Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!
The PDB_REDO entry for 3bdn was pretty old, so I replaced it using a newer version of PDB_REDO that can use nucleic acid restraints from LibG: http://www.cmbi.ru.nl/pdb_redo/bd/3bdn/index.html. Obviously, the new structure model is far from brilliant (PDB_REDO doesn't rebuild at this resolution), but Molprobity seems to like it quite a bit more. I agree with the replies so far in that: - The topic starter was rather blunt and could have been more subtle. He should probably go work in the Netherlands ;) - Building structure models at 3.9A is incredibly difficult. - The tools we have now are much better than in 2008. However, we should not act like 2008 were still in the dark ages of crystallography. There are a lot of good structures available from that time (and also from long before) even at that resolution. That is not surprising seeing that we also already had very good building and refinement tools available. We also had enough validation tools available to tell us that this particular structure model isn't very good. I really believe that a good crystallographer that was not pressed for time (or at least didn't rush) could have done better with the data and the tools available. I'm now going to hide behind an asbestos wall to say this: The manuscript was submitted in July 29th 2007, the PDB entry was deposited November 15th 2007. That means that the referees probably did not have a chance to see the finished structure model, at least not in the first pass. This implies that the authors didn't want to deposit the model on time. There are a whole lot of excuses for this, that are fortunately dealt with now (http://onlinelibrary.wiley.com/doi/10.1107/S0907444913029168/abstract), but the referees could have been a bit more critical. They should have at least seen that the supplemental table 1 did not show any Ramachandran statistics. We can only speculate what happened. I'm guessing that the authors didn't finish the structure yet and rushed the publication through to avoid being scooped or for the general glory of a Nature paper. To bad that came at the expense of the crystallography. Cheers, Robbie Date: Thu, 23 Apr 2015 18:43:13 + From: kell...@janelia.hhmi.org Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers! To: CCP4BB@JISCMAIL.AC.UK Is it in pdb redo? Take a look here: http://www.cmbi.ru.nl/pdb_redo/bd/3bdn/index.html JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Misbah ud Din Ahmad Sent: Thursday, April 23, 2015 2:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers! Dear Phoebe A. Rice, I didn't mean to discredit the work but the statistics of the structure just shocked me at the first instance. I could for example point out to another structure 1ZR2, which has the same resolution (the protein Molecular weight is almost the same) and the statistics are: R-work: 0.27 Rfree: 0.319 Ramachandran outliers: 2.8% The structure was solved by a combination of MIR and MAD three years earlier in 2005 and definitely they didn't had better softwares. As per your advice, I tried one cycle of refinement of this structure in Phenix and the statistics are: Start R-work = 0.2816 ; Start R-free = 0.3551 Final R-work = 0.2887 ; Final R-free = 0.3671 Ramachandran outliers: 18.91% Rotamer Outliers: 25.19% Being a crystallographic community, isn't it our responsibility that if better softwares are available now, we try to re-refine our older structures and deposit better models in the PDB. That would help the users immensely. Misbha On Thu, Apr 23, 2015 at 7:06 PM, Mark J van Raaij mjvanra...@cnb.csic.es wrote: The abstract of the papers says they used MIR. Mark J van Raaij 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 23 Apr 2015, at 18:57, Todd Jason Green wrote: My guess is they had the best data they could get, did molecular replacement with the two halves of the repressor and the dna, got a solution and didn't use appropriate restraints in the refinement. Like Phoebe mentioned, we have better tools for this these days. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Mark J van Raaij [mjvanra...@cnb.csic.es] Sent: Thursday, April 23, 2015 11:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers! How reliable is too general a question - it depends on what you want to know. At 3.9Å they could probably place the phosphate atoms quite well and see the general fold of the protein. Finer details will be less reliable, i.e. where the exact side-chains are etc. They could probably have forced more amino acids into favourable
Re: [ccp4bb] Maximising anomalous signal in helical/line scan
