[ccp4bb] AW: [ccp4bb] twinning fun
Dear Bert, The first thing I would do is to calculate the Matthews number: Does at least one monomer fit in the P622 asymmetric unit? If not, your crystals are definitively twinned. As mentioned below, I would also check the I^2/I^2 ratio, but I would do it with the data processed in P6, since processing true P6 data in P622 will produce a twinned ratio even when the P6 data was not twinned. If it turns out, that some crystals are twinned and others not, I would look at the diffraction patterns to see if something funny is going on (ice rings, high background, strange spot shape etc.). In this case, I would try to solve the structure with untwinned crystals. Maybe less fun, but also less hassle, frustration and cleaner maps. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dirk Kostrewa Gesendet: Dienstag, 28. Januar 2014 22:01 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] twinning fun Dear Bert Van-Den-Berg, as far as I understand this, if you have true P622, process the data in P6 and then test for twinning, both the Britton-test and H-test will indicate perfect merohedral twinning. This is because the Britton-test checks for a sudden increase of negative intensities after de-twinning, which happens only at twin fractions close to 0.5 if the intensities used for de-twinning are the same. But this is true if they are related by crystallographic symmetry. The H-test relates the absolute difference to the sum of the presumably twinned intensities, which gives 0 for intensities related by crystallographic symmetry, again resulting in twin fractions close to 0.5. In other words, intensities related by crystallographic symmetry would indicate perfect twinning in both of these tests. A better test for perfect merohedral twinning would be the ratio of I^2/I^2 which should be 2 for untwinned and 1.5 for perfectly twinned data, tested in the higher space group. These values are reported by data processing programs like XDS. Please, be aware that these ratios have rather strange values if you have an unusually high background (loop fiber diffraction, ice rings, etc.) or extremely weak data. For a really good discussion of twin tests, see Yeates, Methods. Enzymol. 276, 344-358, 1997. Best regards, Dirk. Am 28.01.14 18:26, schrieb Bert Van-Den-Berg: Dear all, I recently collected several datasets for a protein that needs experimental phasing. The crystals are hexagonal plates, and (automatic) data processing suggests with high confidence that the space group is P622. This is where the fun begins. For some datasets (processed in P622), the intensity distributions are normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is no twinning (twinning fractions 0.05). However, for other datasets (same cell dimensions), the intensity distributions are not normal (eg Z-scores 10). Given that twinning is not possible in P622, this suggests to me that the real space group could be P6 with (near) perfect twinning. If I now process the normal L-test P622 datasets in P6, the twin-law based tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), suggesting all my data is twinned. Does this make sense (ie can one have twinning with normal intensity distributions)? If it does, would the normal L-test datasets have a higher probability of being solvable? Is there any strategy for experimental phasing of (near) perfect twins? SAD would be more suitable than SIR/MIR? (I also have potential heavy atom derivatives). Thanks for any insights! Bert -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.demailto:kostr...@genzentrum.lmu.de WWW: www.genzentrum.lmu.dehttp://www.genzentrum.lmu.de ***
Re: [ccp4bb] twinning fun
Dear Bert, as Dirk has pointed out, if P622 is the correct space group, then the twinning statistics printed out if you process in P6 are meaningless. Intensity statistics, like the ratio of I^2/I^2 , can be misleading if there is (e.g. pseudo-translational) NCS in the crystal; however, the effect of NCS on the value of the ratio of I^2/I^2 is opposite to that of twinning. Thus if a crystal is twinned and has NCS, you might not notice any problem in the ratio of I^2/I^2 . The other statistics, like Britton and H-test, present the intensity statistics in a different way, but from my understanding do not give substantially different information. The L-test does look at a different kind of information and therefore gives additional insight. If your measurements suffer from high background, diffuse scatter, ice rings, smeared reflections, additional crystals in the beam, or any other pathology, then all these tests may give distorted answers. In other words, even if twinning is not really present, any test designed to convert the deviation of data from ideality into an estimate of the twinning fraction will give you an alpha 0. So my experience is: if your data are very good, then the tests give good answers; if the