[ccp4bb] AKTA Prime dead/delay volume...and a transmembrane protein...
Hello all, Does anyone have a rough estimate (or has anyone actually determined) an average dead/delay volume between buffers run on an AKTA Prime FPLC? We are attempting to overexpress/isolate a smaller His-tagged transmembrane protein, and require running several detergent buffers in succession over the column. This obviously creates fractions that are just dead volume/ddH2O between buffers, and we would like to narrow in on where to look for the protein prior to analyzing the fractions (i.e. how many fractions can we discard?). Should we run ddH2O and then the first mL into 'waste' before running each buffer over the column (and collecting fractions)? Just not used to this system yet! Thank you! Jim
[ccp4bb] Last issue - Lower completeness, decent R factors, but low B factor...
If I go back to my SCALA mtz and run 'Truncate', I get a completely different B value at 27.3, which is obviously much better. However, why did I have to do this, and where would I find this value at the end of my refinement? i.e. did I miss a line in PROCHECK or my final REFMAC log file? Thank you all, Jim --- On Wed, 8/20/08, Lijun Liu <[EMAIL PROTECTED]> wrote: > From: Lijun Liu <[EMAIL PROTECTED]> > Subject: Re: [ccp4bb] Lower completeness, decent R factors, but low B > factor... > To: CCP4BB@JISCMAIL.AC.UK > Date: Wednesday, August 20, 2008, 1:32 PM > What is the reason for lower completeness is important. > 1) rotation range is not wide enoughyou lost frames? > What about > the completeness against > resolution shell? > 2) too many rejections? > a) symmetry error > b) really bad crystal (this seems not the case) > c) too many overlaps during collection, mosaicity > Guess 1) is your case, if I can. > > Also, good R factors from low-completeness (either reason > above) is > less reliable. > > Good luck. > > Lijun > > > On Aug 20, 2008, at 9:24 AM, James Pauff wrote: > > > Hello all, thank you for the responses. Just to clear > up a couple > > of things... > > 1) My dataset was acquired on a synchrotron and > scaled/truncated > > there. To my knowledge they used the same procedure > as for other > > structures that we have obtained there...which have > had no B factor > > issues. Granted, our modified protein is of a > different spacegroup > > than previous. > > > > 2) I have not used/touched the TLS parameters at all. > > > > The idea that our 73% completeness (thus lacking 27% > of the > > 'weakest' reflections) has lead to an > artificially low B factor > > sounds most appealing at this point? > > > > As usual, I greatly appreciate all of your insights > here! > > > > Best, > > Jim > > > > > > --- On Wed, 8/20/08, Eleanor Dodson > <[EMAIL PROTECTED]> wrote: > > > >> From: Eleanor Dodson <[EMAIL PROTECTED]> > >> Subject: Re: [ccp4bb] Lower completeness, decent R > factors, but low > >> B factor... > >> To: [EMAIL PROTECTED] > >> Cc: CCP4BB@jiscmail.ac.uk > >> Date: Wednesday, August 20, 2008, 4:30 AM > >> James Pauff wrote: > >>> Hello all, > >>> > >>> I have a refined structure at 2.6 angstroms > that at > >> about 73% completeness at this resolution. The > I/sigma is > >> about 2.0 at 2.6 angstroms, and the omit density > for my > >> ligands is great contoured at 3.0sigma. My Rcryst > is 19 or > >> so and the Rfree is 24.5 or so. > >>> > >>> HOWEVER, my mean B value is 13.9, whereas my > other 2 > >> structures (at 2.2 and 2.3 angstroms, same > protein, >95% > >> completeness) have mean B values of 22+. Any > suggestions as > >> to what is going on here? I'm having trouble > explaining > >> this. > >>> > >>> Thank you, > >>> Jim > >>> > >>> > >>> > >>> > >>> > >>> > >> Have you used TLS - listed B factors will then be > given > >> relative to the > >> TLS parameters. You need to run tLSANL to get a > more > >> realistic value. > >> Eleanor > >> > >> > >> But in fact temperature factors are rather harder > to > >> estimate at lower > >> resolutions than higher. Look at your > and > >> curves v resolution > >> ( part of a REFMAC loggraph) and you can see that > sometimes > >> the overall > >> scaling struggles to get a reasonable fit.. > > > > > > > > Lijun Liu, PhD > Institute of Molecular Biology > HHMI & Department of Physics > University of Oregon > Eugene, OR 97403 > 541-346-4080