Dear William, at the ESRF we have a Helical characterisation workflow that will calculate a strategy based on the observed diffraction patterns and the distance between 2 points selected on a crystal, best wishes, Matt. On 2015-04-24 12:00, William Chao wrote: Dear all, Does anyone have experience in maximising anomalous signal during a helical/line scan? Most (all?) automated data collection strategies in synchrotrons seem to work on a single point of a crystal but don't give strategies for line scan. Would it be advisable to increase the recommended transmission as radiation is spread along the crystal? If the strategy suggests us to use 10% transmission (100% = 2x10^11 ph/s) for a 360-degree collection on a single point, to what amount should I increase say along a 200 micron scan with a 20 micron beam (crystal dimension 200x100x20)? Thank you very much in advance for the suggestions! William --- The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 215 Euston Road, London NW1 2BE. -- Matthew Bowler Synchrotron Diffraction Group European Molecular Biology Laboratory 71, avenue des Martyrs CS 90181 F-38042 GRENOBLE Cedex 9 FRANCE France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ ===
Re: [ccp4bb] Maximising anomalous signal in helical/line scan
Hi Matthew, William seems to want to increase the x-ray intensity gradually during the experiment by augmenting the transmission gradually. It that possible? If so, would it be advisable? Mark J van Raaij 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 24 Apr 2015, at 12:45, Matthew BOWLER wrote: Dear William, at the ESRF we have a Helical characterisation workflow that will calculate a strategy based on the observed diffraction patterns and the distance between 2 points selected on a crystal, best wishes, Matt. On 2015-04-24 12:00, William Chao wrote: Dear all, Does anyone have experience in maximising anomalous signal during a helical/line scan? Most (all?) automated data collection strategies in synchrotrons seem to work on a single point of a crystal but don't give strategies for line scan. Would it be advisable to increase the recommended transmission as radiation is spread along the crystal? If the strategy suggests us to use 10% transmission (100% = 2x10^11 ph/s) for a 360-degree collection on a single point, to what amount should I increase say along a 200 micron scan with a 20 micron beam (crystal dimension 200x100x20)? Thank you very much in advance for the suggestions! William --- The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 215 Euston Road, London NW1 2BE. -- Matthew Bowler Synchrotron Diffraction Group European Molecular Biology Laboratory 71, avenue des Martyrs CS 90181 F-38042 GRENOBLE Cedex 9 FRANCE France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ ===
[ccp4bb] aimless error
Hi, I try to run aimless (on OS10.6, CCP4v6.5.007) using as input a xds output. Aimless actually finishes (and scales fine), but then I get the error below and truncate does not start: --- /CCP4/S1_9_Kin_VSCFAKK7_6_correlplot.xmgr ROGUEPLOT /Users/CSAG/Structural/PROC/2015_04_19_ALBA/Kinase/SRM_1_9_Kinase_VSCFAKK7_d/CCP4/S1_9_Kin_VSCFAKK7_6_rogueplot.xmgr has failed with error message OMP: Warning #181: OMP_STACKSIZE: ignored because KMP_STACKSIZE has been defined *** #CCP4I TERMINATION STATUS 0 OMP: Warning #181: OMP_STACKSIZE: ignored because KMP_STACKSIZE has been defined #CCP4I TERMINATION TIME 24 Apr 2015 12:20:54 #CCP4I TERMINATION OUTPUT_FILES /Users/CSAG/Structural/PROC/2015_04_19_ALBA/TEST/S1_9_Kin_VSCFAKK7_6_2_mtz.tmp TEMPORARY #CCP4I MESSAGE Task failed --- Indeed, I do have the KMP_STACKSIZE defined to 8m in my .cshrc file (for XDS) but even if I don’t I get the same error. Strangely, if I run aimless with the same input file and options on another computer (OS10.8, CCP4 v6.5.006) it finishes fine without error. Any help to prevent the error is greatly appreciated. Thanks in advance, Daniel
Re: [ccp4bb] Maximising anomalous signal in helical/line scan
There's some discussion of this in here: http://journals.iucr.org/d/issues/2013/07/00/ba5199/index.html section 6.5. But basically you figure out how much dose a volume can take, and then change collection parameters so that each volume gets that full dose. The strategy is just one way to quantify the dose; I personally like to convert things back to unattenuated seconds, i.e. how many seconds will the crystal survive with the unattenuated beam (in your case, anomalous signal rather than crystal). phx On 24/04/2015 11:00, William Chao wrote: Dear all, Does anyone have experience in maximising anomalous signal during a helical/line scan? Most (all?) automated data collection strategies in synchrotrons seem to work on a single point of a crystal but don't give strategies for line scan. Would it be advisable to increase the recommended transmission as radiation is spread along the crystal? If the strategy suggests us to use 10% transmission (100% = 2x10^11 ph/s) for a 360-degree collection on a single point, to what amount should I increase say along a 200 micron scan with a 20 micron beam (crystal dimension 200x100x20)? Thank you very much in advance for the suggestions! William --- The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 215 Euston Road, London NW1 2BE.