data are mediocre or bad, don't necessarily believe the numbers. Finally, it's not only twinning of P6 that would give you P622, it's also twinning of P3x21, P3x12 that gives P6y22. Hope this helps, Kay On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: Dear all, I recently collected several datasets for a protein that needs experimental phasing. The crystals are hexagonal plates, and (automatic) data processing suggests with high confidence that the space group is P622. This is where the fun begins. For some datasets (processed in P622), the intensity distributions are normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is no twinning (twinning fractions 0.05). However, for other datasets (same cell dimensions), the intensity distributions are not normal (eg Z-scores 10). Given that twinning is not possible in P622, this suggests to me that the real space group could be P6 with (near) perfect twinning. If I now process the normal L-test P622 datasets in P6, the twin-law based tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), suggesting all my data is twinned. Does this make sense (ie can one have twinning with normal intensity distributions)? If it does, would the normal L-test datasets have a higher probability of being solvable? Is there any strategy for experimental phasing of (near) perfect twins? SAD would be more suitable than SIR/MIR? (I also have potential heavy atom derivatives). Thanks for any insights! Bert
Re: [ccp4bb] Examples of multiple ASU copies with different conformations
Hi Shane, some crystal forms of trimeric AcrB (a multi-drug resistance secondary transporter) have 3 (or 6) monomers in the ASU and these are substantially different, which suggests how the protein functions. One reference is e.g. Seeger et al. (2006) Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism Science 313, 1295-1298 DOI: 10.1126/science.1131542 (sorry for the self-plug!) best, Kay On Mon, 27 Jan 2014 13:08:33 -0500, Shane Caldwell shane.caldwel...@gmail.com wrote: Hi ccp4bb, I'm putting together a talk for some peers that highlights strengths and weaknesses of structural models for the outsider. For one point, I'd like to find some examples of proteins that show very different conformations between different copies in the ASU. One example I know of is c-Abl (1OPL), which crystallizes with both autoinhibited and active forms in the ASU, with dramatically different domain organization. I'd like to find some additional examples - can anyone suggest some other structures that have multiple copies with large structural variations? Thanks in advance! Shane Caldwell McGill University
Re: [ccp4bb] Examples of multiple ASU copies with different conformations
On 27 Jan 2014, at 19:08, Shane Caldwell shane.caldwel...@gmail.com wrote: Here is another interesting one: Perez, C., Koshy, C., Yildiz, Ö., Ziegler, C. (2012). Alternating-access mechanism in conformationally asymmetric trimers of the betaine transporter BetP. Nature. best wishes, Tobias Hi ccp4bb, I'm putting together a talk for some peers that highlights strengths and weaknesses of structural models for the outsider. For one point, I'd like to find some examples of proteins that show very different conformations between different copies in the ASU. One example I know of is c-Abl (1OPL), which crystallizes with both autoinhibited and active forms in the ASU, with dramatically different domain organization. I'd like to find some additional examples - can anyone suggest some other structures that have multiple copies with large structural variations? Thanks in advance! Shane Caldwell McGill University
Re: [ccp4bb] refining nucleic acids with Coot
For reference, the CCP4 tool is called 'pdb2to3' -- David On 14 January 2014 18:32, Almudena Ponce Salvatierra maps.fa...@gmail.comwrote: Thank you all very much for your very helpful advices! I managed to solve my problem! :-) Best, Almudena. 2014/1/14 Ashley Pike ashley.p...@sgc.ox.ac.uk Hi Almudena - I came across this issue recently as well with an old pdb file. As Paul says you need to convert from v2.3 to v3.2 pdb formats. In addition to the link he gave, you can also accomplish this using the iotbx toolbox that comes with phenix. iotbx.pdb_remediator file_name=in.pdb output_file=out.pdb will rename * atom names to ' (eg O4* to O4') which can then be regularised in coot. Ashley *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Almudena Ponce Salvatierra *Sent:* 14 January 2014 15:52 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] refining nucleic acids with Coot Dear all, I am trying to refine an oligonucleotide chain in Coot and I get the following message when I try: Fail to match (to the dictionary) the following model atom names: G O5* C5* O4* etc That would cause exploding atoms so the refinement did not start Does anyone know how to solve this? Best wishes, Almudena. -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] twinning fun
