many drops, changing several conditions (pH, salt...) but nothing conclusive appeared. I know that crystallogenesis in presence of glycerol works (Sousa, Acta Cryst (1995), ...) however, because of the aspect of the drops (precipitates that seem close to the nucleation phase), I suspect that the glycerol can be one of the limiting factors of the protocol. Has anybody else been already confronted to the same problem? Does someone know if there is an alternate additive to glycerol? Thanks in advance for suggestions/help With best wishes Toby
[ccp4bb] xds - problem with reference profile
Dear All, I am getting a warning message in XDS I have not seen before, when trying to integrate a low resolution (~ 7 A) dataset. !!! WARNING !!! REFERENCE PROFILE # 1 IS EMPTY. THE AVERAGE PROFILE IN THIS BATCH IS USED INSTEAD. and so forth for the other reference profiles. Indexing works alright with XDS; with likely point group C2. Scaling the data integrated with XDS, with Scala fails; apparently most of the reflections are rejected. What are probable causes for the above error message? Cheers, Florian
Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
On Jan 30, 2012, at 10:28 AM, Jacob Keller wrote: I'm intrigued: how come this apparently excellent idea has not become standard best practice in the 14 years since it was published? It would seem because too few people know about it, and it is not implemented in any software in the usual pipeline. Maybe it could be? Phenix.model_vs_data calculates a sigmaA_ vs resolution plot (in comprehensive validation in the GUI). Pavel would probably have replied by now, but I don't think the discussion has been cross-posted to the phenix bb. Cheers, Florian Perhaps the way to do it would be always to integrate to ridiculously-high resolution, give that to Refmac, and starting from lower resolution, to iterate to higher resolution according the most recent sigma a calculation, and cutoff according to some reasonable sigma a value? JPK phx On 30/01/2012 09:40, Randy Read wrote: Hi, Here are a couple of links on the idea of judging resolution by a type of cross-validation with data not used in refinement: Ling et al, 1998: http://pubs.acs.org/doi/full/10.1021/bi971806n Brunger et al, 2008: http://journals.iucr.org/d/issues/2009/02/00/ba5131/index.html (cites earlier relevant papers from Brunger's group) Best wishes, Randy Read On 30 Jan 2012, at 07:09, arka chakraborty wrote: Hi all, In the context of the above going discussion can anybody post links for a few relevant articles? Thanks in advance, ARKO On Mon, Jan 30, 2012 at 3:05 AM, Randy Read rj...@cam.ac.uk wrote: Just one thing to add to that very detailed response from Ian. We've tended to use a slightly different approach to determining a sensible resolution cutoff, where we judge whether there's useful information in the highest resolution data by whether it agrees with calculated structure factors computed from a model that hasn't been refined against those data. We first did this with the complex of the Shiga-like toxin B-subunit pentamer with the Gb3 trisaccharide (Ling et al, 1998). From memory, the point where the average I/sig(I) drops below 2 was around 3.3A. However, we had a good molecular replacement model to solve this structure and, after just carrying out rigid-body refinement, we computed a SigmaA plot using data to the edge of the detector (somewhere around 2.7A, again from memory). The SigmaA plot dropped off smoothly to 2.8A resolution, with values well above zero (indicating significantly better than random agreement), then dropped suddenly. So we chose 2.8A as the cutoff. Because there were four pentamers in the asymmetric unit, we could then use 20-fold NCS averaging, which gave a fantastic map. In this case, the averaging certainly helped to pull out something very useful from a very weak signal, because the maps weren't nearly as clear at lower resolution. Since then, a number of other people have applied similar tests. Notably, Axel Brunger has done some careful analysis to show that it can indeed be useful to take data beyond the conventional limits. When you don't have a great MR model, you can do something similar by limiting the resolution for the initial refinement and rebuilding, then assessing