[ccp4bb] Cys modification - deciding between CSS and SMC
I am working on a model at a resolution of 2.1 A. The active site Cys in both copies have a positive density towards the S end of the residue and these blobs are there in FEM and Polder maps. When I replace these residues with either CSS or SMC, I get the following statistics. What is the best way of deciding which one is correct? CSS: R/Rfree - 0.2016/0.2299 Local CC (chain A/B) - 0.950/0.937 Bfactor (chain A N/CA/C/O/CB/SG/SD) - 42.8/48.6/46.7/42.8/57.3/66.2/98.8 Bfactor (chain B N/CA/C/O/CB/SG/SD) -42.0/44.5/47.2/45.7/57/60.3/95.3 No residual positive density left. SMC: R/Rfree - 0.2087/0.2299 Local CC (chain A/B) - 0.903/0.886 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 43.9/47.9/46.5/42.1/51.8/64.4/66.5 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 43.4/45.0/47.3/45.4/51.8/59.4/66.1 Crescent shaped positive density left towards the CS atom end. Although CSS seems more plausible, the high B factors of SD make me wonder if they are correct. Thanks. Mohamed
[ccp4bb] Ligand building in real space
Dear all Is it possible to directly build a ligand in real space (in Coot?) and then generate a SMILES string for restraint generation. I have some unknown blobs in my density where they look like PEG molecules but these do not really fit the density (local CC of 0.7). I am already using Polder and omit maps that confirm these are not noise. Thanks. Mohamed
[ccp4bb] Accidental crystallization of E. coli protein
During the crystallization of a totally unrelated protein from a different bacterium in E. coli, we managed to somehow crystallize an E. coli protein. It turned out to be only the catalytic domain of an enzyme. Two previous reports both used recombinant expression of this enzyme followed by limited proteolysis in order to crystallize this domain. Has something like this been reported before? I know the stories of AcrB etc., but I am looking specifically for a 'naturally proteolyzed' crystallization. Looking at our structure and the previously published one, there is not much difference, with an rmsd of about 0.3 A but our data resolution is a bit higher. This is perhaps unsurprising as the unit cells are very similar (in fact, I just did a quick RBR). Should we bother depositing and publishing this observation? Thanks. Mohamed
[ccp4bb] tNCS and PISA
I am turning again to the wisdom of the community. In a paper, the authors used a dataset in P 1 2(1) 1 displaying tNCS (not mentioned anywhere in the paper) to model a dimeric structure. At the refinement stage, they used a twin law. Looking at the PDB-REDO entry, there is a warning line - "Used extra refinement runs to compensate for possible R-free bias". When I ran the data from PDB in Xtriage, I get the following: 1. translational NCS is present at a level that may complicate refinement 2. severe anisotropy (is this unsurprising because when I re-refined it, the CCwork/free didn't display a classical drop at the high resolution? artificial truncation?) 3. one or more twin operators show a significant twin fraction but since the intensity statistics does not indicate twinning, you may have an NCS rotation axis parallel to crystallographic axis 4. one or more symmetry operators suggest that the data has a higher symmetry 5. the intensity statistics look normal, indicating the data are not twinned. How should I proceed in re-refining this structure? Reindex to P222 and re-do molecular replacement? The reason I am interested in this model is that the authors claim their structure is a homodimer but PISA deltaG(diss) is around 4 kcal/mol. In a related structure where the protein was co-crystallized with another protein, the homodimer deltaG(diss) is about 7. Is this a significant difference? Thanks. Mohamed
[ccp4bb] Estimate NCS copies
Dear all Is it possible to estimate the number of NCS copies by looking at reflections in the reciprocal space? If so, where can I find the details? Thanks. Mohamed
Re: [ccp4bb] How to convince oneself (and others) of ligand presence
Dear all First of all, thanks for the replies here and off-list. I reprocessed the data to 2 A, and modeled two copies of the ligand in each of the two protein NCS molecules, without using NCS restraints for refinement. I also calculated an SA omit map and a polder map. In the end, the ligand molecules have a relatively low B factor and CC of 0.8 and 0.9. The fact that these two ligands are in the same place (active site) in both NCS copies suggests they are not some random noise. However, I also could model two more of the ligand outside of the active site, also with low B factor and CC > 0.8. Can I call these 'adventitious' molecules? Thanks. Mohamed On Thu, 23 Feb 2017 13:56:05 +0000, Mohamed Noor <mohamed.n...@staffmail.ul.ie> wrote: >Dear all > >I think I have a ligand (substrate) placed in the active site of my model >correctly. The CC is 0.785, B factor of 60 A^2 which is roughly similar to the >neighboring residues and there was no ligand in my search model*. The data >extends to 2.2 A, although I think I can get something higher with manual >processing**. How can I be really sure that it is the ligand that I think it >is? And assuming you are a reviewer, what would you expect to see in a >manuscript? > >* The unliganded model was obtained by molecular replacement using a search >model that did not have the ligand. I then used this model to simply do one >cycle of rigid-body refinement and another one cycle of coordinate/ADP >refinement. > >** The data was auto-processed during data collection using xia2/XDS. I have a >dozen of datasets so I am just taking the autoprocessed ones and see if my >ligand is there. > >Thanks. >Mohamed
[ccp4bb] How to convince oneself (and others) of ligand presence
Dear all I think I have a ligand (substrate) placed in the active site of my model correctly. The CC is 0.785, B factor of 60 A^2 which is roughly similar to the neighboring residues and there was no ligand in my search model*. The data extends to 2.2 A, although I think I can get something higher with manual processing**. How can I be really sure that it is the ligand that I think it is? And assuming you are a reviewer, what would you expect to see in a manuscript? * The unliganded model was obtained by molecular replacement using a search model that did not have the ligand. I then used this model to simply do one cycle of rigid-body refinement and another one cycle of coordinate/ADP refinement. ** The data was auto-processed during data collection using xia2/XDS. I have a dozen of datasets so I am just taking the autoprocessed ones and see if my ligand is there. Thanks. Mohamed
[ccp4bb] Pocket identification and substrate tunnelling
Dear all I think this was discussed before on this board but I can't find the relevant thread(s), so apologies. I have a crystal structure on an enzyme with the NAD+ cofactor bound but no substrate, despite trying both co-crystallization and soaking methods and collecting datasets from 20-30 crystals. Is there a way of identifying the approximate path that the substrate takes from the outside into the active site computationally using electrostatics, residue chemistry etc.? Thanks Mohamed
[ccp4bb] Salt bridge-hydrogen bonds
Dear all Is it possible for two residues to form a salt bridge between them, and at the same time**, each of them form a hydrogen bond with another residue? In other words: Arg1 - Glu10 (salt bridge) Arg1 - Tyr600 (H bond) Glu10 - Thr590 (H bond) ** I understand proteins are not static structures, so I am referring to an exact time and space here and not something formed and broken throughout a sampled period of time.
[ccp4bb] autoSHARP: Am I on the right track?
Dear all Following on the previous thread on Fe-SAD, there is a solution(?) from autoSHARP with an OK-ish map and model. A quick refinement with phenix.refine gave me an R/Rfree of 28/31 % (2 A, although for the phasing run I told autoSHARP to use only up to 2.5 A). However, the model was filled in with about 6000 dummy atoms (DUM) plus some regions with just Gly. I did provide a sequence file, so not sure why it didn't build them in. When I deleted all the DUM atoms, the R/Rfree increased to about 48 %. I am wondering, are the positions of DUM atoms correct and I just need to build it manually somehow? Thanks. Mohamed
[ccp4bb] Metrics for hkl2map (Fe SAD)
Dear all I am trying to solve a structure using Fe SAD collected with mini-kappa from several non-isomorphic crystals (following on from the previous thread https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1608=CCP4BB=0=35088). Xtriage suggests weak anomalous signal to 4 A. For the SHELX pipeline using hkl2map, are there any metrics/numbers that I can use to judge the quality of each dataset? For example, what should be the value of CCall/CCweak to decide between strong/promising and useless cases? Is there any difference between using CRANK2 and hkl2map, considering that the underlying software are the same? Will the wavy Rmerge affect the 'solvability'? Thanks. Mohamed
[ccp4bb] Off-topic: Request for DNA
Dear all I am looking for a small amount of Aquifex aeolicus DNA or cell pellet for PCR. Unfortunately neither ATCC nor DSMZ holds this bacterium. Thanks.
[ccp4bb] Point group
Dear all I can process my 3.8 A dataset in either P4 or P422 point groups. MR searches and refinement in SG P41 and P41212 results in R/Rfree of around 30/35 % with 8 and 4 NCS copies, respectively. Pointless doesn't seem to complain but Xtriage suggests 25 % twinning in the former (refinement was done without twin law) and that the true point group could be P422. So do I go with P422? Secondly, where can I find the DIALS equivalent to XDS FRAME.cbf? The statistics are slightly better but I prefer to confirm it visually. I recently had a case of pseudosymmetry which makes me suspicious of automated processing suggestions. Thanks.
[ccp4bb] PISA values
Dear all I have a dimer that is related to each other by a crystallographic symmetry. My questions are: a. how reliable are the delta G values given by PISA? Has there been an experimental study? b. For a dissociation delta G of say, around 30 kcal/mol, is it possible to estimate the sort of chemical forces (pH or ionic strength) that will be needed to experimentally produce the monomer? Thanks. Mohamed
[ccp4bb] Refinement with phase/FOM and HL coefficients
Dear all In a few places (Refmac and Phenix, maybe there are also others), there is an option to use either phase/FOM or HL for refinement and DM. Is there any difference between these two? The dataset in question is a Fe SAD dataset. Thanks. Mohamed
[ccp4bb] PDB deposition - sequence file
Dear all The protein that was crystallized is only the first 105 residues of a 230-residue protein. In the structure, I can see density for residues 6-72. For deposition, should the whole native/biological sequence be deposited? Thanks. Mohamed
[ccp4bb] How far does rad dam travel?
Dear all In a metal-containing crystal of (say) 200 um x 200 um, and a beam size of 10 um x 10 um, how far will I need to move away from an irradiated part to a fresh part to obtain an undamaged dataset? Exposure conditions: 100 % transmission at 10^12 ph/s, 0.1 s exposure, fine sliced at 0.1 degree/frame with a total 180 degrees. Obviously it will be crystal dependent but I would like to have a rule of thumb. I could use fresh crystals altogether, but not all crystals diffract well unfortunately. Thanks. Mohamed
[ccp4bb] Intensities and amplitudes
Dear crystallographers Is there any reason for using one data type over the other? Are there any errors associated with the French and Wilson I-to-F conversion step? Thanks. Mohamed
[ccp4bb] Lysozyme crystals
Dear all Would anyone have nice images of lysozyme crystals in different space groups (monoclinic, triclinic and tetragonal)? Google wasn't particularly helpful... Thanks. Mohamed