Try looking into tetartohedral twinning as well--I think I may have such a crystal, and it's tough going. And as Kay pointed out, try the various P3's. Since I have not yet been successful in figuring my similar case out, what do people on the list recommend as an approach to figuring this out--just trying every possible space group with various parameters? I would think there should be some actual advantages at the phasing step to having twins, such as a sort of NCS of intensities rather than amplitudes, weighted by twin fraction, but it doesn't seem that any software uses this. Perhaps there is a reason for that? A paper on tetartohedral twinning I saw: Acta Crystallogr D Biol Crystallogr. 2012 Apr;68(Pt 4):418-24. doi: 10.1107/S0907444912006737. Epub 2012 Mar 16. Tetartohedral twinning could happen to you too. Roversi P, Blanc E, Johnson S, Lea SM. Author information Abstract Tetartohedral crystal twinning is discussed as a particular case of (pseudo)merohedral twinning when the number of twinned domains is four. Tetartohedrally twinned crystals often possess pseudosymmetry, with the rotational part of the pseudosymmetry operators coinciding with the twinning operators. Tetartohedrally twinned structures from the literature are reviewed and the recent structure determination of tetartohedrally twinned triclinic crystals of human complement factor I is discussed. PMID: 22505261 [PubMed - indexed for MEDLINE] PMCID: PMC3322600 Free PMC Article JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay Diederichs Sent: Wednesday, January 29, 2014 4:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] twinning fun Dear Bert, as Dirk has pointed out, if P622 is the correct space group, then the twinning statistics printed out if you process in P6 are meaningless. Intensity statistics, like the ratio of I^2/I^2 , can be misleading if there is (e.g. pseudo-translational) NCS in the crystal; however, the effect of NCS on the value of the ratio of I^2/I^2 is opposite to that of twinning. Thus if a crystal is twinned and has NCS, you might not notice any problem in the ratio of I^2/I^2 . The other statistics, like Britton and H-test, present the intensity statistics in a different way, but from my understanding do not give substantially different information. The L-test does look at a different kind of information and therefore gives additional insight. If your measurements suffer from high background, diffuse scatter, ice rings, smeared reflections, additional crystals in the beam, or any other pathology, then all these tests may give distorted answers. In other words, even if twinning is not really present, any test designed to convert the deviation of data from ideality into an estimate of the twinning fraction will give you an alpha 0. So my experience is: if your data are very good, then the tests give good answers; if the data are mediocre or bad, don't necessarily believe the numbers. Finally, it's not only twinning of P6 that would give you P622, it's also twinning of P3x21, P3x12 that gives P6y22. Hope this helps, Kay On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: Dear all, I recently collected several datasets for a protein that needs experimental phasing. The crystals are hexagonal plates, and (automatic) data processing suggests with high confidence that the space group is P622. This is where the fun begins. For some datasets (processed in P622), the intensity distributions are normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is no twinning (twinning fractions 0.05). However, for other datasets (same cell dimensions), the intensity distributions are not normal (eg Z-scores 10). Given that twinning is not possible in P622, this suggests to me that the real space group could be P6 with (near) perfect twinning. If I now process the normal L-test P622 datasets in P6, the twin-law based tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), suggesting all my data is twinned. Does this make sense (ie can one have twinning with normal intensity distributions)? If it does, would the normal L-test datasets have a higher probability of being solvable? Is there any strategy for experimental phasing of (near) perfect twins? SAD would be more suitable than SIR/MIR? (I also have potential heavy atom derivatives). Thanks for any insights! Bert
Re: [ccp4bb] twinning fun