Re: [ccp4bb] Thin plate crystals
Lysine-methylation has proved to be helpful to change the molecular packing in protein crystals (i.e. you might obtain a completely different crystal form). _Gang From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Prerana G. Sent: Friday, April 24, 2015 5:01 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Thin plate crystals Dear all, I am working on a protein (40kDa) which forms very thin plate shaped crystals which diffracts at very low resolution. Protein concentration that i have used for crystallisation is approx. 8mg/ml. I have attached the picture of the protein crystal. How can I improve upon the shape of the crystal?
Re: [ccp4bb] Cleaved peptide density!
Hi all, late to the discussion as usual... Related to the issue of why does my single point mutant appear to have residual activity, what do people here make of this warning from 1989? http://pubs.acs.org/doi/pdf/10.1021/ar00163a001 Some highlights: Substitution of the Ser68 codon with a codon for glycine (Gly) yields a beta-lactamase which has a low but reproducible activity that is about 0.1% of that of the wild-type enzyme The underlying assumption in all experiments of the sort described above is that knowledge of the nucleotide sequence of the gene for a protein is tantamount to knowledge of the amino acid sequence. There is ample evidence to support this assumption. However, there is also much evidence that the biochemical machinery which converts DNA sequences into proteins makes occasional mistakes. These can be errors in transcription and in translation, although the latter are probably more common. These mistakes can lead to microscopic heterogeneity of the proteins which are produced from a fixed DNA sequence, with the protein that is specified by the gene mixed with minute amounts of sequence variants. A variant produced by an error of translation, for example, can have an intrinsic activity that is different from that of the predominant species. This is the likely explanation for the putative activity of a beta-lactamase Gly68 mutant which turns out to have no measurable activity whatsoever. Because of the degeneracy of the genetic code, most amino acids have more than one codon. The glycine codons are GCX, where X is A, G, C, or U. When the Ser68 AGC codon is changed to GGC, a low beta-lactamase activity is observed, as described above. When AGC is replaced instead by GGA, however, the mutant beta-lactamase is made in the same amount, but it has no detectable activity. Thus, the Gly68 mutant protein per se has no inherent activity. Instead, activity is correlated with the presence in the gene of a specific codon for glycine. The probable explanation is that serine is occasionally inserted instead of glycine at the GGC codon (at position 68) of the messenger RNA that is transcribed from the mutant gene. I don't think that is the case here, as there is no obvious path from Ala codons back to Cys codons (although Ser or Thr would be possible). This is meant as a more general question of particular interest in crystallography as we like to co-crystallize our enzymes in tiny solutions at several mg/ml for several weeks, which is akin to asking 0.1% activities to show their ugly heads. Cheers, Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dkmailto:josea.cuesta.se...@carlsberglab.dk From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Monday, April 20, 2015 8:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cleaved peptide density! Dear Crystallographers, I am working with a cysteine protease. I co-crystallized the protease with some small chemically synthesised peptides of 7 amino acid residues. I mutated the active Cysteine residue with Alanine to avoid the peptide cleavage so that I can get the whole peptide bound with my protein. But interesting I got the density for a cleaved peptide with 4 amino acids instead of the whole peptide. The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred exactly at the same peptide bond where it should. But I do not know how! Now my question is why I am getting the cleaved peptide as I already mutated the active residue Cysteine with Alanine (this mutant did no show any activity when I checked with SDS-PAGE). If anybody has the same kind of experience please advice me. Thanks in advance. Best, Dipankar -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
[ccp4bb] Maximising anomalous signal in helical/line scan
Dear all, Does anyone have experience in maximising anomalous signal during a helical/line scan? Most (all?) automated data collection strategies in synchrotrons seem to work on a single point of a crystal but don't give strategies for line scan. Would it be advisable to increase the recommended transmission as radiation is spread along the crystal? If the strategy suggests us to use 10% transmission (100% = 2x10^11 ph/s) for a 360-degree collection on a single point, to what amount should I increase say along a 200 micron scan with a 20 micron beam (crystal dimension 200x100x20)? Thank you very much in advance for the suggestions! William --- The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 215 Euston Road, London NW1 2BE.
[ccp4bb] structural biology postdoc position at the Wellcome Trust Centre for Cell Biology
Applications are invited for a Postdoctoral Research Associate in the laboratory of Dr. Julie Welburn, funded by Cancer Research UK. The laboratory investigates fundamental mechanisms of chromosome segregation at the molecular and cellular level. The focus of our laboratory is the function and properties of microtubules in mitosis and how microtubule-binding proteins, motors and signaling molecules can utilize and modify the dynamics of microtubule subpopulations. Our laboratory use biochemical and structural approaches on mitotic complexes and molecular motors to understand their contribution to the regulation of microtubule dynamics and chromosome segregation. I am now looking for an enthusiastic and talented researcher to join our team investigating the molecular mechanisms that control microtubule-based processes in mitosis, focusing in particular on kinesins. You should have, or will shortly obtain, a PhD in a relevant subject area and will have experience in structural biology or/and biochemistry. An outstanding academic track record is essential and at least one first author publication in a prominent journal is expected. Interested candidates should email their CV and a summary of research experience/accomplishments to Julie Welburn (julie.welb...@ed.ac.uk) My lab website is: http://welburn.bio.ed.ac.uk Dr. Julie Welburn CRUK Career Development Fellow Wellcome Trust Centre for Cell Biology University of Edinburgh Mayfield Road Edinburgh EH9 3JR julie.welb...@ed.ac.uk 0131 650 7778 The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.
