Re: [ccp4bb] Expert opinion for optimizing ethylene glycol crystallization condition
Shankar, I would suggest to also set up a few plates with your protein in HEPES instead of Tris. I once worked for months trying to improve tiny crystals while my protein was in Tris, pH 7.4, to no avail. I got beautiful crystals when I purified my protein in HEPES, pH 7.4, with everything else the same. HTH, Cale On Fri, Jan 4, 2013 at 11:11 AM, Sankaranarayanan Srinivasan texs...@gmail.com wrote: Dear all, Thank you very much for all your helpful comments. I will try them and post on the BB my results. Best regards Shankar On Wed, Jan 2, 2013 at 11:59 AM, Sankaranarayanan Srinivasan texs...@gmail.com wrote: Dear all, A very happy new year to all. I would appreciate some expert advice on optimizing a crystallization condition in which the initial hits were obtained with ethylene glycol as the main precipitant. Here is the summary of things tried. We have a protein, size (31Kda) and the starting protein buffer is 0.1M Tris pH7.5, 0.1M NaCl, 10% glycerol. The initial crystal hit was obtained from the emerald cryo kit condition that has 0.1M imidazole pH 8.0 , 50% (v/v) ethylene glycol. The crystals were tiny (10-20um). A crystallization matrix to obtain better crystals by varying the imidazole pH and ethylene glycol concentrations was tried from which the best condition obtained was 0.1M imidazole pH 6.5 , 30% (v/v) ethylene glycol. The crystals were slightly bigger 50um. On trying the additive screen, bigger crystals (200um) were obtained, but putting them under the x-ray beam with direct freezing did not yield any diffraction spots. Trying other cryo-conditions like glycerol and 50-50 paratone/oil mixture also yielded similar results. Low resolution spots near the beam stop were also not seen. Similarly spots indicative of salt was also not seen. It just had hazy ice rings kind of stuff. (The beam was definitely on the crystal) To check if what we have was salt, a control condition with no protein was tried. Also the crystals were run on a gel after thorough washing. Both these tests, show that they are definitely protein crystals and not salt. Seeding also did not yield any improved crystals. I was suggested using di-ethylene glycol, propane diol as alternatives. I would greatly appreciate if you can give your opinion on using other di-alcohols as precipitants or other ways to improve these crystals. I tried searching the PDB to see if someone had actually used ethylene glycol as a precipitant, most of them were used as a cryo condition than actually as a precipitant. Thank you very much in advance. Regards Shankar Srinivasan
Re: [ccp4bb] do you think it is interesting?
If we are to strictly adhere to polymer as describing few, then were do we stand with DNA/RNA being described as a polymer - a long polymer made up from repeating units of nucleotides, as has been used in textbooks for ages?! Is DNA/RNA too now a myriomer? Cale
Re: [ccp4bb] protein lost on membrane of centricon!!
Hello Rashmi, How large are your protein monomer units? Are you expecting these units to have formed an oligomer? As a general rule of thumb, you want your protein molecules to be no smaller than 3x the membrane MWCO. Perhaps all you need is to try concentrating with a lower MWCO membrane, e.g. 3,000. Alternatively, you could try: - change membrane manufacturers (this sometimes does make a difference), - pretreating your membrane (e.g. with BSA) to block all binding sites (but then you'll have to worry about BSA contamination of your sample), - concentrating your protein in under nitrogen pressure (I forget right now what the device is called but I used to use it all the time), - concentrating your protein in a speed vac to evaporate off some of the water, - ammonium sulphate precipitation, - or even TCA precipitation. HTH, C On Sat, Jan 28, 2012 at 10:54 AM, rashmi panigrahi rashmi.panigrah...@gmail.com wrote: Hi all, I tried to concentrate my protein using vivaspin 20 10,000 MWCO PES. The protein was in 50mM Hepes pH 7.5, 500mM KCl and 10% glycerol. I lost about 90% of my protein on the membrane of the centricon. Please suggest some way of concentrating this protein. Will concentrating using peg 20K be a good alternative?? regards rashmi
Re: [ccp4bb] protein lost on membrane of centricon!!