Dont forget that with twinning in apparent point group PG6/mmm the true SG may be P6i or P3i21 See the twinning notes: http://www.ccp4.ac.uk/dist/html/twinning.html Detecting twinning can be problematic - My rule of thumb, following the procedure od ctruncate:: 0) Check the matthews coefficient for likely number of molecules. Half a molecule must mean you are assigning too high a symmetry count. Lots of molecules means you need to check for non-crystallographic translation etc. 1) Look at the I^2/I^2 plot after correction for anisotropy If it isnt reasonably straight with resolution you probably have some data problems, and these can make all the tests pretty useless. 2) Is there a NC translation - truncate tells you that. If not, and the data is OK, you are unlikely to have twinning if I^2/I^2 for acentrics is ~ 2, and the L test looks OK. H test and Britten tests a bit more influenced by other NC symmetry considerations 3) If there IS NC translation I^2/I^2 for acentrics will probably be 2 but the L test is still pretty reliable. Good luck Eleanor experimental phasing is tricky with perfect twinning but it has been done. Sorry I have forgotten reference though.. Eleanor On 29 January 2014 09:17, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: Dear Bert, as Dirk has pointed out, if P622 is the correct space group, then the twinning statistics printed out if you process in P6 are meaningless. Intensity statistics, like the ratio of I^2/I^2 , can be misleading if there is (e.g. pseudo-translational) NCS in the crystal; however, the effect of NCS on the value of the ratio of I^2/I^2 is opposite to that of twinning. Thus if a crystal is twinned and has NCS, you might not notice any problem in the ratio of I^2/I^2 . The other statistics, like Britton and H-test, present the intensity statistics in a different way, but from my understanding do not give substantially different information. The L-test does look at a different kind of information and therefore gives additional insight. If your measurements suffer from high background, diffuse scatter, ice rings, smeared reflections, additional crystals in the beam, or any other pathology, then all these tests may give distorted answers. In other words, even if twinning is not really present, any test designed to convert the deviation of data from ideality into an estimate of the twinning fraction will give you an alpha 0. So my experience is: if your data are very good, then the tests give good answers; if the data are mediocre or bad, don't necessarily believe the numbers. Finally, it's not only twinning of P6 that would give you P622, it's also twinning of P3x21, P3x12 that gives P6y22. Hope this helps, Kay On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: Dear all, I recently collected several datasets for a protein that needs experimental phasing. The crystals are hexagonal plates, and (automatic) data processing suggests with high confidence that the space group is P622. This is where the fun begins. For some datasets (processed in P622), the intensity distributions are normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is no twinning (twinning fractions 0.05). However, for other datasets (same cell dimensions), the intensity distributions are not normal (eg Z-scores 10). Given that twinning is not possible in P622, this suggests to me that the real space group could be P6 with (near) perfect twinning. If I now process the normal L-test P622 datasets in P6, the twin-law based tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), suggesting all my data is twinned. Does this make sense (ie can one have twinning with normal intensity distributions)? If it does, would the normal L-test datasets have a higher probability of being solvable? Is there any strategy for experimental phasing of (near) perfect twins? SAD would be more suitable than SIR/MIR? (I also have potential heavy atom derivatives). Thanks for any insights! Bert
[ccp4bb] Post-doctoral positions in Structural Microbiology in Stockholm, Sweden
Post-doctoral positions in Structural Microbiology with a focus on Streptococcus pneumonia-associated virulence factors at the Science for Life Laboratory in Stockholm, Sweden. We are currently seeking for two highly motivated and multi-talented post-doctoral fellows to work on a collaborative project in the Achour laboratory within SciLifeLab (SciLifeLab.sehttp://SciLifeLab.se). This is an ideal position for a post-doc who is looking to bolster experience and/or publication record for several years before moving to a faculty position. The selected post-doctoral fellow will focus on determining the three-dimensional structures of Streptococcus pneumonia-associated virulence factors alone and/or in complex with ligands/inhibitors (Schulte et al, Open Biology 2014; Mellroth et al, mBio 2014). A successful PhD in a relevant scientific discipline and proficiency in structural and molecular biology are required. The post-doc fellows will have a lot of autonomy, but are also expected to provide theoretical and technical assistance with overall laboratory computational and crystallographic projects. The candidates should have a strong will to work on challenging and significant protein complexes in a multidisciplinary setting. Main skill sets required are facility in cloning and protein production, practical aspects of protein crystallization, structure determination and computation. Familiarity with protein expression systems, cell culture, various biochemical and biophysical techniques to study protein-protein interactions are also highly desirable. Besides determining the three-dimensional structures of protein complexes, the candidates will also be provided with the possibility to assess biophysically their interactions using e.g. Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC) or microscale thermophoresis. A large panel of other biophysical technologies is also available within SciLifeLab. The positions are initially offered for a period of two years (1+ 1year), but if successful, the post-doctoral fellows will also be offered a portfolio of their own projects in the areas of structure and/or protein engineering, as well as possibility to establish an own research group. The applicants should send a CV, with a complete list of publications (only published manuscripts) as well as a letter in which they describe the basis of their interest in the proposed project. Three professional references should also be sent in which the scientific and social qualities of the candidate are described. All required information should be sent by e-mail to adnane.ach...@ki.semailto:adnane.ach...@ki.se. More information about the Achour group can be found in scilifelab.sehttp://scilifelab.se. The positions are available from the beginning of April 2014, and will remain open until filled. The initial appointment is for 1 year. Consideration of applications starts when this advertisement appears. This call closes on February 26th, 2014. The newly created SciLifeLab, which is a joint effort between four Swedish universities, including the Royal Institute of Technology (KTH), the Karolinska Institute (KI), Stockholm University (SU) and Uppsala University (UU), serves as a Swedish national infrastructure to support infrastructure and technically advanced research in the life science area. The infrastructure at SciLifeLab includes state-of-the-art technologies for high-throughput molecular biosciences. Adnane Achour
[ccp4bb] Open Post-doc positions at the Science for Life Laboratory (scilifelab.se) in Stockholm, Sweden.
Open Post-doc positions in Structural Immunology with a focus on enhanced TCR recognition of MHC molecules at the Science for Life Laboratory (scilifelab.sehttp://scilifelab.se) in Stockholm, Sweden. We are currently seeking for two highly motivated and multi-talented post-doctoral fellows to work on a collaborative project in the Achour laboratory within SciLifeLab (SciLifeLab.sehttp://SciLifeLab.se). This is an ideal position for a post-doc who is looking to bolster experience and/or publication record for several years before moving to a faculty position. The selected post-doctoral fellow(s) will focus on determining the three-dimensional structures of T cell receptors in complex with MHC class I or class II, presenting wild-type and altered versions of epitopes related to autoimmune and/or cancer diseases. The overall aim of the project is to establish and understand the mechanisms underlying enhanced TCR recognition of infected and/or cancer cells, as well as to provide a structural basis for the induction of autoimmune responses. A successful PhD in a relevant scientific discipline and proficiency in structural and molecular biology are required. The post-doc fellows will have a lot of autonomy, but are also expected to provide theoretical and technical assistance with overall laboratory computational and crystallographic projects. The candidates should have a strong will to work on challenging and significant protein complexes in a multidisciplinary setting. Main skill sets required are facility in cloning and protein production, practical aspects of protein crystallization, structure determination and computation. Familiarity with protein expression systems, cell culture, various biochemical and biophysical techniques to study protein-protein interactions are also highly desirable. Besides determining the three-dimensional structures of protein complexes, the candidates will also be provided with the possibility to assess biophysically their interactions using e.g. Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC) or microscale thermophoresis. A large panel of other biophysical technologies is also available within SciLifeLab. The positions are initially offered for a period of two years (1+ 1year), but if successful, the post-doctoral fellows will also be offered a portfolio of their own projects in the areas of structure and/or protein engineering, as well as possibility to establish an own research group. The applicants should send a CV, with a complete list of publications (only published manuscripts) as well as a letter in which they describe the basis of their interest in the proposed project. Three professional references should also be sent in which the scientific and social qualities of the candidate are described. All required information should be sent by e-mail to adnane.ach...