[ccp4bb] MRC-funded PhD studenthip at the University of Leicester
Dear All, An MRC-funded PhD position (starting in September 2015) is available in my group at the University of Leicester (UK) to target protein-protein interactions in poor prognosis T-cell lymphomas, in collaboration with Profs Simon Wagner and Richard Bayliss. This is an exciting opportunity to work within the newly established Cancer Research UK Centre Leicester that partners with the University of Leicester and the Leicester NHS Hospitals to enhance translational research. Applications are invited from talented graduates who hold or expect to gain a first or upper second class honours degree or equivalent, or a Masters degree, in a relevant subject. Successful candidates will undertake a four-year PhD studentship under the guidance of a team of structural biologists and oncologists. How to apply: Full details about this PhD studentship project, the application guidelines and the application form, are available on our website at: http://www2.le.ac.uk/study/research/funding/biochemistry. Informal project enquiries can be addressed to Dr A. Echalier (aude.echal...@le.ac.uk) Application deadline: Friday 15th May 2015 With best wishes, Aude - Aude Echalier, Ph.D. Lecturer in Structural Biology Departments of Biochemistry and of Cancer Studies, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester, LE1 9HN, UK T. +44(0)116 229 7120 E. aude.echal...@le.ac.uk [https://email.le.ac.uk/owa/attachment.ashx?id=RgBM4xSId5YtSpz6cDaVIIwsBwA%2bfW0enQplRo0dZMAx9BCyq2kCAAA%2bfW0enQplRo0dZMAx9BCyq47YAAAJattcnt=1attid0=BAAAattcid0=image001.jpg%4001CFA4D7.0C95C6E0] [CRUKcentre] Elite without being elitist Follow us on Twitterhttps://twitter.com/uniofleicester or visit our Facebookhttp://www.facebook.com/uniofleicester page
Re: [ccp4bb] Maximising anomalous signal in helical/line scan
Hi Mark, I think William was asking by how much he could increase the transmission by given a certain crystal size - the rough answer being he could increase to 100% as he has ~10 positions given the beam and crystal size... But a proper calculation is always better and of course one has to assume that a crystal diffracts homogeneously, which is not always the case. To answer your question the transmission could be changed if the collection is multiple position and not a continuous scan (ie the crystal is not translated during a wedge). There are certain conditions where this is advantageous and Sasha Popov's BEST strategy calculation program has options to do this Best wishes, Matt. On 2015-04-24 13:18, Mark J van Raaij wrote: Hi Matthew, William seems to want to increase the x-ray intensity gradually during the experiment by augmenting the transmission gradually. It that possible? If so, would it be advisable? Mark J van Raaij 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 24 Apr 2015, at 12:45, Matthew BOWLER wrote: Dear William, at the ESRF we have a Helical characterisation workflow that will calculate a strategy based on the observed diffraction patterns and the distance between 2 points selected on a crystal, best wishes, Matt. On 2015-04-24 12:00, William Chao wrote: Dear all, Does anyone have experience in maximising anomalous signal during a helical/line scan? Most (all?) automated data collection strategies in synchrotrons