whether there's useful information at higher resolution by using the improved model (which hasn't seen the higher resolution data) to compute Fcalcs. By the way, it's not necessary to use a SigmaA plot -- the correlation between Fo and Fc probably works just as well. Note that, when the model has been refined against the lower resolution data, you'll expect a drop in correlation at the resolution cutoff you used for refinement, unless you only use the cross-validation data for the resolution range used in refinement. - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 29 Jan 2012, at 17:25, Ian Tickle wrote: Jacob, here's my (personal) take on this: The data quality metrics that everyone uses clearly fall into 2 classes: 'consistency' metrics, i.e. Rmerge/meas/pim and CC(1/2) which measure how well redundant observations agree, and signal/noise ratio metrics, i.e. mean(I/sigma) and completeness, which relate to the information content of the data. IMO the basic problem with all the consistency metrics is that they are not measuring the quantity that is relevant to refinement and electron density maps, namely the information content of the data, at least not in a direct and meaningful way. This is because there are 2 contributors to any consistency metric: the systematic errors (e.g. differences in illuminated volume and absorption) and the random errors (from counting statistics, detector noise etc.). If the data are collected with
Re: [ccp4bb] detect dsDNA
There exists a less toxic chemical than EtBr to stain DNA: SYBR safe DNA stain (a fluorescence dye sold by a certain vendor). Another benefit is to be able to use blue light, reducing UV/VIS light exposure when handling gels. Florian On Oct 2, 2011, at 11:49 AM, Edward A. Berry wrote: Jacob Keller wrote: I actually looked at an EtBr MSDS a while ago, and was shocked at how benign it was. I also heard from someone that they used to feed it to Argentinian cows routinely a few years back... Wikipedia says it was used as a trypanosomacidal - It's being discontinued not because of toxicity to beast or man, but because of insufficient toxicity to trypanosomes- the little buggers are developing resistance. Of course resistance would develop earlier if EtBr is mutagenic. Maybe they overexpress DNA repair enzymes.
[ccp4bb] Off topic: vector map editing and DNA sequence alignment software
Dear All, What type of software are people commonly using these days for vector/ plasmid map editing, making/visualizing vector maps, and aligning (small to medium size) DNA sequencing data? Preferably, it should not be too expensive and be able to write text files, readable by other programs. I am familiar with VectorNTI, which is great for vector visualization and editing; but I find it somewhat expensive. Sequencher seems good to quickly align DNA sequences (such as from DNA sequencing) with templates, but is not free. I have been using ApE for while for alignments, but aligning many sequences is more cumbersome than in Sequencher; I have not tested if Sequencher is good at visualizing and editing plasmid maps. Ideally, I would like to have a single program for both purposes (vector editing and DNA sequence comparison). Does something like that exist? What are the alternatives to above programs? Thank you in advance. Florian --- Florian Schmitzberger, PhD Biological Chemistry and Molecular Pharmacology Harvard Medical School 250 Longwood Avenue, Seeley G. Mudd 123 Boston, MA 02115, US Tel: 001 617 432 5603
Re: [ccp4bb] Electostatic surface at various pH
Hi John, I would probably use Pdb2pqr, to assign charges and radii for the atoms at different pHs, and then APBS integrated in Pymol software for visualization: http://kryptonite.nbcr.net/pdb2pqr/ http://www.poissonboltzmann.org/apbs/ Hope this helps, Florian On Aug 19, 2011, at 2:35 PM, john peter wrote: Hi All, Apologies for this slight off topic, however this could very well be the best bulletin board to seek help. I need to calculate the electrostatic surface and make surface figures for a protein at various pHs, say 4.0, 7.0 9.0. I'm doing this kind of wok for the first time. Could you suggest me a user-friendly software, tutorials and tips. Sincere thanks and appreciation. John
Re: [ccp4bb] XDS problem: REMOVE.HKL ignored?
