[ccp4bb] Off Topic- Fe-S electron transfer
Dear Community, I apologize for the off topic question but it is hard to resist since so many of our colleagues are incredibly knowledgeable. I am looking for a way to block or even remove the 2Fe-2S cluster in my enzyme. This cluster is involved in transferring electron from the Alpha to the Beta subunit and I wish to block this for an experiment. Does anyone know of a reagent or procedure that is known to do this (elegance is optional)? I first thought of EDTA but I don't think it will work given the covalent character of the Fe-S bonds. Thank you
[ccp4bb] Off Topic- Cystine Detection
Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP
[ccp4bb] How to merge data from 2 separate sections of same crystal
hello everyone, I have collected data on a problematic crystal. (first mistake...) Images spanning phi angles 45-80 look ok and usable, also images 229-279 are usable (index well and merge well too). How can I combine the 2 separate .mtz files from Mosflm when I scale them? Attempts to process them in the same session keep maling the program crash! Thank you very much
Re: [ccp4bb] How to merge data from 2 separate sections of same crystal
Thanks for all the suggestions on and off BB. I used the GUI Sort/Reindex to combine 6 sections then ran Pointless and Scala. Got a data set to 2.6A Rmerge 0.12 (0.06 inner shell) and solved it in C2221. It is a good night! Cheers,
[ccp4bb] To cryo or not to cryo...
Dear community, I have what seems to be a pretty decent single crystal that grew from a screen set up 2 weeks ago. I am trying to reproduce it but so far I have not succeeded. I am however afraid the crystal that did form will start to deteriorate. So this brings me to dilemma, I feel like I should try and mount this crystal and shoot it. But since I only have 1 sample, I do not want to mess this up... I am inclined to try cryo conditions, but I am afraid the addition of a cryo such as glycerol could destroy the little guy. The crystal formed in 30% PEG 4000, 0.1M NaCitrate pH5.6 and 0.2M NH4AcO, I wonder if this is a cryo condition already? Any suggestions would be appreciated. best,
[ccp4bb] B-iso vs. B-aniso
Dear community, The protein model I am refining has 400 amino acids (3320 atoms). Some real quick calculations tell me that to properly refine it anisotropically, I would need 119,520 observations. Given my unit-cell dimension and space-group it is equivalent to about a 1.24 A complete data set. However, I have had a couple of cases where anisotropic B-factor refinement significantly improved R-work and R-free, while maintaining a reasonable gap for lower resolution models (1.4-1.5 A, around 70,000 reflections). What is the proper way of modelling the B-factors? Any thoughts and/or opinions from the community are welcome. Cheers,
[ccp4bb] Ligand geometry obs. vs. ideal
Hi everyone, I am trying to show that a ligand underwent catalysis during a soaking experiment. One of the things I would like to show is the geometry of the ligand, bond angles/lengths, dihedrals, etc... One of my models has a hi-res of 1.18A and the ligand density is really clear and complete. What is the best way to refine the ligand unrestrained and then generate measurements? Also, the idea is to finally compare to ideal geometry. How should I generate these values (any softwares in mind)? ANy idea is welcome. Thanks a lot
Re: [ccp4bb] Evaluating crystallogarphic experiment
Thanks for all the replies {off list}.
Now it's wait and hope that this crystallization experiment is succesful.
Re: [ccp4bb] Optimizing xtals conditions
Shoot the existing crystals. Who says you will need optmization? lol
Re: [ccp4bb] Ferredoxin Containing Crystal Bleaching
Actually with haeme, and or flavin containing enzymes it is known for the cofactor to undergo reduction. There is a recent JACS paper that explores these changes as a function of X-ray exposure. (2012) J.Am.Chem.Soc. 134: 2823-2834 Excellent work done in part at Allen Orville's X6A As far as quality of your data goes, unless your crystal is suffering from radiation damage (they all do!) from those free electrons/radicals then your data should not be affected by the bleaching. HTH. Yuri
Re: [ccp4bb] FE-Sulfur proteins
Hi Jan, I wonder if the protein has a hexaHis tag. I recently was working on a Fe-S containing protein and noticed significant aggregation/precipitation. After I cleaving the His tag, the enzyme seems stable for days in the same buffer. HTH, Yuri
Re: [ccp4bb] Questions on unknown density?