seem to work on a single point of a crystal but don't give strategies for line scan. Would it be advisable to increase the recommended transmission as radiation is spread along the crystal? If the strategy suggests us to use 10% transmission (100% = 2x10^11 ph/s) for a 360-degree collection on a single point, to what amount should I increase say along a 200 micron scan with a 20 micron beam (crystal dimension 200x100x20)? Thank you very much in advance for the suggestions! William --- The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 215 Euston Road, London NW1 2BE. -- Matthew Bowler Synchrotron Diffraction Group European Molecular Biology Laboratory 71, avenue des Martyrs CS 90181 F-38042 GRENOBLE Cedex 9 FRANCE France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ === -- Matthew Bowler Synchrotron Diffraction Group European Molecular Biology Laboratory 71, avenue des Martyrs CS 90181 F-38042 GRENOBLE Cedex 9 FRANCE France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ ===
[ccp4bb] Fwd: [ccp4bb] Structural classification
Dear Deepa, When classifying structures you need to first ask why?. For example, if you want to get an idea about evolutionary relations, you must try to find their DNA sequences and analyse those in the light of a multiple sequence alignment (MSA) that follows the protein alignment. In pharma you might want to superpose them based on just the residues around the pocket of interest. If interested in regulatory processes, you map the regulation-related sequence motifs on a protein sequence MSA (that might be supported by a multiple structure superposition). Your question contains insufficient detail for better answers. One question, though: What is wrong with DALI giving you 50-60 groups? You can combine groups in bigger groups, cann't you? If something crashes on 300+ structures then make a phylogenetic tree first (based on protein sequences) and remove doubles or near-doubles till you have less then 300 structures. Proteins that are 95% sequence identical normally are 'the same anyway'. Gert Forwarded Message Subject:[ccp4bb] Structural classification Date: Fri, 24 Apr 2015 13:36:42 +0100 From: Deepa Raju deepakmraj...@gmail.commailto:deepakmraj...@gmail.com Reply-To: Deepa Raju deepakmraj...@gmail.commailto:deepakmraj...@gmail.com To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Dear all, I am Deepa, MSc final year student. For my final year project, I am working on protein structure classification. I have a set of 300 methyltransferase protein structures. I wish to classify the proteins in to different groups based on their secondary structures and sequence. I have tried to classify the proteins based on sequence but it is not satisfactory in view point of structures. I have used the DALI server results like Z-score, RMS deviation for grouping. I ended up with 50-60 groups. I have used the PIRSF, but in that most of the structures are not available. I have also used Chimera - structure based sequence alignment, but the program is crashing for 300 structures. Any suggestions will be highly beneficial to me. Thank you in advance. With regards Deepa Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The Radboud university medical center is listed in the Commercial Register of the Chamber of Commerce under file number 41055629.