Hi Engin, I encountered the same issue a couple of months ago. As I understand, the REMOVE.HKL file will only be used if you specify the spacegroup and unit cell in the XDS.INP file. Cheers, Florian On Jul 25, 2011, at 1:14 PM, Engin Özkan wrote: Hi all, After about a year of not working with XDS, I was a little surprised to see that adding a REMOVE.HKL file to the current directory and running CORRECT does not remove outliers. I still see the same reflections reported at the end of my new CORRECT.LP file and I have grepped the XDS_ASCII.HKL file to observe, to my dismay, that the removed are still present. Since I do not see this reported and this must be a daily used option by many of you out there, I am guessing that I must be doing something wrong, but it escapes me. For my and anybody else's sanity, here is my REMOVE.HKL file: $ more REMOVE.HKL 21 15 -172.10 963.70 0.1638E+06 0.9804E+04 alien 39 -272.17 58.11 0.2972E+04 0.1788E+03 alien 3 11 -262.17 32.12 0.1643E+04 0.1002E+03 alien 259 -202.04 11.89 0.3647E+03 0.1498E+02 alien 24 -331.87 10.88 0.1215E+03 0.8789E+01 alien 139 -192.71 10.69 0.1397E+04 0.4445E+02 alien 177 -291.99 10.65 0.2353E+03 0.1573E+02 alien 16 14 -291.83 10.58 0.8817E+02 0.9706E+01 alien 18 12 -281.90 10.16 0.1566E+03 0.1325E+02 alien I am using the most recently updated version of xds on a Scientific Linux 6.0 (64-bit) machine. Hey, I just figured out that using the December 6, 2010 version installed back in January on a different 64-bit linux machine, the REMOVE.HKL file was successfully used. So this may be a problem with the recent bug-fix update (which we have through SBGrid). All clues, hits, pointers are appreciated. Best, Engin
[ccp4bb] merge datasets
Dear All, I have two questions: 1) I have collected multiple, native datasets (5) from the same crystal (different parts of the crystals exposed with different transmission and oscillation angles). Each dataset on its own is close to complete (96-98 %). Naturally, differences in exposure, onset of radiation damage (datasets were collected with high transmission), local differences in the crystal, will affect the variance of the errors in the measurements for the reflections between the different datasets; but I would think the redundancy and increased number of measurements from all datasets should outweigh this. My tendency is to include all datasets. I am working at 3.8 A resolution (structure is solved; 80 % solvent content). Missing even a few reflection will probably have more of an impact at this lower than at higher resolution. Essentially, I am trying to obtain better signal in the resolution range 4-3.8 A, where there is also diffuse scattering from the solvent between 4 and 3 A and ice rings and the signal from the individual datasets is weak. Obviously the criterion will be the calculated map quality, but wanted to know what some experiences of people have been in such cases. Should I merge the datasets or rather use them individually for map calculations? 2) What's the quickest/easiest way to ensure equivalent indexing in ccp4/imosflm/scala, when merging different datasets together (space group P6222) (in XDS there is reference_data_set). Use pointless then cad+scala? Cheers, Florian
[ccp4bb] Effect of NCS on estimate of data:parameter ratio
Dear All, I would have a question regarding the effect of non-crystallographic symmetry (NCS) on the data:parameter ratio in refinement. I am working with X-ray data to a maximum resolution of 4.1-4.4 Angstroem, 79 % solvent content, in P6222 space group; with 22 300 unique reflections and expected 1132 amino acid residues in the asymmetric unit, proper 2-fold rotational NCS (SAD phased and no high- resolution molecular replacement or homology model available). Assuming refinement of x,y,z, B and a polyalanine model (i.e. ca. 5700 atoms), this would equal an observation:parameter ratio of roughly 1:1. This I think would be equivalent to a normal protein with 50 % solvent content, diffracting to better than 3 Angstroem resolution (from the statistics I could find, at that resolution a mean data:parameter ratio of ca. 0.9:1 can be expected for refinement of x,y,z, and individual isotropic B; ignoring bond angle/length geometrical restraints at the moment). My question is how I could factor in the 2-fold rotational NCS for the estimate of the observations, assuming tight NCS restraints (or even constraint). It is normally assumed NCS reduces the noise by a factor of the square root of the NCS order, but I would be more interested how much it adds on the observation side (used as a restraint) or reduction of the parameters (used as a constraint). I don't suppose it would be correct to assume that the 2-fold NCS would half the number of parameters to refine (assuming an NCS constraint)? Regards, Florian --- Florian Schmitzberger Biological Chemistry and Molecular Pharmacology Harvard Medical School 250 Longwood Avenue, SGM 130 Boston, MA 02115, US Tel: 001 617 432 5602