Hi, Without looking at your model/maps it is difficult to make a really educated guess. By looking at the single screen shot you attach, I would say it could be a couple of different things, in order of likelihood based on a single screenshot (at whatever level of confidence this assures): 1-Zn coordinate to His, and some waters around it. 2-Phosphorylation of the His. Is this expected or known for this enzyme? Although I dont really see nice tetrahedral geometry, and I dont know that you really would at your resolution. 3-Alternate His side chain conformation. I cant really say if this fits your density with just that one screen shot. It doesnt really look like it to me. I would inspect distances and angles for each of the scenarios presented above and then build a model that makes good chemical and physical sense. Hope his helps. yuri
[ccp4bb] Protein-Ligand Binding Energetics
Hi everyone, I have several protein-ligand crystal structures of different ligands bound to a combination of mutants of the same enzyme. I wanted to look at the energetics of these complexes based on the crystal structures. Is there a program or suite of programs that could calculate DeltaG of complex formation, given the high resolution crystal structures. I would be looking for a trend, and possibly an explanation for why some mutations do not seem to form a complex. Thanks for your input. Yuri
[ccp4bb] Off-topic Thrombin cleavage
Dear community, I am trying to cleave a hexaHis tag from my protein prior to crystallization. As I was setting up my digestion, my protein started to precipitate as soon as I added the recommended thrombin buffer. My question is, if anyone has encountered this, how well does it cleave without thrombin buffer? or even, without the CaCl2? Any other buffer/conditions known to work? thanks in advance
[ccp4bb] .CBF raw images in iMOSFLM- error
Hello everyone, I am trying to process ###.cbf raw images (BNL X25) using the iMOSFLM utility and I get the following error message: Distance has refined to an unreasonable value This causes the program to freeze and not open the rest of the frames. Also it is not picking up the X and Y beam positions. It looks to me like its just simply not reading the image file HEADER properly. Anyone has encountered this before and has any ideas on how to get around/fix this? Thanks a lot! Yuri
[ccp4bb] Error in Scala
Hi everyone, Sorry for the newbie type problems, but I am just starting to use ccp4 for data processing. Here is my problem. After I index and integrate my images using iMOSFLM, I end up with the .mtz files that contains, AIUI, unmerged reflections. Next I should try to merge and scale experimental intensities, using SCALA. I am getting an error saying: Giving up Scala: Negative Scales task failed. Anything obvious I am missing? thanks a lot.
[ccp4bb] MOSFLM- Image compatibility
Can MOSFLM work with image files of type .x (BNL X6A) ? I am having no luck... I know it can do .cbf (BNL X25) for instance. Thanks a lot
Re: [ccp4bb] MOSFLM- Image compatibility
thanks for kindly pointing that out. (despite the level of stupidity on my part) Those were not the raw imgs... They were denzo output files. Its been a while.
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
could it be that the scattering table would be slightly different for the sulfur atoms at the collected wavelength? Are they Cys or Met residues? if Cys is there a possibility of oxidation to the disulfides?
