[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

[ccp4bb] Active site volume calculator

Dear BB, I am sorry for posting off-topic but it is hard not to ask when you know you can get a good answer ;-) I need to calculate the volume of several active sites. Nothing fancy, just a number for comparison sake. I understand there are probably 10 different ways/programs and I would apprec

[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

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] 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 the

[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 sho

[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.

[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.

Re: [ccp4bb] Evaluating crystallogarphic experiment

Thanks for all the replies {off list}. Now it's wait and hope that this crystallization experiment is succesful.

[ccp4bb] Evaluating crystallogarphic experiment

Hello everyone, I have just made a curious observation. I purified an enzyme domain and concentrated it to around 16 mg/mL without much mischief. I then proceeded to set-up some drops. After 5 min most of the drops had a muddy appearance to them which led me to think I was probably too concentrat

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 qual

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

[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 co

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

[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 thr

Re: [ccp4bb] Strange Density

Grinter, I would be very careful when comparing atomic distances (or even considering them) using a 3.1 A data set. Take a look at what error estimates are for this resolution, given your R values ;). Also, I second what has been said, that you should build a model that makes "physical" sense,

Re: [ccp4bb] how to ignore spot overlap in imosflm?

I think "IGNORE" is the wrong word here, but as many have pointed out, you may be able to deal with this situation. Besides the many good tips that been given, I think EVAL15 (I started reading the paper on it) should be able to deal with some overlaps pretty succesfully. I don't know where the

Re: [ccp4bb] question on metal refinement in a protein structure

Hi Dave, I sounds to me like you are worried about 2 separate things here. A: Am I affecting the geometry of the coordination sites with a restraint file that is innacurate? B:Are my electron density maps biased, and what I am seeing is not really there? AFAIU, if you have a restraint file that

Re: [ccp4bb] Deposition of riding H

Dear community, I am probably disturbing a sleeping bear, but I'll post anyways... Reading the thread on hydrogen deposition with the model, I came accross several arguments that make sense on their own, but when put together are puzzling and dont seem to converge to an answer. (I do realize howe

[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 p

[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 experiment

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.

[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] 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 bu

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) gr

[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

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

Re: [ccp4bb] Protein Fold Motifs- off-topic

Thanks guys

[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] 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

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 l

Re: [ccp4bb] Structure Determination combining X-ray Data and NMR

correction: You should NOT have Rwork>Rfree

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.

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] 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 s

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 jus

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 fl

[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 al

[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 inst

[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] Anomalous signal for chlorides

Hello everyone, I have a good data set to 1.17A that I solved by MR. I come accross some sites that appear to be chlorides. I was wondering if they could have some anomalous signal. I also have a well ordered phosphate and a couple of S from Met´s. How do I go about probing the signal from these?

[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, pe

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

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.

[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

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...

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

These papers describe something similar to what I see. Acta Cryst. (2001). D57, 1829-1835 Acta Cryst. (2009). D65, 388-392

[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

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

[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

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 ma

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

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 i

[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 quatern

[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

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...

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.

[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__(

[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