[ccp4bb] Problem with NCS detection in Parrot
Dear All, I am encountering a problem when using Parrot (for combined density modification and non crystallographic symmetry (NCS) averaging) in ccp4 6.1.13, run via ccp4i. Parrot does not detect the (2-fold) NCS present among my heavy atom substructure with 20 seleniums (the pdb was output by Phaser-EP, single chain ID, and is read by Parrot from what I can tell). I have tried a to split the NCS related heavy atoms into separate chains, but Parrot does still not appear to detect any NCS (error message: WARNING: No NCS found from heavy atoms). The Professs program seems to detect the NCS readily. Unfortunately, I don't think it is possible to input externally determined NCS operators into Parrot. Regards, Florian --- Florian Schmitzberger Biological Chemistry and Molecular Pharmacology Harvard Medical School 250 Longwood Avenue, SGM 130 Boston, MA 02115, US Tel: 001 617 432 5602
[ccp4bb] Format conversion of Shelx coordinate file
Dear All, What is currently the quickest/easiest way to convert a .hat file with fractional coordinates of heavy atoms generated by ShelxE to PDB format and/or a file format accepted by Sharp? I tried to use coordconv from ccp4, but it failed to make the conversion. Thank you. Regards, Florian --- Florian Schmitzberger Biological Chemistry and Molecular Pharmacology Harvard Medical School 250 Longwood Avenue, SGM 130 Boston, MA 02115, US Tel: 001 617 432 5602
[ccp4bb] Reindex and Rfree column
Dear All, I am wondering whether the Free_R column of an mtz file is altered by using Reindex (and/or Cad). The way I understand it, reindexing does not affect the Free_R column, correct? I have solved a structure with a reindexed dataset (P21212 from P22121). Now I have datasets of ligand-bound forms of the protein, which I would like to reindex (in the same way as the initial apo- datasets) and copy the Free_R from the initial dataset. What I am concerned about is whether I will get the same reflections Rfree flagged in the ligand-bound dataset, as I did not resort reflections with Cad after the reindexing of the initial apo- dataset. The procedure I chose now is to merge and scale, then reindex the ligand-datasets (with Reindex), and then copy the Rfree from the original dataset (with Uniqueify); (i.e. no resorting of the reflections after reindex). Perhaps, I should have sorted the reflections after the reindexing in both apo- and ligand datasets. Thank you very much in advance for any comments! Florian
[ccp4bb] Reindex and Rfree column
Dear All, I am wondering whether the Free_R column of an mtz file is altered by using Reindex (and/or Cad). The way I understand it, reindexing does not affect the Free_R column, correct? I have solved a structure with a reindexed dataset (P21212 from P22121). Now I have datasets of ligand-bound forms of the protein, which I would like to reindex (in the same way as the initial apo- datasets) and copy the Free_R from the initial dataset. What I am concerned about is whether I will get the same reflections Rfree flagged in the ligand-bound dataset, as I did not resort reflections with Cad after the reindexing of the initial apo- dataset. The procedure I chose now is to merge and scale, then reindex the ligand-datasets (with Reindex), and then copy the Rfree from the original dataset (with Uniqueify); (i.e. no resorting of the reflections after reindex). Perhaps, I should have sorted the reflections after the reindexing in both apo- and ligand datasets. Thank you very much in advance for any comments! Florian
[ccp4bb] arp/warp in p22121
Dear All, I am trying to build a molecular replacement model in arp/warp in space group P22121. Refmac alone seems to be fine with refining the model in P22121; but arp/warp fails, as far as I can see at the first Refmac refinement stage. In the log-file it says this space group is not supported. I am wondering whether arp/warp needs the Hermann-Mauguin convention space group P21212. I suppose I will need to reindex in P21212 in order to use arp/warp? (the diffraction data were indexed in XDS, scaled in SCALA, and then run through CAD to change the space group from P222 to P22121). I am using arp/warp via the ccp4i interface. Also, arp/warp gives the following message when I load the mtz file cannot extract arp/warp asymmetric unit limits, the job will fail if run. (i did run arp/warp successfully with other mtz-files). Thank you in advance for any comments! Florian