@ki.semailto:adnane.ach...@ki.se. The newly created SciLifeLab, which is a joint effort between four Swedish universities, including the Royal Institute of Technology (KTH), the Karolinska Institute (KI), Stockholm University (SU) and Uppsala University (UU), serves as a Swedish national infrastructure to support infrastructure and technically advanced research in the life science area. The infrastructure at SciLifeLab includes state-of-the-art technologies for high-throughput molecular biosciences. More information about the Achour group can be found in scilifelab.sehttp://scilifelab.se. The positions are available from the beginning of April 2014, and will remain open until filled. The initial appointment is for 1 year. Consideration of applications starts when this advertisement appears. This call closes on February 26th, 2014. Adnane Achour
Re: [ccp4bb] twinning fun
Also, if you have translational NCS then recent versions of Phaser can correct for the statistical effects and give you I^2/I^2 moment tests that are diagnostic of twinning. This works pretty well for 2-fold tNCS (i.e. one major Patterson peak corresponding to one or more pairs of molecules separated by the same translation). If there's higher order tNCS, then this works less well in the current version. We give some examples in the paper describing the algorithm: http://journals.iucr.org/d/issues/2013/02/00/dz5268/dz5268.pdf. Best wishes, Randy Read On 29 Jan 2014, at 13:30, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: Dont forget that with twinning in apparent point group PG6/mmm the true SG may be P6i or P3i21 See the twinning notes: http://www.ccp4.ac.uk/dist/html/twinning.html Detecting twinning can be problematic - My rule of thumb, following the procedure od ctruncate:: 0) Check the matthews coefficient for likely number of molecules. Half a molecule must mean you are assigning too high a symmetry count. Lots of molecules means you need to check for non-crystallographic translation etc. 1) Look at the I^2/I^2 plot after correction for anisotropy If it isnt reasonably straight with resolution you probably have some data problems, and these can make all the tests pretty useless. 2) Is there a NC translation - truncate tells you that. If not, and the data is OK, you are unlikely to have twinning if I^2/I^2 for acentrics is ~ 2, and the L test looks OK. H test and Britten tests a bit more influenced by other NC symmetry considerations 3) If there IS NC translation I^2/I^2 for acentrics will probably be 2 but the L test is still pretty reliable. Good luck Eleanor experimental phasing is tricky with perfect twinning but it has been done. Sorry I have forgotten reference though.. Eleanor On 29 January 2014 09:17, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: Dear Bert, as Dirk has pointed out, if P622 is the correct space group, then the twinning statistics printed out if you process in P6 are meaningless. Intensity statistics, like the ratio of I^2/I^2 , can be misleading if there is (e.g. pseudo-translational) NCS in the crystal; however, the effect of NCS on the value of the ratio of I^2/I^2 is opposite to that of twinning. Thus if a crystal is twinned and has NCS, you might not notice any problem in the ratio of I^2/I^2 . The other statistics, like Britton and H-test, present the intensity statistics in a different way, but from my understanding do not give substantially different information. The L-test does look at a different kind of information and therefore gives additional insight. If your measurements suffer from high background, diffuse scatter, ice rings, smeared reflections, additional crystals in the beam, or any other pathology, then all these tests may give distorted answers. In other words, even if twinning is not really present, any test designed to convert the deviation of data from ideality into an estimate of the twinning fraction will give you an alpha 0. So my experience is: if your data are very good, then the tests give good answers; if the data are mediocre or bad, don't necessarily believe the numbers. Finally, it's not only twinning of P6 that would give you P622, it's also twinning of P3x21, P3x12 that gives P6y22. Hope this helps, Kay On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: Dear all, I recently collected several datasets for a protein that needs experimental phasing. The crystals are hexagonal plates, and (automatic) data processing suggests with high confidence that the space group is P622. This is where the fun begins. For some datasets (processed in P622), the intensity distributions are normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is no twinning (twinning fractions 0.05). However, for other datasets (same cell dimensions), the intensity distributions are not normal (eg Z-scores 10). Given that twinning is not possible in P622, this suggests to me that the real space group could be P6 with (near) perfect twinning. If I now process the normal L-test P622 datasets in P6, the twin-law based tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), suggesting all my data is twinned. Does this make sense (ie can one have twinning with normal intensity distributions)? If it does, would the normal L-test datasets have a higher probability of being solvable? Is there any strategy for experimental phasing of (near) perfect twins? SAD would be more suitable than SIR/MIR? (I also have potential heavy atom derivatives). Thanks for any insights! Bert -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +
Re: [ccp4bb] making high res image in pymol
Thank you all for the suggestions. I lowered the hash-max and could get ray tracing to complete for line or even ribbon modes (to be honest line mode worked even without lowering the hash-max). But as Matthew pointed out, I could not get it to work in the cartoon mode. I think I am restricted here with my hardware limitations... Alex On Tue, Jan 28, 2014 at 2:20 PM, Yong Wang wang_yon...@lilly.com wrote: Hi Alex, If you don't mind forgoing the ray tracing, you may try the draw command to specifically set the resolution (and antialiasing) to your needs, and then save the image. Yong -- *Yong Wang, Ph.D. Research Advisor, Discovery Chemistry Research* Eli Lilly Company Phone: 317-655-9145 Lilly Corporate Center DC 0403 Fax: 317-651-6333 Indianapolis, IN 46285 wang_y...@lilly.com CONFIDENTIALITY NOTICE: This e-mail message from Eli Lilly and Company (including all attachments) is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *A K *Sent:* Tuesday, January 28, 2014 10:27 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] making high res image in pymol Hi all, I am trying to generate a high resolution figure of a molecule together with its symmetry mates (250 A readius) for a poster. If I try to ray it, the pymol session crashes (perhaps too many molecules are open). Using png xxx.png, dpi=300 or dpi=600 command doesn't make any difference; the image is still kind of low resolution for A0 or A1. Any idea how I can generate this in pymol? (I am using the free version of pymol) Thank you in advance, Alex
Re: [ccp4bb] twinning fun
Two more papers on twinning I found informative: === Acta Cryst. (2003). D59, 2004-2016[ doi:10.1107/S0907444903021085 ] Twinned crystals and anomalous phasing Z. Dauter Abstract: Merohedral or pseudomerohedral twinning of crystals cannot be identified from inspection of the diffraction patterns. Several methods for the identification of twinning and the estimation of the twin fraction are suitable for macromolecular crystals and all are based on the statistical properties of the measured diffraction intensities. If the crystal twin fraction is estimated and is not too close to 0.5, the diffraction data can be detwinned; that is, related to the individual crystal specimen. However, the detwinning procedure invariably introduces additional inaccuracies to the estimated intensities, which substantially increase when the twin fraction approaches 0.5. In some cases, a crystal structure can be solved with the original twinned data by standard techniques such as molecular replacement, multiple isomorphous replacement or multiwavelength anomalous diffraction. Test calculations on data collected from a twinned crystal of gpD, the bacteriophage [lambda] capsid protein, show that the single-wavelength anomalous diffraction (SAD) method can be used to solve its structure even if the data set corresponds to a perfectly twinned crystal with a twin fraction of 0.5. Keywords: twinning; merohedral; pseudomerohedral; anomalous scattering; SAD. === Acta Crystallogr D Biol Crystallogr. 2008 Jan;64(Pt 1):99-107. Epub 2007 Dec 5. Surprises and pitfalls arising from (pseudo)symmetry. Zwart PH, Grosse-Kunstleve RW, Lebedev AA, Murshudov GN, Adams PD. Author information Abstract It is not uncommon for protein crystals to crystallize with more than a single molecule per asymmetric unit. When more than a single molecule is present in the asymmetric unit, various pathological situations such as twinning, modulated crystals and pseudo translational or rotational symmetry can arise. The presence of pseudosymmetry can lead to uncertainties about the correct space group, especially in the presence of twinning. The background to certain common pathologies is presented and a new notation for space groups in unusual settings is introduced. The main concepts are illustrated with several examples from the literature and the Protein Data Bank. PMID: 18094473 [PubMed - indexed for MEDLINE] PMCID: PMC2394827 Free PMC Article
[ccp4bb] coloring by RMSD
I was wondering if anybody knows of a program or script that you can run that will calculate local RMSDs between 2 superposed pdbs and color based on those local RMSDs or allow for the ability to color based on RMSD? I have a molecule that is similar in some aspects to another molecule but also very different in others and thought this would be a good way to show this graphically. Tim Colussi