Re: [ccp4bb] Thin plate crystals
Following up on Dave's suggestion, you could try using crystal screens as additives. This has worked well in my lab. The idea is to mix the condition that you currently have (the base) with all the crystal screen reagents you have in stock (CS, Index, etc.). As a first trial, use reservoirs containing 3 parts base and 1 part screen. This generates a new matrix of hits. You might find a condition that produces thicker crystals or perhaps a new new crystal form. I think this approach is described in the literature but don't remember the citation. Jack On Apr 24, 2015, at 12:31 AM, David Briggs wrote: Hi, In my experience, additive screens (e.g Hampton's) can change crystal morphology. You could also re-screen for new conditions either using matrix micro seeding, or change the protein buffer. Perhaps adding a ligand or a component from your current crystallisation conditions to your protein stock? HTH, Dave On Fri, 24 Apr 2015 04:02 Prerana G. tracy...@gmail.commailto:tracy...@gmail.com wrote: Dear all, I am working on a protein (40kDa) which forms very thin plate shaped crystals which diffracts at very low resolution. Protein concentration that i have used for crystallisation is approx. 8mg/ml. I have attached the picture of the protein crystal. How can I improve upon the shape of the crystal? John J. Tanner, PhD Professor of Biochemistry and Director of Graduate Admissions and Recruitment Professor of Chemistry (Joint Appointment) University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 email: tanne...@missouri.edumailto:tanne...@missouri.edu phone: 573-884-1280 fax: 573-882-2754 http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html
[ccp4bb] Structural classification
Dear all, I am Deepa, MSc final year student. For my final year project, I am working on protein structure classification. I have a set of 300 methyltransferase protein structures. I wish to classify the proteins in to different groups based on their secondary structures and sequence. I have tried to classify the proteins based on sequence but it is not satisfactory in view point of structures. I have used the DALI server results like Z-score, RMS deviation for grouping. I ended up with 50-60 groups. I have used the PIRSF, but in that most of the structures are not available. I have also used Chimera - structure based sequence alignment, but the program is crashing for 300 structures. Any suggestions will be highly beneficial to me. Thank you in advance. With regards Deepa
Re: [ccp4bb] Thin plate crystals
Along those lines suggested by David I would say these crystals are way too big. Try freezing some when they are smaller. It would also be worth trying to freeze in meshes to support the fragile plate instead of conventional loops. J?rgen .. J?rgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Streetx-apple-data-detectors://4, W8708 Baltimore, MD 21205x-apple-data-detectors://5/0 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.eduhttp://lupo.jhsph.edu/ On Apr 24, 2015, at 03:52, Hargreaves, David david.hargrea...@astrazeneca.commailto:david.hargrea...@astrazeneca.com wrote: The crystals don't look that bad in my opinion. Maybe the cryo step is sub optimal? Try using large loops (reduces surface tension forces during transfer). Butane 2,3 diol is a good cryo to try. Maybe shoot them at room temp (in situ) to get an idea of how they diffract before you manipulate them. Good luck! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Prerana G. Sent: 24 April 2015 04:01 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Thin plate crystals Dear all, I am working on a protein (40kDa) which forms very thin plate shaped crystals which diffracts at very low resolution. Protein concentration that i have used for crystallisation is approx. 8mg/ml. I have attached the picture of the protein crystal. How can I improve upon the shape of the crystal? AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
Re: [ccp4bb] Lipid molecule