[ccp4bb] Wilson plot from truncated.mtz
If I've lost my SCALA MTZ, and have only the truncated.mtz for my dataset, which program is the quickest means of obtaining a Wilson plot? Thank you again, Jim --- On Wed, 8/20/08, Eleanor Dodson <[EMAIL PROTECTED]> wrote: > From: Eleanor Dodson <[EMAIL PROTECTED]> > Subject: Re: [ccp4bb] Lower completeness, decent R factors, but low B > factor... > To: CCP4BB@JISCMAIL.AC.UK > Date: Wednesday, August 20, 2008, 4:30 AM > James Pauff wrote: > > Hello all, > > > > I have a refined structure at 2.6 angstroms that at > about 73% completeness at this resolution. The I/sigma is > about 2.0 at 2.6 angstroms, and the omit density for my > ligands is great contoured at 3.0sigma. My Rcryst is 19 or > so and the Rfree is 24.5 or so. > > > > HOWEVER, my mean B value is 13.9, whereas my other 2 > structures (at 2.2 and 2.3 angstroms, same protein, >95% > completeness) have mean B values of 22+. Any suggestions as > to what is going on here? I'm having trouble explaining > this. > > > > Thank you, > > Jim > > > > > > > > > > > > > Have you used TLS - listed B factors will then be given > relative to the > TLS parameters. You need to run tLSANL to get a more > realistic value. > Eleanor > > > But in fact temperature factors are rather harder to > estimate at lower > resolutions than higher. Look at your and > curves v resolution > ( part of a REFMAC loggraph) and you can see that sometimes > the overall > scaling struggles to get a reasonable fit..
[ccp4bb] Lower completeness, decent R factors, but low B factor...
Hello all, thank you for the responses. Just to clear up a couple of things... 1) My dataset was acquired on a synchrotron and scaled/truncated there. To my knowledge they used the same procedure as for other structures that we have obtained there...which have had no B factor issues. Granted, our modified protein is of a different spacegroup than previous. 2) I have not used/touched the TLS parameters at all. The idea that our 73% completeness (thus lacking 27% of the 'weakest' reflections) has lead to an artificially low B factor sounds most appealing at this point? As usual, I greatly appreciate all of your insights here! Best, Jim --- On Wed, 8/20/08, Eleanor Dodson <[EMAIL PROTECTED]> wrote: > From: Eleanor Dodson <[EMAIL PROTECTED]> > Subject: Re: [ccp4bb] Lower completeness, decent R factors, but low B > factor... > To: [EMAIL PROTECTED] > Cc: CCP4BB@jiscmail.ac.uk > Date: Wednesday, August 20, 2008, 4:30 AM > James Pauff wrote: > > Hello all, > > > > I have a refined structure at 2.6 angstroms that at > about 73% completeness at this resolution. The I/sigma is > about 2.0 at 2.6 angstroms, and the omit density for my > ligands is great contoured at 3.0sigma. My Rcryst is 19 or > so and the Rfree is 24.5 or so. > > > > HOWEVER, my mean B value is 13.9, whereas my other 2 > structures (at 2.2 and 2.3 angstroms, same protein, >95% > completeness) have mean B values of 22+. Any suggestions as > to what is going on here? I'm having trouble explaining > this. > > > > Thank you, > > Jim > > > > > > > > > > > > > Have you used TLS - listed B factors will then be given > relative to the > TLS parameters. You need to run tLSANL to get a more > realistic value. > Eleanor > > > But in fact temperature factors are rather harder to > estimate at lower > resolutions than higher. Look at your and > curves v resolution > ( part of a REFMAC loggraph) and you can see that sometimes > the overall > scaling struggles to get a reasonable fit..