P.S. I haven't personally tried concentrating with PEG 20K but I suppose it could work. On Sat, Jan 28, 2012 at 12:41 PM, Cale Dakwar c.dak...@gmail.com wrote: Hello Rashmi, How large are your protein monomer units? Are you expecting these units to have formed an oligomer? As a general rule of thumb, you want your protein molecules to be no smaller than 3x the membrane MWCO. Perhaps all you need is to try concentrating with a lower MWCO membrane, e.g. 3,000. Alternatively, you could try: - change membrane manufacturers (this sometimes does make a difference), - pretreating your membrane (e.g. with BSA) to block all binding sites (but then you'll have to worry about BSA contamination of your sample), - concentrating your protein in under nitrogen pressure (I forget right now what the device is called but I used to use it all the time), - concentrating your protein in a speed vac to evaporate off some of the water, - ammonium sulphate precipitation, - or even TCA precipitation. HTH, C On Sat, Jan 28, 2012 at 10:54 AM, rashmi panigrahi rashmi.panigrah...@gmail.com wrote: Hi all, I tried to concentrate my protein using vivaspin 20 10,000 MWCO PES. The protein was in 50mM Hepes pH 7.5, 500mM KCl and 10% glycerol. I lost about 90% of my protein on the membrane of the centricon. Please suggest some way of concentrating this protein. Will concentrating using peg 20K be a good alternative?? regards rashmi
Re: [ccp4bb] Sub-angstrom resolution
In theory, no: sub-angstrom resolution can be obtained for any and all proteins, including membrane proteins, and for large complexes. In reality, it becomes technically very difficult to achieve; you would need ever-colder temperatures and ever-stronger irradiation sources. P.S. In theory, the only limit to describing the location of the atoms would be described by the heisenberg uncertainty principle. On Mon, Jan 9, 2012 at 1:15 PM, Theresa H. Hsu theresah...@live.com wrote: Dear crystallographers A theoretical question - can sub-angstrom resolution structures only be obtained for a limited set of proteins? Is it impossible to achieve for membrane proteins and large complexes? Theresa
Re: [ccp4bb] Refmac and metal on a two-fold?
Good to know, thanks. I was actually wondering about this recently. On Sun, Jan 1, 2012 at 1:14 PM, Dima Klenchin klenc...@facstaff.wisc.eduwrote: I've seen this happening to water molecules as well (in a somewhat unpredictable fashion). In the latest refmac versions, you can try harmonic restraints, although these will only slow down the atom drift, as the target position is updated every cycle. Perhaps you can use distance restraints against a dummy atom to fix the metal ion in place. Thanks, Ed! Just in case anyone else has the same issue: With Garib's help, I have forced the atom into its position by using external distance restrains of zero length against the same symmetry-related atom. The cause is unclear because the same program handles special positions in another structure just fine. Here is the exact command I used: EXTERNAL DISTANCE first chain M residue 3 atom MN - second chain M residue 3 atom MN value 0.0 sigma 0.00 symm Y Occupancy set manually to 0.5. - Dima
Re: [ccp4bb] How to calculate the percent of the buried hydrohobic surface area in a protein with known structure
Jiyuan, I believe PISA will easily do this for you. C On Thu, Nov 10, 2011 at 5:10 PM, Ke, Jiyuan jiyuan...@vai.org wrote: Dear All, ** ** I have a protein that exists as a dimer in the crystal structure. I want to calculate and compare the area of the buried hydrophobic core of a monomer with that of a dimer? Does anyone know how to do this? Thanks in advance! ** ** Jiyuan Ke, Ph.D. Research Scientist Van Andel Research Institute 333 Bostwick Ave NE Grand Rapids, MI 49503 ** ** -- *The information transmitted is intended only for the person or entity to which it is addressed and may contain confidential and/or privileged material. Any review, retransmission, dissemination or other use of, or taking of any action in reliance upon, this information by persons or entities other than the intended recipient is prohibited. If you received this in error, please contact the sender and delete the material from any computer.* --
[ccp4bb] Assigning secondary structure
Hello all, Given a PDB file of a newly solved protein structure, what is the standard procedure for assigning regions of secondary structure? And by this I mean to ask, how does one decide which residues form beta strands, which alpha helices, and so on? Is DSSP sufficient for this? Are we supposed to manually walk through the entire molecule and assign secondary structure as we deem appropriate based on hydrogen bonding behaviour? Some other procedure? And what of structures solved to ~2.7 A (or worse) where we can't be sure of H-bonding. Cheers, Cale
Re: [ccp4bb] PDB data mining
Thank you to everyone who replied. I went through all the suggestions and in the end used Jason's PyMOL script, using Thomas' cpv.distance suggestion (which did make it much faster for me) and a few more modifications to eliminate redundant pairing listings. Bellow is the modified script, saved as dist_set1.py, run in terminal using /Applications/PyMOLX11Hybrid.app/Contents/MacOS/MacPyMOL -cq dist_set1.py It works on all pdb files in the directory /Users/cale/pdb/set1 It works on gziped pdb files so that the folder pdb can be compressed to be smaller All pdb files must be in the same folder (set1) as it does not traverse subdirectories It is limited to at most ~30,000 files in one folder so I had to split the ~72,000 files mirrored off the PDB into three folders (set1, set2 and set3) and generate an output file for each individually. the files can then be merged together into one using cat. # begin script # import glob, os, pymol, sys from pymol import cmd from chempy import cpv the_pdb=/Users/cale/pdb/set1 files = glob.glob(the_pdb+os.sep+*.ent.gz) if not len(files): print Please set 'the_pdb' variable to a valid path containing PDB files. sys.exit(1) else: print Processing %d files. % len(files) s, outFile = resn HIS and name ND1, dist_set1.csv f = open(outFile, 'wb') # write the header f.write(PDB\tCHAIN\tRESI\tATOM-A\tCHAIN\tRESI\tATOM-B\tDISTANCE\n) # for each file in the mirror for x in files: cmd.load(x,finish=1) n = cmd.get_names()[0] m = cmd.get_model(s).atom # pairwise for each atom for aa in m: for bb in m: # avoid distances to self if aa==bb: continue # avoid duplicates if aabb: continue distance = cpv.distance(aa.coord, bb.coord) # don't list if distance is above 10 angstroms # if distance 10 : continue f.write( %s\t%s\t%s\t%s\t%s\t%s\t%d\t%f\n % (n, aa.chain, aa.resi, aa.index, bb.chain, bb.resi, bb.index, distance)) cmd.delete(n) f.close() print Processed %d files. Please see %s for results. % (len(files), outFile) # end script # Cheers, Cale
[ccp4bb] PDB data mining
Hello all, For any given structure in the PDB, I want to identify all the Histidine ND1 atoms. I then want to consider these atoms in pairs, measure the distance in Angstroms between the ND1 atoms in each pair, and compile these distances (along with residue numbers of the pair) in a table. I then want to repeat this procedure for each unique structure in the PDB and generate a table containing all occurrences of HisND1 pairs with their corresponding separation distance. Amongst other things, I want e.g. generate a histogram from this table and determine e.g. the shortest HisND1 pair distance observed and the structure in which this happens. Does anyone have any suggestions for any tools I might be able to use to perform this search? Thanks in advance, C
Re: [ccp4bb] PDB data mining
Thanks for the quick reply. This is a very good resource but not quite what I need. As far as I can tell, this resource is based on a dataset from 10-08-1998 and then only included x-ray crystal structure with better than 2 angstrom data. I also can't in this database a mention of the PDB structures in which e.g. the shortest His-His distances have been observed. - C On Tue, Mar 8, 2011 at 6:19 PM, Ed Pozharski epozh...@umaryland.edu wrote: On Tue, 2011-03-08 at 17:44 -0500, Cale Dakwar wrote: Amongst other things, I want e.g. generate a histogram from this table and determine e.g. the shortest HisND1 pair distance observed and the structure in which this happens. For this specific question this can be useful http://www.biochem.ucl.ac.uk/bsm/sidechains/index.html -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] PDB data mining
Hello Bob, I am familiar a little with scripting but was hoping someone knew of an already existing tool. Alright, its off to perl then. Thanks, -c On Tue, Mar 8, 2011 at 7:28 PM, Robert Immormino immorm...@gmail.comwrote: Hi Cale, My advice would be to find someone local with experience in scripting possibly in python or perl. This is a tractable problem with a bit of scripting and if you aren't already experienced this may be a good opportunity to delve into a bit of programing. Good Luck, -bob On Tue, Mar 8, 2011 at 7:20 PM, Cale Dakwar c.dak...@gmail.com wrote: Thanks for the quick reply. This is a very good resource but not quite what I need. As far as I can tell, this resource is based on a dataset from 10-08-1998 and then only included x-ray crystal structure with better than 2 angstrom data. I also can't in this database a mention of the PDB structures in which e.g. the shortest His-His distances have been observed. - C On Tue, Mar 8, 2011 at 6:19 PM, Ed Pozharski epozh...@umaryland.edu wrote: On Tue, 2011-03-08 at 17:44 -0500, Cale Dakwar wrote: Amongst other things, I want e.g. generate a histogram from this table and determine e.g. the shortest HisND1 pair distance observed and the structure in which this happens. For this specific question this can be useful http://www.biochem.ucl.ac.uk/bsm/sidechains/index.html -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
[ccp4bb] PDB Validation Server
Dear colleagues, Please excuse these naive questions but I am new to the field of structural biology and need to ask it. 1) By submitting my structure to the PDB Validation Server for precheck and validation, will my structure become publicly available in any way? 2) When I finally submit my structure using the ADIT deposition tool, which would be the appropriate structure factor amplitudes file to include in the deposition: an mtz version of the original sca file obtained from HKL2000 not having gone through any refinements, or the final refined mtz file outputted by Refmac? Thank you. Regards, Cale
[ccp4bb] Refmac 5.5 gives very low B-factors
Dear