Re: [ccp4bb] twinning fun
Dear Bert, In my own review:- http://www.tandfonline.com/doi/abs/10.1080/08893110802360925?journalCode=gcry20#.UulGyGtYCSM molecular replacement emerged in my mind as the most robust option for structure determination in such a case, apart from finding an untwinned crystal form of course. Best wishes, John Prof John R Helliwell DSc FInstP CPhys FRSC CChem F Soc Biol. Chair School of Chemistry, University of Manchester, Athena Swan Team. http://www.chemistry.manchester.ac.uk/aboutus/athena/index.html On 28 Jan 2014, at 17:26, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: Dear all, I recently collected several datasets for a protein that needs experimental phasing. The crystals are hexagonal plates, and (automatic) data processing suggests with high confidence that the space group is P622. This is where the fun begins. For some datasets (processed in P622), the intensity distributions are normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is no twinning (twinning fractions 0.05). However, for other datasets (same cell dimensions), the intensity distributions are not normal (eg Z-scores 10). Given that twinning is not possible in P622, this suggests to me that the real space group could be P6 with (near) perfect twinning. If I now process the normal L-test P622 datasets in P6, the twin-law based tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), suggesting all my data is twinned. Does this make sense (ie can one have twinning with normal intensity distributions)? If it does, would the normal L-test datasets have a higher probability of being solvable? Is there any strategy for experimental phasing of (near) perfect twins? SAD would be more suitable than SIR/MIR? (I also have potential heavy atom derivatives). Thanks for any insights! Bert
Re: [ccp4bb] coloring by RMSD
Hi Tim - There is a script on the PyMOL Wiki that does just this: http://pymolwiki.org/index.php/ColorByRMSD. Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center 550 First Avenue New York, NY 10016 212-263-7898 http://kong.med.nyu.edu/ On Jan 29, 2014, at 12:43 PM, Colussi, Timothy timothy.colu...@ucdenver.edumailto:timothy.colu...@ucdenver.edu wrote: I was wondering if anybody knows of a program or script that you can run that will calculate local RMSDs between 2 superposed pdbs and color based on those local RMSDs or allow for the ability to color based on RMSD? I have a molecule that is similar in some aspects to another molecule but also very different in others and thought this would be a good way to show this graphically. Tim Colussi This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. =
[ccp4bb] Post-doc opening at MacCHESS
Postdoctoral Associate: Applications of Pressure to Structural Biology The Macromolecular Diffraction Facility of the Cornell High-Energy Synchrotron Source (MacCHESS) has an opening for a post-doctoral associate to continue development of the pressure cryocooling method (Kim et al. Acta Cryst. D61, 881-890 (2005), Kim et al. J. Appl. Crystallog. 46, 234-241 (2013)) and to apply pressure cryocooling to areas such as trapping of intermediates in biochemical reactions, preparation of samples for diffraction and imaging experiments, and elucidation of the effects of pressure on macromolecular structure. Applicants should have a Ph.D. degree in structural biology, biophysics, or a related field. Experience in hands-on development of sample-handling methods is desirable, and experience working at a synchrotron source is a plus. In addition to pursuing his/her own study of pressure effects, the successful candidate will be expected to collaborate with research groups wishing to apply pressure cryocooling to their samples. The Cornell High-Energy Synchrotron Source (CHESS) serves a world-wide user base of structural biologists, chemists, physicists, and engineers. MacCHESS is an NIH-supported National Resource providing support for structural biology at CHESS. MacCHESS is a heavily team-oriented environment. Good clear communication skills are a must, including fluency in the English language. This position is renewable for up to 3 years total, contingent upon availability of funds and employee performance. Direct inquiries and applications (include cover letter, CV including publications, and detailed summary of research experience and interests, and arrange to have at least three letters of reference sent) to: Dr. Marian Szebenyi CHESS, 200L Wilson Lab Cornell University Ithaca, NY 14853 E-mail: dm...@cornell.edu Applications must be received by March 31, 2014. Starting date is negotiable. Cornell is an equal opportunity employer.
[ccp4bb] preparation of citrate buffer pH3-6.5
Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama
