Re: [ccp4bb] large R-Rfree difference in final structure
Hi, Ian Tickle has asked the most pertinent question - there are many reasons for highish FreeR factors. Only some are due to errors or missing molecules or other problems. How many Free reflections are there? When the sample is very small ( 500 say) you can get different results from a different Free R set. And remember that the numbers you quote are overall values, It is worth looking at the plot of R and Free R against resolution. They should stay reasonably parallel, but if the data becomes very weak at the resolution limit the the FreeR can shoot up relative to the overall Rvalue. I use COOT validation to search the difference maps for peaks and holes. If there is something missing from your model you should see it in that list.. Eleanor On Wed, 13 Jul 2011 20:37:51 +0100, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de wrote: Hi Careina, our lab we once had the problem, that the asymmetric unit contained 8 molecules, whereas 7 had only been modeled. Somehow the 8th monomer had evaded detection. So be careful not to miss density. Matthias On 7/13/2011 7:54 PM, Robbie Joosten wrote: Hi Careina, Assuming you don't suffer from a very poor data parameter ratio that would lead to such a large R-free/R, you need to improve your refinement. If you have NCS you should use local NCS restraints. You could also try jelly-body restraints, although they may not work at your resolution. Cheers, Robbie Date: Wed, 13 Jul 2011 08:38:38 -0700 From: careinaedgo...@yahoo.com Subject: [ccp4bb] large R-Rfree difference in final structure To: CCP4BB@JISCMAIL.AC.UK Dear ccp4 bulletin board I just have a slight concern regarding my Rwork Rfree difference. I have a structure that I have solved. I am reasonably content that it is complete because it has refined well, it no longer has bad geometries and contacts and all the rotamers, ramachandra, bond lengths etc are good. It gives favourable scores on molprobity and procheck. My only concern is the R factor difference. The resolution of the structure is 2.3A. The R factor is 0.24 after refinement but the Rfree is 0.33 which seems to me to be rather high. Should I be concerned? During refinement Rfree only drops from about 0.36 to 0.33 while the R factor drops from 0.31 to 0.24.. I have removed automatic weighting in refmac in order to constrain my bond lengths and angles during a couple of rounds of refinement. This did not have any effect on the R factors, however. I am fairly content that the space group I have chosen is correct so I am not sure what else could cause the big difference in R factors? There is no twinning. Can I be satisfied that my structure is correct despite the high R free or should I be doing other checks/ trying other things before I can submit this structure? Thank you for any help Careina
Re: [ccp4bb] Off topic - transformation problems
You may also want to try 0.5 - 2% (w/v) glucose in your plates alongside transformation into the NEB T7 Express strains: http://www.neb.com/nebecomm/products/productC3013.asp http://www.neb.com/nebecomm/products/faqproductC3013.asp LysY gives you higher final ODs [no lysozyme activity] than using pLysS/E and is compatible with the T7 promoter in your pET20b. Renos On 14/07/2011 03:33, Dima Klenchin wrote: In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htmhttp://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low leak expression from PTAC promoter. Definitely lower than the relatively leaky T7 promoter in pET20, which does not express laqI^q. Switching to a plasmid from higher pET series that do have laqI^q (e.g. pET31) should reduce the leakiness. I also thought that target protein may have tocicity on host strain. But, why pGEX4T3-target protein was not show the same phenomena. Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and BL21(DE3)pLysS. pLysS should help with repression under non-inducing conditions. If it is true toxicity issue, you might need to switch to plates with synthetic medium that does not contain any traces of lactose. - Dima
[ccp4bb] Influence of symmetry related molecules on a protomer conformation in crystal structures
I know, in PyMOL using 'symexp' possible to generate symmetry related molecules for a given crystal structure. But I'm looking for some program/software (for batch) by which I can find out the number of symmetry related molecules (distance cutoff = 5A) interacting with a given chain in a crystal structure. Thanking you, suku NIBIO, Osaka
Re: [ccp4bb] large R-Rfree difference in final structure
Dear Careina, As other have mentioned, my feeling is that something is suspicious. However the problem might be very difficult or impossible to catch and other structures have been deposited with similar R/Rfrees... What I would check in addition to what has been mentioned, is if some twinning is present. With say a twin-factor of 10-20% processing may proceed sort of ok and refinement as well with a somewhat higher Rfree. You may also suffer from disorder. You mention that you model is complete: does that mean that the complete sequence of your protein has been fitted, or does this mean that no more positive difference density is left? If a significant portion of your protein is not visible, this may still compromise your Rfactors. In the latter case, I don't think you can do much about it, except offering this as an explanation for your high R/Rfree. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Careina Edgooms Sent: Wednesday, July 13, 2011 5:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] large R-Rfree difference in final structure Dear ccp4 bulletin board I just have a slight concern regarding my Rwork Rfree difference. I have a structure that I have solved. I am reasonably content that it is complete because it has refined well, it no longer has bad geometries and contacts and all the rotamers, ramachandra, bond lengths etc are good. It gives favourable scores on molprobity and procheck. My only concern is the R factor difference. The resolution of the structure is 2.3A. The R factor is 0.24 after refinement but the Rfree is 0.33 which seems to me to be rather high. Should I be concerned? During refinement Rfree only drops from about 0.36 to 0.33 while the R factor drops from 0.31 to 0.24.. I have removed automatic weighting in refmac in order to constrain my bond lengths and angles during a couple of rounds of refinement. This did not have any effect on the R factors, however. I am fairly content that the space group I have chosen is correct so I am not sure what else could cause the big difference in R factors? There is no twinning. Can I be satisfied that my structure is correct despite the high R free or should I be doing other checks/ trying other things before I can submit this structure? Thank you for any help Careina
Re: [ccp4bb] Influence of symmetry related molecules on a protomer conformation in crystal structures
hi , in CCP 4 package u can run CONTACT program use NCONT to find symmetry contacts only. u can Fix contact distance minimum and maximum also . On Thu, Jul 14, 2011 at 5:18 PM, sukanta mondal sukanta.mon...@gmail.comwrote: I know, in PyMOL using 'symexp' possible to generate symmetry related molecules for a given crystal structure. But I'm looking for some program/software (for batch) by which I can find out the number of symmetry related molecules (distance cutoff = 5A) interacting with a given chain in a crystal structure. Thanking you, suku NIBIO, Osaka -- Vandana Kukshal Postdoc Fellow structure biology group international Center For Genetic Engineering and Biotechnolgy India
Re: [ccp4bb] Off topic - transformation problems
On Thu, 14 Jul 2011, Wonjin Bae wrote: Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. Simplest explanation - wrong antibiotic used on the pET20b case? What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie, Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu
Re: [ccp4bb] Off topic - transformation problems
oops - I just caught that the empty pET20 did transform well so the other suggestions about toxicity are probably more accurate. On Thu, 14 Jul 2011, Eric Larson wrote: On Thu, 14 Jul 2011, Wonjin Bae wrote: Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. Simplest explanation - wrong antibiotic used on the pET20b case? What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie, Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu
[ccp4bb] Multi crystal averaging : data on same scale before averaging?
Hi all Walking through multi xtal averaging with RAVE. I finally got a good mask and optimized NCS for my xtal forms. However, in the CRAVE manual I see this - the reflections in the input MTZ files *MUST* have been put on the same temperature factor scale prior to cross-crystal averaging (see the DATAMAN manual on how to do this) !!! Scale the separate datasets together? Wouldn't this just be a mess since the crystal should be non isomorphous to each other? Or does this say that within each dataset, all the data should be on the same scale (which would be if I used scala to scale) ? Am I interpreting this correctly? Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Multi crystal averaging : data on same scale before averaging?
If I recall correctly DATAMAN does Wilson scaling in which the scale and B-factor are adjusted so the average reflection intensity in resolution bins are the same. I suspect it may not be required if all the data have been put on an approximately absolute scale by e.g. truncate (although that doesn't adjust the B-factor). If you do end up scaling your data in dataman, be sure to go back to the original data for refinement once you solve the structure, or your B-factors will not be right. eab Francis E Reyes wrote: Hi all Walking through multi xtal averaging with RAVE. I finally got a good mask and optimized NCS for my xtal forms. However, in the CRAVE manual I see this - the reflections in the input MTZ files *MUST* have been put on the same temperature factor scale prior to cross-crystal averaging (see the DATAMAN manual on how to do this) !!! Scale the separate datasets together? Wouldn't this just be a mess since the crystal should be non isomorphous to each other? Or does this say that within each dataset, all the data should be on the same scale (which would be if I used scala to scale) ? Am I interpreting this correctly? Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] NMR resources at PDBe (pdbe.org/nmr)
Hi all, The Protein Data Bank in Europe (PDBe; http://pdbe.org/) continues to improve its services to the scientific community. As part of our recent website update, we have reorganised and simplified the way information about NMR entries is shown. We have also released Vivaldi (Visualisation and Validation Display; http://pdbe.org/vivaldi), an interactive tool that allows you to inspect available validation reports and check for restraint violations or RDC fits. For an example, try http://pdbe.org/vivaldi/2keo The pages describing the NMR resources at PDBe (http://pdbe.org/nmr) have been revised and streamlined. They allow you to search the VASCO, OLDERADO, RECOORD and logRECOORD databases (see below) and also provide information about deposition of NMR structures and accompanying data, as well as some work related to the CCPN framework. The NMR statistics page shows the current status of NMR-related information in the PDB and at PDBe (http://www.ebi.ac.uk/pdbe-apps/nmr/statistics). On the PDBe atlas pages of a particular NMR entry (e.g., http://pdbe.org/2keo - cytochrome-b5-like domain of the HERC2 E3 ligase - or try http://pdbe.org/hasnmr for a randomly picked NMR entry), you can access available experimental information by clicking on the Experiment link in the navigation menu on the left of the page. Alternatively, whenever you see a PDBprint (http://pdbe.org/pdbprints) for an NMR entry, you can click on the NMR icon to get to the NMR experimental information page. Finally, if you know the PDB code, you can go there directly (e.g., http://pdbe.org/2keo/experimental). When you click a View link in the table on the experimental information page, it will redirect you to a predefined Vivaldi view (e.g., restraints or validation reports). The experimental information page allows you to navigate to the corresponding Biological Magnetic Resonance Bank (BMRB) entry containing assigned chemical shifts. (Note: experimentally determined chemical shifts are now mandatory when depositing NMR structures). Sometimes, more than one BMRB entry relates to the same PDB entry, for instance in the case of close homologues or alternate protein constructs (97% sequence identity). You can download deposited restraints from the PDB archive or navigate to the remediated restraints from the NMR Restraints Grid (NRG) at BMRB. For most NMR entries there is an in-depth validation report from the NRG-CING database, hosted at the Radboud University of Nijmegen. VASCO - Validation of assigned chemical shifts through coordinates - is a database of 2000+ validation reports, which can be downloaded as text files or viewed interactively in Vivaldi. OLDERADO reports on domain composition, clustering and representative models of a deposited ensemble are available for most NMR entries that contain proteins. They can be viewed in Vivaldi or in a tabular format. Two databases of recalculated ensembles are hosted by PDBe: RECOORD, with 500+ entries, which compares four common structure-determination protocols, and logRECOORD, with 300+ entries, which uses a log-normal potential for interpreting NOE data. Finally, you may be interested in some recent NMR-related papers co-authored by current and former PDBe staff: 1. Lemak et al., A novel strategy for NMR resonance assignment and protein structure determination, Journal of Biomolecular NMR 49, 27-38, 2011 (http://dx.doi.org/10.1007/s10858-010-9458-0) 2. Bernard et al., Bayesian estimation of NMR restraint potential and weight: a validation on a representative set of protein structures, Proteins 79, 1525-1537, 2011 (http://dx.doi.org/10.1002/prot.22980) 3. Penkett et al., Straightforward and complete deposition of NMR data to the PDBe, Journal of Biomolecular NMR 48, 85-92, 2010 (http://dx.doi.org/10.1007/s10858-010-9439-3) 4. Fogh et al., MEMOPS: data modelling and automatic code generation, Journal of Integrative Bioinformatics 7, 123, 2010 (http://dx.doi.org/10.2390/biecoll-jib-2010-123) 5. Rieping Vranken, Validation of archived chemical shifts through atomic coordinates, Proteins 78, 2482-2489, 2010 (http://dx.doi.org/10.1002/prot.22756) We hope that expert and non-expert users alike will find the NMR resources at PDBe useful. As always, we welcome constructive criticism, comments, suggestions, bug reports, etc. through the feedback button at the top of any PDBe web page. --Gerard --- Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK ger...@ebi.ac.uk . pdbe.org Secretary: Pauline Haslam pdbe_ad...@ebi.ac.uk
Re: [ccp4bb] Multi crystal averaging : data on same scale before averaging?
This is correct - see http://xray.bmc.uu.se/usf/dataman_man.html#S27 At the time I wrote this (1993...), I was a recovering NMRtist and The Other Gerard was doing a sabbatical in Uppsala and in fact in the office next to mine. He pointed out that I had to do this and also provided code. --The other other Gerard On Thu, 14 Jul 2011, Edward A. Berry wrote: If I recall correctly DATAMAN does Wilson scaling in which the scale and B-factor are adjusted so the average reflection intensity in resolution bins are the same. I suspect it may not be required if all the data have been put on an approximately absolute scale by e.g. truncate (although that doesn't adjust the B-factor). If you do end up scaling your data in dataman, be sure to go back to the original data for refinement once you solve the structure, or your B-factors will not be right. eab Francis E Reyes wrote: Hi all Walking through multi xtal averaging with RAVE. I finally got a good mask and optimized NCS for my xtal forms. However, in the CRAVE manual I see this - the reflections in the input MTZ files *MUST* have been put on the same temperature factor scale prior to cross-crystal averaging (see the DATAMAN manual on how to do this) !!! Scale the separate datasets together? Wouldn't this just be a mess since the crystal should be non isomorphous to each other? Or does this say that within each dataset, all the data should be on the same scale (which would be if I used scala to scale) ? Am I interpreting this correctly? Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder Best wishes, --Gerard ** Gerard J. Kleywegt http://xray.bmc.uu.se/gerard/ mailto:ger...@xray.bmc.uu.se ** The opinions in this message are fictional. Any similarity to actual opinions, living or dead, is purely coincidental. **
[ccp4bb] output individual redundancies
I am looking for a way to output redundancy per individual reflection, preferably for scala but if that is not possible then maybe for scalepack. From my (admittedly quick) look at the scala manual it seems that I can use something like UNMERGED output option to exclude outliers and then would need to write a bit of code to calculate the redundancies. But I hope that I missed something and there is a secret keyword that would add redundancies to the merged mtz file. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] output individual redundancies
On Thursday, July 14, 2011 02:55:26 pm Ed Pozharski wrote: I am looking for a way to output redundancy per individual reflection, preferably for scala but if that is not possible then maybe for scalepack. If you read the unmerged file from scalepack into ccp4 using combat, it creates a data column with label M/ISYM that I think is what you are asking for. You can use the Import Unmerged Data (Combat) tab in the ccp4i GUI. Ethan From my (admittedly quick) look at the scala manual it seems that I can use something like UNMERGED output option to exclude outliers and then would need to write a bit of code to calculate the redundancies. But I hope that I missed something and there is a secret keyword that would add redundancies to the merged mtz file. Cheers, Ed. -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] occupancy-refinement
You are right that refining occupancy and B value at the same time cant be done with REFMAC, and probably wouldnt work anyway at 2.9A However, if you set the ligand occupancy to 0.7 say, and refine the residue B factors, the occupancy should not be set back to 1.0. It certainly isnt in the local version of REFMAC. You can judge to some extent what occupancy gives the cleanest map by inspection. But at 2.9A the correlation between B and occupancy is very high and it wilnswer. l be hard to get a definitive answer. Eleanor On Wed, 13 Jul 2011 10:31:59 -0500, dhurjati putcha antharvaah...@gmail.com wrote: Dear CCP4ers,ncy and B value While trying to refine a protein-ligand structure (reso=2.9A) I notice that the density (2Fo-Fc)for the ligand is discontinuous. I also notice that the density for the residues in the ligand binding pocket (LBP) is also very feeble. And when I refine the ligand occupancy the density for the ligand appears and covers almost all the molecule, and the occupancy refines to 1.0. However, I cannot refine the occupancy/B factors for the LBP residue side chains because the occupancy stays at 1.0 (and if I change it manually, it returns to 1) and the B factors (group individual) remain close to the average for the remainder of the molecule. If I omit the LBP residue side chains, I get some Fo-Fc density suggesting that these side chains are real. Is there previous evidence for such phenomena suggesting multiple conformations/dynamics/loose binding? Is there a way to quantify the dynamics associated with the side chains (as with the ligand where the occupancy is 1.0) so I can argue it out with potential manuscript reviewers? The R-factor/R free #s (22%/19%) suggest that the refinement is nearing convergence. Best regards, Kumar.
Re: [ccp4bb] Same protein, different molecule numbers per ASU
They must represent a different packing - the volume of Shape 1 is 10% than 2*Vol-Shape2 But it isnt uncommon to get different forms - check your symmetry contacts (PISA will do that) and see how the two forms pack. eleanor On Mon, 11 Jul 2011 18:09:52 +0800, ferrol shariff ferrol2...@gmail.com wrote: Hi Alenxander, Thanks for the reply. Here's the details on the unit cell parameters [image: image.png] [image: image.png] [image: image.png] Thank you. :) Regards, Fairolniza On Mon, Jul 11, 2011 at 2:47 PM, Alexandre OURJOUMTSEV sa...@igbmc.frwrote: Dear Ferrol, ** ** Could you let us know the unit cell parameters for both these crystals ? (did I miss them somewhere in your previous mails?). I wonder if in fact this is not the same packing with minor variation. Sacha ** ** *De :* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *De la part de*ferrol shariff *Envoyé :* dimanche 10 juillet 2011 10:10 *À :* CCP4BB@JISCMAIL.AC.UK *Objet :* [ccp4bb] Same protein, different molecule numbers per ASU ** ** Hello and good day to everyone! :) ** ** I have some general questions on crystallography work. I hope you don't mind giving me some ideas. I have solved my lipase protein both ground-grown crystals and space-grown crystals with good resolutions (1.4A and 2.2A). They are the same protein from the same source, same purification methods, and produced crystals from the same crystallization conditions (except the gravity part). From the data, it shows that both of them belong to the same space group P212121. But they have different number of molecule per asymmetric unit. Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the moment i have problem explaining this issue. Is it normal to have such results? Same protein with different number of molecule/ASU? I've been trying to get some references on this matter but so far i don't really get anything that can directly explain it. Furthermore, do i need to relate this with the gravity effect? I hope you don't mind sharing some experiences on crystallography especially regarding this matter. Thank you very much -- FAIROLNIZA
Re: [ccp4bb] abnormal I/Sigma over phi rotation range
This seems somewhat weird - your Rmerge values increase with more frames as explained by Tim, but I cant see why the I/SigI should increase sharply for the 180 degree set then fall off again. if you have run Scala look at the scaling plots v batch, and resolution to see if there are any weird outliers. Eleanor On Mon, 11 Jul 2011 10:23:00 +0200, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Vennila, my guess is that you had a larger number of images in the batch/set rge starting at 180 degrees (hence the slightly incereased Rmerge for that batch) and that by the end of that run your crystal had suffered from radiation damage (hence the large Rmerge for the set starting at 360degrees). You should report Rmeas instead of Rmerge and let us know about the number of frames and the frame width in each set. Cheers, Tim On Sun, Jul 10, 2011 at 01:54:07PM +0100, Vennila Natesan wrote: Dear CCP4BB users, In order to determine the redundancy at which the structure can be solved, I divided the master data set of my protein into five sets with phi rotations of 45,65,90, 180 and 360 degrees. I got the mean I/Sigma value as 22.4(11.4), 21.7(11.0), 22.7(11.2), 60.9(45.2) and 15.6(8.1) respectively. I will be very helpful if i get any idea/possible reasons for the abnormal value for 180 degree dataset. For the information, the completeness values are 75.7(78.5),92.6(92.7),99.5(97.7),99.6(97.7)and 99.5(96.4) respectively and values inside brackets are for highest resolution shell. There was no noticable change in mosaicity value. The Rmerge values are 2.2,2.4,2.7,3.2, and 5.7 (8.7) respectively. Thanks in advance
Re: [ccp4bb] low resolution refinement
Roberto steiner has told you how to use these new REFMAC5.6 features, Rob Nicholls has suggested how to generate secondary structure restraints, and Martyn Winn given a page to install a new interface to make it easier to use them.. But with such limited data it isnt surprising that the FreeR climbs steadily. Scaling Fcalcs and Fobs together at this resolution is tricky, and you should look at your plots of Rfactor and Fo/Fc at the end of refinement. There may be anomalies at the top or bottom resolution.. There really isnt a general rule for a fix. You need to know your data. You can still get useful information from the maps. Eleanor On Sat, 9 Jul 2011 16:59:29 +0800, Qixu Cai caiq...@gmail.com wrote: Dear all, Recently, I refine two low resolution structures in refmac 5.5. Their resolutions are 3A and 3.5A respectively. For 3A structure, after MR by phaser and rigidbody refinementrestraint refinement by refmac5.5, I got R factor 25% and R free 35%. And then each time, after my model building in coot and restraint refinement by refmac 5.5, the R factor stays 25%, but R free increases to 38%, even 39%. For 3.5A structure, the R factor stays 27%, but R free increases from 37% to 42% after my slightly model building in coot. Could you help me to find the reason? Maybe the reason is the overfit of the structure? I found that new version of refmac 5.6 has many new features for low resolution refinement, such as jelly boy, secondary structure restraints. But I don't know how to use these new features in old version ccp4i (6.1.13)? I also used phenix.refine with the reference model ( I have high resolution model for one domain of the low resolution protein) and secondary structure restraints, but it seams the same. Any suggestion? BTW, is that simulator annealing not suitable for low resolution structure? I used the simulator annealing method of CNS and phenix.refine, but the geometry of the structure is always destroyed seriously. Could you help me? Thank you very much!