You could try an LLG anomalous map from Phaser as well to clarify the matter: in my experience, even sulfurs are found, and your Rb should have f” ~0.75 even at 1 Ang xrays. Also, you could refine in Refmac including anomalous data, and refine occupancy of Rb. Having both modeled into the data seems fine to me, and I think fairly common. Just set occupancies accordingly. Incidentally, it looks to me like a weird place for a cation to dwell, but if the data support it… JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jarrod Mousa Sent: Friday, April 24, 2015 9:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Lipid molecule Hi all, Picture attached. I solved the structure of a membrane protein using LCP. When trying to identify the cation-binding site, I co-crystallized with Rb+. I see strong density corresponding to Rb+, and it was confirmed in an isomorphous difference map. One problem: in the native structure I see strong density for a lipid molecule from the LCP, in which I can model in very well. In the Rb+ structure, I see more of a blob of density at low rmsd, but when at higher the spherical Rb begins to show. Based on the isomorphous differnece map showing nice spherical denstiy for Rb+, it seems the crystal I shot contains protein molecules with the lipid and others with Rb+. I currently have Rb+ modeled in, but there is lots of extra density surrounding it, corresponding to the lipid molecule. Should I put both molecules in the structure, even though it doesn't make much sense and would results in odd lipid-cation clashes? Or should I just leave the Rb+ without lipid since this makes sense chemistry-wise? Thanks, Jarrod - Jarrod J. Mousa, Ph.D. Bruner Laboratory Department of Chemistry University of Florida email: mo...@chem.ufl.edumailto:mo...@chem.ufl.edu
[ccp4bb] Lipid molecule
Hi all, Picture attached. I solved the structure of a membrane protein using LCP. When trying to identify the cation-binding site, I co-crystallized with Rb+. I see strong density corresponding to Rb+, and it was confirmed in an isomorphous difference map. One problem: in the native structure I see strong density for a lipid molecule from the LCP, in which I can model in very well. In the Rb+ structure, I see more of a blob of density at low rmsd, but when at higher the spherical Rb begins to show. Based on the isomorphous differnece map showing nice spherical denstiy for Rb+, it seems the crystal I shot contains protein molecules with the lipid and others with Rb+. I currently have Rb+ modeled in, but there is lots of extra density surrounding it, corresponding to the lipid molecule. Should I put both molecules in the structure, even though it doesn't make much sense and would results in odd lipid-cation clashes? Or should I just leave the Rb+ without lipid since this makes sense chemistry-wise? Thanks, Jarrod - Jarrod J. Mousa, Ph.D. Bruner Laboratory Department of Chemistry University of Florida email: mo...@chem.ufl.edu
[ccp4bb] Post doctoral position in molecular membrane biology
Dear all, the lab of Hartmut Michel at the Max Planck Institute of Biophysics has an opening for a postdoctoral scientist. For more informations see: http://www.biophys.mpg.de/en/open-positions/postdocs.html Best wishes Julia _ Dr. Julia Preu Department of Molecular Membrane Biology Max Planck Institute of Biophysics Max-von-Laue-Strasse 3 60438 Frankfurt am Main Germany office: +49 69/6303-1048 fax:+49 69/6303-1002 julia.p...@biophys.mpg.de
[ccp4bb] PDBcur fails with error message
Dear All, Here is the CCP4 version 6.5 installed, where I want to remove the ANISOU atoms from the pdb file, but its shows failed job with an error message : has failed with error message child process exited abnormally So I am not able to figure it out, how to rectify. Please suggest. Thank you Regards Jeorge
[ccp4bb] anisotropic data F(+)_ISOB, F(-)_ISOB output
Hi all, I have an anisotropic native data sets with heavy atoms and I want to use the anomalous signal for the final refinement. Does anybody know how to run the anisotropic sharpening and output the F(+)_ISOB, F(-)_ISOB? It seems both the anisotropy server (UCLA) and phaser_anisotropy analysis can only output F_ISOB. Thank you Jiangtao Guo UT Southwestern Medical Center