Re: [ccp4bb] weird--No protein expression using pET30a
Hello, Probably a stupid comment on my part, but anyways, make sure your strain has a T7 polymerase! (I have forgotten before). And I agree with the last idea that sometimes it is worth to just start from scratch. New construct new vector. It may simply just work. HTH, Yuri
Re: [ccp4bb] Membrane Protein Crystallization Plates
Thanks for all the replies. I will try a couple of different plates/set-ups. My favorite will be the one that gives me a crystal ;)
[ccp4bb] Membrane Protein Crystallization Plates
Hello Everyone, I am considering the purchase of crystallization plates for membrane proteins. I would love to hear what some of the community thinks or has experienced with these. Particulalrly the monoolein and monoolein/cholesterol coated plates ( I am not sure I can mention the vendor here but it should not matter) So fire away. Is it worth it? Any succes stories? Bad experiences? I appreciate the input Best, Yuri
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Maybe someone has suggested this already... If so, I am re-enforcing it. If the cracking is coming from actual molecular movement induced by binding (and not other reason like differing ionic strength in your soaking conditions) you could try setting up some co-crystallization and (hopefully) grow some enzyme-substrate complex crystals... hth yuri
Re: [ccp4bb] Problem with getting Rfree and Rf down
Hi Sam, some obvious questions: 1-Space group right? ( i´d say so from your R values...) 2-Is your data good throughout all of the 180 frames? whats Rsym if take only 100? 3-how good/complete is model? Missing parts, residues, base pairs?? 4-evaluate your refinement strategy... HTH
[ccp4bb] writing scripts-off topic
Hello Everyone, I want to play around with some coding/programming. Just simple calculations from an input PDB file, B factors averages, occupancies, molecular weight, so forth... What should I use python,C++, visual basic? thanks
[ccp4bb] Protein Fold Motifs- off-topic
Hello Everyone, Does anyone know of a quick (yet somewhat reliable) sort of cheat-sheet/quick reference sheet with the more common folds with an illustrative example?
Re: [ccp4bb] Protein Fold Motifs- off-topic
Thanks guys
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
to echo Tim's question: If by pattern you mean the position of the spots on the film, I dont think they would change based on the complexity of the macromolecule being studied. As far I know it, the position of the spots are dictated by the reciprocal lattice points (therefore the real crystal lattice) (no?) The intensity will, obviously, vary dramatically... ps. Very interesting (cool) images James!!!
Re: [ccp4bb] on the electronic density of several maps
Fenghui, What is your resolution? If your having trouble distinguishing between pro and leu I am guessing it is worse than 2.8 A. You may not be able to model side chains confidently with lower resolution data. You may have to make a call on wether or not to model side chains, and if your model is interesting enough to pursue even without side chains.
Re: [ccp4bb] live streaming of ccp4 study weekend
I also had some trouble streaming live. So I am going to go ahead and also suggest/ask please that the video files be made available for subscribers and/or all academic users. Cheers,
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
First thing I would try to shoot more crystals. Easy way out. I once struggled with a 2.7A data set for weeks only to find out I had a 1.5A diffracting crystal taking a bath in some storage buffer right next to my bench. You mention you have at this point you are looking at 25% rotamer outliers. I wonder if at 3.2A if you really have density for these side chains. You may be trying to fit these side chains in very weak unreliable (e.g. noise) electron density. As Ed Pozharski suggested, omitting these may be the right thing to do. How certain are you of your space group and how did you generate you Rfree test set. You should have RworkRfree after refining your model. HTH Yuri
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
correction: You should NOT have RworkRfree
Re: [ccp4bb] MERGING THREE DATASETS
I was actually trying to do the same thing. I have to data sets processed in p21 that I would like to merge as one contains higher resolution data. They were processed and merged using the HKL suite. So I have the two .sca files I guess the first step for me would be to create a fake unmerged set of both using Combat, as suggested by prof. Dodson and others. Practically speaking, do I accomplish that by changing the space group to P1 while keeping my cell dimensions constant in combat? Thanks a lot
Re: [ccp4bb] Jrh correction Re: [ccp4bb] Mg or water?
In my personal opinion, whatever that is worth, I would question why you are modelling a Mg2+ ion if you are having to go through some trouble to prove it is there. If you dont see octahedral coordination to waters and or Asp/Glu it probably is not a Mg. HTH
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
Hi Ed, I just had a chance of looking at your comment more closely. You are right it only uses PHIC if in refmacs set up you choose to refine with prior phase information -AFAIU. So what exactly is the info contained in the output refmacX.mtz besides map coefficients for COOT? If it is not just the raw xray data Fo, is it Fc only, or Fc that are filled in for missing Fo? I guess I am not really sure. I was under the impression that model´s PHIC would cause the problems, if they were present. Best,
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
PHENIX has an otpion under the reflection editor program that will create R flags that are compatible with ccp4 programs. Another point worth mentioning is in phenix.refine it is appropriate to use the data.mtz files generated each round of refinement, as these are the raw data plus the Rfree flags. In refmac however the newly generated refmacX.mtz file contains phase info as PHIC calculated from your model. Using this for subsequent rounds of refinement results in terrific looking maps as they are now biased (even more so) by the input model.
[ccp4bb] Efficient way of showing residue conservation
I once saw a figure showing the protein as surface, but instead of having it coloured by atom type or potential, it was shown by percent conservation in the family. Something like red highly conserved, all the way to white, not conserved at all... Now, I assume the figure was done by uploading aligned sequnces of several members of a family, and the colouring the generated surface accordingly. Does anyone know a way to do this more elegantly than what I tried doing? ps. I quit colouring them manually after I remebered my protein was 407 aa long...
[ccp4bb] ccp4 on Win7 'clipper::Message_fatal'
Dear Developers, I was trying to get ccp4 up and running on a Windows7 pc, and even though the installation proceeds smoothly, I cant get any task to finish succesfully. Below is what the log file has to say about it. I saw someone having trouble with the windows install as well. I have the installation packaged posted by Marcin installed. Thanks for any help * Information from CCP4Interface script *** The program run with command: ctruncate -hklin C:/Ccp4Temp/oye1_77-73_3_1_mtz.tmp -hklout C:/Ccp4Temp/oye1_77-73_3_3_mtz.tmp -colin /*/*/\[IMEAN,SIGIMEAN\] -colano /*/*/\[I(+),SIGI(+),I(-),SIGI(-)\] -colout yap-ii-77-73 -nres 400 has failed with error message CCP4MTZfile - internal error This application has requested the Runtime to terminate it in an unusual way. Please contact the application's support team for more information. terminate called after throwing an instance of 'clipper::Message_fatal' *** #CCP4I TERMINATION STATUS 0 CCP4MTZfile - internal error This application has requested the Runtime to terminate it in an unusual way. Please contact the application's support team for more information. terminate called after throwing an instance of 'clipper::Message_fatal' #CCP4I TERMINATION TIME 05 Dec 2011 20:19:00 #CCP4I MESSAGE Task failed
[ccp4bb] Software for showing crystal packing
Hello everyone, Whats a good software for showing crystal packing and unit cell, axes , etc... I know pymol and coot will do it but would love to hear other possibilities/ideas. Cheers,
Re: [ccp4bb] Anomalous signal for chlorides
Hi Boaz, Yes indeed, the phosphate group of the molecule looks quite beautiful at 1.17A and it has a really big peak 18sigma! Is there a utility for calculating anomalous maps, or is it simply an option in the refinement program?
[ccp4bb] Merging with CAD fails
Hello, I am trying to merge two data sets on from 28 to 2.3 A 99% comlpeteness with another one from 26 to 1.95A 82% completeness. I keep getting an error saying Duplicate labels in the output file. I am sure its something simple but I cannot seem to figure it out Any ideas? Thanks
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Hi Napo, i am sorry if you already answered this, why are so sure your solutions are wrong? Your maps do not look that bad. What kind of R's do you get? Are you not happy with the packing of your coils?
[ccp4bb] NCS 2-fold perpendicular to crystal 2-fold
Hello Everyone, I have data sets that scaled fairly welll in C2 2 21, but intensity statistics suggested twinning. I was able to solve the structure in P1 21 1 (Rwork0.19/Rfree0.24 at 2.3A) with OK looking maps. I notice that I have 2-fold NCS that is paralell to the crystallographic A axis, perpendicular to my crystallographic 2-fold B axis. Can anyone ellaborate on the effect of this on the data (indexing/scaling and refining)? Best,
Re: [ccp4bb] Weird blob appears
Jacob, By simply looking at the figures you show, it does look like you have some type of long, maybe polymeric, molecule bound. With that being said: 1- It is in the symmetry axis so maybe be a little noisy there 2-If you are in doubt about it being real or not check the density and how it fits into your protein and (symm. related neighbours of course). If the desity appears to be in position for some real interactions- i.e, there are some peaks at H-bond distances of polar groups etc...- it may be real. If its all random and through your protein atoms, probably not Keep in mind alternate conformers HTH
Re: [ccp4bb] help me after several refmac
Echoing whats been said: 1- Are you sure your crystal really is in C 2 2 21? If so How good is your data (completeness, Rmerge, etc...) 2-Could have twinning? I recently just got done working on a structure that could be scaled in C 2 2 21 but turned out to really be an almost perfect P21 twin. (of course in monoclinic there are certain conditions for twinning...) 3- How good is your model, is it complete, all the waters? Missing protein or DNA? HTH Yuri
[ccp4bb] Ramachandran Restraints in refmac
Hello everyone, I am refining a structure to 2.4A with 2-fold NCS and twinned. Maps look ok and Rfree is 0.27however as I start checking my validations I notice that after refinemnt my geomtry gets significantly worse. especially the rama plot. Initially I have 2 outliers and I end up with 32 (5%)!!! I played with the Xray weight term but alll it helped me with was rmsd bond/angles, rama is still messing up... Can I impose some ramachadran restraints or maybe have a reference model? best,
Re: [ccp4bb] Apparent twinning in P 1 21 1
After I ran DETWIN with the estimated 0.46 alpha, my completeness for the detwinned data is now down to 54%!!! Is this normal behavior? (I am guessing yes since the lower symmetry untwinned dat is P1 21 1)
Re: [ccp4bb] Apparent twinning in P 1 21 1 (refmac)
after 1 round of refmac rigid body and restrained refinement with twin law (estimated alpha 0.47) I am looking at 0.25 -0.29 Rwork Rfree and overall FOM 0.72. I also defined NCS restraints...
[ccp4bb] Apparent twinning in P 1 21 1
Hello everyone, I have a 2.3A data set that could be scaled in C 2 2 21 and P 1 21 1 Intensity statistics tests indicate twinning (pseudo-merohedral h,-k,-h-l in P 1 21 1) I find a good MR solution and when I try to refine it with the twin law I get fairly good maps and decent Rs 21-28%. I can see features tha were not in the search model Which leads me to think that this a valid solution. The one thing that bothers me however is the fact that my beta angle in P 1 21 1 is 104 (not close to 90) and that the geometry gets worse after refinement? Any suggestions? cheers
Re: [ccp4bb] Apparent twinning in P 1 21 1
These papers describe something similar to what I see. Acta Cryst. (2001). D57, 1829-1835 Acta Cryst. (2009). D65, 388-392
[ccp4bb] Direct method solution at 1.15A
Hello everyone, I have a data set 99% completeness to 1.15A This is a 400 amino acid long protein and it has 7 Met (Sulfur peaks around 20sigma) And a tightly bound phosphate (P peak around 22sigma) Could I try and solve this directly or is it crazy idea? If so what program should I try? thanks Yuri
Re: [ccp4bb] Direct method solution at 1.15A
I solved the structure using molecular replacement. Those sigmas are simply from my sigmaa 2mFo-DFc maps. I was wondering if I could try and solve sort of like small molecules are
Re: [ccp4bb] Structure problem
Just echoing what has been said. I would make sure you have the right space group. It may be worthwhile tyring to find a MR solution in different space groups with different compositions. Another imporatant thing is how complete is your model? Do you have all the protein and DNA modeled in? How many waters (you should see plenty at 1.5 A) How good is the difference map? These are all things that should be checked before panic sets in Cheers
Re: [ccp4bb] COOT on CentOS 6 (x86_64)
Hi Paul, I am running the centos5 build. After a couple of yum installs it seems to be happy... Except I cannot maximize the window for some reason. Thanks for the reply. Yuri
[ccp4bb] COOT on CentOS 6 (x86_64)
Hello everyone, Could anyone tell me (or point me to) how to get COOT running on CentOS6 64-bit? It doesnt launch due to failed dependencies, it requires packages that CentOS6 has replaced... At least that is what it looks like to me... Cheers,
Re: [ccp4bb] EDS server
A simple way to validate real space density fit is to look at it under validate---density fit anlysis in COOT. I think even the GUI of phenix.refine at the end of a run the results give a list of all residues that are under the user-definied RSCCoeficient. With those two tools you should be able to see what you need to see
Re: [ccp4bb] Trying to digest PISA results
This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
[ccp4bb] Trying to digest PISA results
I was playing around with PDBe PISA and came across the following: For pdb entry 1OYA. The most promising interface has an area bury of around 720A^2 and DeltaG of -10.6Kcal/mol. sym_op(y,x,-z+1) and CSS of 0.039! Assembly analysis says it has no strong indications that point to stable quaternary structure. This protein has been extensively studied and determined to be a dimer. Entry 3RND is the same protein with one single conservative mutation deep in the active site. They align with a RMSD of 0.3 A, 99.8% sequence identity. Superposition and inspection of the regions that contact the adjacent monomer shows they are basically identical. The interface here shows Area bury of 760 A^2 and DeltaG = -6.6Kcal/mol. sym_op (-y,-x,-z-1/2) CSS=0.00 ! Assembly analysis basically says no stable oligomers form. This enzyme also is dimer according to gel filtration. Could anyone ellaborate on this please, if they feel like they have the time... Cheers
[ccp4bb] Temperature Factor statistics
Quick newbie question, After i get my output file from baverage containing the average b-factor and rms by residues, How can I calculate and display the average (and or mean) B-factors? Is there a way of calculating it by protein, ligands and solvent separately? thank you
[ccp4bb] PyMol plugin
I installed the plugin superSym but when I triesd to run it, ended up with the following message: File /home/local/UFAD/yuri.pompeu/Desktop/PHENIX_installation/phenix-installer-dev-833/phenix-dev-833/pymol/modules/pmg_tk/PMGApp.py, line 156, in initialize_plugins __builtin__.__import__(mod_name) File /home/local/UFAD/yuri.pompeu/Desktop/PHENIX_installation/phenix-installer-dev-833/phenix-dev-833/pymol/modules/pmg_tk/startup/SuperSymPlugin12.py, line 18, in ? Color: map_colour defined as [ 0.000, 0.500, 1.000 ]. Color: iso_diff_map_colour defined as [ 0.000, 1.000, 0.250 ]. quit(Oops! SuperSym requires cctbx and numeric python to function. Please install these.) I then installed cctbx+Python bundle from http://cci.lbl.gov/cctbx_build/ and sourced it. I still get the same result in PyMol though.. Does anyone know what to do? Thank you for your time Best, Yuri
Re: [ccp4bb] PyMol plugin
Jurgen, I was able to make the picture i wanted the poor man´s way. I saved the symmetry coordinates in COOT and open all of them as objects independently. I just wanted to know what else I need to do to get the plugins going. I have tried two differents ones with no success.
Re: [ccp4bb] PyMol plugin
Yes I just tried symex and it is a LOT faster... especially with 12 symm operators... yes I want to show crystal packing, but I know SuperSym can show the three axis and fancy representations of unit cells, etc...
[ccp4bb] Mixed Iso/Aniso in refmac5
Hello everyone, How does refmac5 pick atoms for B-factor refinement, particularly with the mixed option enabled? I dont see a place for entering a manual selection, eg resname FMN... Thank you