Re: [ccp4bb] PDBcur fails with error message
Hi Jeorge: My Luddite approach to such things: grep -v ANISOU original.pdbiso_only.pdb HTH, Bill William G. Scott http://scottlab.ucsc.edu/~wgscott On Apr 24, 2015, at 8:54 AM, jeorgemarley thomas kirtswab...@gmail.com wrote: Dear All, Here is the CCP4 version 6.5 installed, where I want to remove the ANISOU atoms from the pdb file, but its shows failed job with an error message : has failed with error message child process exited abnormally So I am not able to figure it out, how to rectify. Please suggest. Thank you Regards Jeorge
Re: [ccp4bb] Postdoctoral position at Harvard Medical School
A *post-doctoral position* is available to study the molecular mechanism for self vs. non-self discrimination by the innate immune system. We use a combination of X-ray crystallography, biochemistry, cell biology and virology to characterize structures and functions of key host molecules that recognize foreign molecules, in particular viral nucleic acids. Applicants should have received (or expect to receive) a PhD and have a strong background in X-ray crystallography, biochemistry, cellular immunology or virology. PhD in multi-disciplinary, mechanistic research is strongly favored. Please send a cover letter, CV, names, e-mail addresses and telephone numbers of three references to Sun Hur at sun@childrens.harvard.edu Please refer to http://hurlab.tch.harvard.edu/ and http://hurlab.tch.harvard.edu/publications/ to learn more about our research. On Tue, Apr 1, 2014 at 11:35 AM, Sun Hur sun.j...@gmail.com wrote: A *post-doctoral position* is available to study the molecular mechanism for self vs. non-self discrimination by the innate immune system. We use a combination of X-ray crystallography, biochemistry and cell biology to characterize structures and functions of key host molecules that recognize foreign molecules, in particular viral nucleic acids. Applicants should have received (or expect to receive) a PhD and have a strong background in X-ray crystallography, biochemistry, cellular immunology or virology. Please send a cover letter, CV, names, e-mail addresses and telephone numbers of three references to Sun Hur at sun@childrens.harvard.edu Please refer to http://hurlab.tch.harvard.edu/ and http://hurlab.tch.harvard.edu/publications/ to learn more about our research.
Re: [ccp4bb] Thin plate crystals
There have been quite a few reports that the addition of a low concentration of alcohol (e.g. isopropanol, a few %) can help to increase the thickness of thin plate crystals like these. However, these are probably in the Hampton additive screen that David Briggs suggested. Good luck, Andrew On 24 Apr 2015, at 04:01, Prerana G. tracy...@gmail.com wrote: Dear all, I am working on a protein (40kDa) which forms very thin plate shaped crystals which diffracts at very low resolution. Protein concentration that i have used for crystallisation is approx. 8mg/ml. I have attached the picture of the protein crystal. How can I improve upon the shape of the crystal? Crystal_1.jpg
Re: [ccp4bb] Thin plate crystals
The crystals don't look that bad in my opinion. Maybe the cryo step is sub optimal? Try using large loops (reduces surface tension forces during transfer). Butane 2,3 diol is a good cryo to try. Maybe shoot them at room temp (in situ) to get an idea of how they diffract before you manipulate them. Good luck! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Prerana G. Sent: 24 April 2015 04:01 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Thin plate crystals Dear all, I am working on a protein (40kDa) which forms very thin plate shaped crystals which diffracts at very low resolution. Protein concentration that i have used for crystallisation is approx. 8mg/ml. I have attached the picture of the protein crystal. How can I improve upon the shape of the crystal? AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