[ccp4bb] Lower completeness, decent R factors, but low B factor...
Hello all, I have a refined structure at 2.6 angstroms that at about 73% completeness at this resolution. The I/sigma is about 2.0 at 2.6 angstroms, and the omit density for my ligands is great contoured at 3.0sigma. My Rcryst is 19 or so and the Rfree is 24.5 or so. HOWEVER, my mean B value is 13.9, whereas my other 2 structures (at 2.2 and 2.3 angstroms, same protein, >95% completeness) have mean B values of 22+. Any suggestions as to what is going on here? I'm having trouble explaining this. Thank you, Jim
[ccp4bb] convenient means to find I/sigma for publications?
Hello all, Silly question, but what is the most convenient means to find the overall I/sigma and the I/sigma for the highest resolution shell in CCP4? At the end of refinement and validation, and after running PROCHECK, I am still using a very convoluted route to obtaining these numbers. Thanks! JMP You rock. That's why Blockbuster's offering you one month of Blockbuster Total Access, No Cost. http://tc.deals.yahoo.com/tc/blockbuster/text5.com
[ccp4bb] CCP4 in Linux Fedora 7
Perhaps a silly question, but I've asked at least a couple of those before... We have been running CCP4 6.0.2 on Windows and Macintosh systems, but have recently purchased two supercomputers that are set up with Linux Fedora 7 for computational work. We are trying to put CCP4 onto these computers, but after going through the install (which I presume worked?), we have no idea how to open the CCP4i interface, or COOT for that matter. Any helpful time-saving suggestions/tips would be greatly appreciated. Best, Jim __ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com
[ccp4bb] HETATM records changed to ATOM records by REFMAC
Good morning/afternoon, evening again all, As sort of a "last question" on this current project, I have noticed that my final input PDB gets altered by REFMAC when it constructs the output PDB. In the output _refmac1.pdb file, all of my HETATM records are changed back to ATOM records, and my TER command lines are gone. What I end up with is simply a list of ATOM records following the PDB header. Is this normal for REFMAC? When I go to submit this PDB to the databank, should I put the HETATM records and the TER commands back in prior to submission? Thank you, Jim Be a better Globetrotter. Get better travel answers from someone who knows. Yahoo! Answers - Check it out. http://answers.yahoo.com/dir/?link=list&sid=396545469
[ccp4bb] HETATM to ATOM change, and atom identifier change in REFMAC...
Good morning/day/evening all, I have a structure that is refined, with the exception of the molybdenum (MoOOS) cofactors (there are two of them), which are square pyramidal and coordinated to 2 sulfurs in an enedithiolate of a pterin ring. I have brought up my issues with refining this before in REFMAC, but something just dawned on me as I work through/with it. Although I define the Mo, O1, O2, S atoms as ATOM records in the PDB, with restrained refinement in REFMAC, the resulting *_refmac1.pdb file converts the MO to a N. If I define the 4 atoms above as HETATM records, the same happens with the output PDB, changing all 4 atoms back to ATOM records and redefining the MO as N. I am also still getting problems with the connectivity of the atoms in the coordination sphere of the molybdenum, but that may be a side issue to this confusing problem with the record lines in my output PDBs here. Thank you, Jim Building a website is a piece of cake. Yahoo! Small Business gives you all the tools to get online. http://smallbusiness.yahoo.com/webhosting
Re: [ccp4bb] needles vs crystals
Shivesh, Having gone down the "needles vs. crystals" route recently, my two suggestions would be: 1) Lower the protein concentration to 10 mg/ml 2) Slightly lower your precipitant percentage, say to 26%. My situation involved PEG4000, and I noticed that modifying the protocol to PEG8000 at a slightly lower percentage worked much better in obtaining rectangular plates versus needle clusters. Best, Jim --- shivesh kumar <[EMAIL PROTECTED]> wrote: > Dear all, > I am trying to crystallize a 7kDa protein with MPD > as a precipitant.I got > the needles with precipitation in two days at 30% > and in 4-5 days,needles > appear in all the drops(pH 3.8-4.8).The > concentration of protein is around > 20mg/ml and the volume of mother liquor in > 500microlt.What should I do to > get good crystals,the temp. I am trying is 16C.Thanx > in advance for the > suggestions. > Shivesh Kumar > Choose the right car based on your needs. Check out Yahoo! Autos new Car Finder tool. http://autos.yahoo.com/carfinder/
[ccp4bb] Metal-ligand(s) positioning following refinement...
Good day all, I have a metal center in my enzyme active site, in a roughly square pyramidal coordination. I know from EXAFS and other work how this coordination sphere should look, but following REFMAC the cofactor here does not end up realistically coordinated/oriented. Although it is in the density and roughly in position, the coordination sphere and ligands are not correct, and not correctly connected. Is there a means by which I can position this cofactor, establish the connectivity I want, and then specifically restrain this cofactor/center while refining? Thank you, Jim Need a vacation? Get great deals to amazing places on Yahoo! Travel. http://travel.yahoo.com/
[ccp4bb] Establishing connecitivity Part II...
Hello again all, sorry to clutter your inboxes... With regards to my problem of connecting a Molybdopterin correctly, does anyone have suggestions as to the use of LINK statements in my coordinate file? Or the use of LINK statements at all? This was an idea when we first started refining the structure, but it never seemed to work right. I have put in LINK statements before, linking the two sulfurs of the pterin to the molybdenum, which has 2 oxygens and one sulfur as ligands, but in my CCP4 log file, I get "Link is found (not be used)" statements. Am I missing a command here? Thank you all again, Jim Choose the right car based on your needs. Check out Yahoo! Autos new Car Finder tool. http://autos.yahoo.com/carfinder/
[ccp4bb] Establishing connecitivity...
Hello again all, We have a molybdopterin active site cofactor. It is a single molybdenum atom, with 2 oxygens and 1 sulfur as ligands, and coordinated to a pterin ring via 2 more sulfur atoms. The geometry of the molybdenum is very roughly square pyramidal. The issue that we are having involves the connectivity of the cofactor. The refinement wants to connect the sulfur ligand to the oxygen ligands, in addition to their respective connections to the molybdenum, and it wants only a single sulfur off the pterin ring connected, not to the molybdenum, but to the sulfur ligand. I have tried playing with the pdb file, but I am basically just unsure as to how best to get the connectivity the way it should be. Is there a way to do this in COOT? Thank you. Jim Park yourself in front of a world of choices in alternative vehicles. Visit the Yahoo! Auto Green Center. http://autos.yahoo.com/green_center/
[ccp4bb] Rfree into an existing mtz...
Good day all, What is the best means of getting a freeR column into an existing mtz file? I've noticed the "Edit MTZ" command bar in CCP4, but there are other options (such as conversion to XPLOR and then back to MTZ) that seem to be able to accomplish the same thing, although there appear to be changes in the reflection parameters upon this conversion. Basically, I just want to add a freeR column to monitor my refinement(s). Thank you as usual. Looking for a deal? Find great prices on flights and hotels with Yahoo! FareChase. http://farechase.yahoo.com/
[ccp4bb] another quick question...
Good day all, In COOT, when I view my coordinate pdb file with my ccp4 map file (using "Auto-open mtz"), I have a couple residues for which only a part of the peptide chain is visible. E.g. for a Lys, only the terminal nitrogens at the end of the R-group chain are visible as bonds. The other parts of the residue (and even parts of adjacent residues) are visible only as stars/stellate points that may or may not be within my blue electron density. If I click on the stars, the atom names that appear are consistent with the missing peptide chain, and correspond to the "missing" amino acid that has the occassional visible R group. Just wondering what this means, that I am seeing stellate points that seem to be acting as place holders for my residues in a couple locations in my structure. The structure was obtained using molrep. Thank you, James Pauff Take the Internet to Go: Yahoo!Go puts the Internet in your pocket: mail, news, photos & more. http://mobile.yahoo.com/go?refer=1GNXIC
[ccp4bb] quick question... thanks
Thank you all, that was much easier than I expected. Best, Jim Boardwalk for $500? In 2007? Ha! Play Monopoly Here and Now (it's updated for today's economy) at Yahoo! Games. http://get.games.yahoo.com/proddesc?gamekey=monopolyherenow
[ccp4bb] quick question...
Good day all, First off, thank you all so much for your help a couple days ago! I just have a quick question regarding the use of CCP4 to generate density maps. Is there a way to generate a map(s) that can be read into PyMol? I understand that PyMol can only read CNS or XPLOR maps, is this correct? Is there a means by which I can output maps (on a Windows platform) in a format that can be read by PyMol? My only option in CCP4 is to create my FFT map in CCP4 format. Thank you, Jim Boardwalk for $500? In 2007? Ha! Play Monopoly Here and Now (it's updated for today's economy) at Yahoo! Games. http://get.games.yahoo.com/proddesc?gamekey=monopolyherenow
Re: [ccp4bb] Ligand insertion and orientation...
Thank you all very much for your help. I am a thousand miles farther than I was at the time I sent out my question(s)!! --- Paul Emsley <[EMAIL PROTECTED]> wrote: > Thanks Juegen, > > > On Wed, May 09, 2007 at 08:45:16AM -0700, Juergen > Bosch wrote: > > Hi James, > > > > start Coot, and read in your cif file plus a > coordinate file containing > > your ligand. Read in your structure and read in > your mtz file as Auto, > > then you end up with two maps FWTxPHWT and > DELFWTxPHDELWT. > > Next go to Calculate / Other Modelling Tools and > select Fit Ligand. > > Since you should have some nice extra density in > you difference map > > select this map to search your ligand in that map. > Let Coot do the rest. > > You can also select flexible ligand docking etc. > > I would also add that for a difference map, I'd > notch up the signigicance > level to 3 sigmas or so. And that should reduce > spurious hits. > > > Should work as advertised. Once your ligand is in > the position you > > expect it to be you can run real space refinement > using the difference > > density map at this point. Then go to > Calculate/Merge Molecules and > > select which one you want to merge, save the new > coordinates including > > your found ligand. > > > > Good luck, > > > > Juergen > > > > P.S. this is assuming Coot 0.31, and there's also > a Coot BB :-) > > 0.3.1 (and not to be > confused with 0.0.31) > > > > > James Pauff wrote: > > > > >Good day all, > > > > > >I have what may be a very simple question. I am > > >trying to insert a substrate/ligand into the > active > > >site of my enzyme using COOT, into electron > density > > >that I have already utilized in "O" for this > purpose. > > >I've created the substrate library file (*.cif) > using > > >a pdb file from PRODRG in the ccp4 sketcher. In > COOT, > > >I went to File->Get Monomer..., but when I type > the 3 > > >letter code, I get nothing. > > OK, Coot should be more informative about what kind > of nothing > you get. > > > >Further, I have imported the substrate as a > separate > > >pdb file, and can move it close to the active > site, > > >but I have no idea how to orient/manipulate the > ligand > > >into the electron density. If I can eventually > get > > >the ligand as a monomer into the screen, I still > don't > > >know how to manipulate its orientation prior to > > >writing it into the enzyme's pdb, so I guess that > I'm > > >just generally stuck here. > > If all else fails, there's alway Rotate/Translate > Zone > (in the Model/Fit/Refine dialog). Using that you can > drag > and rotate the fragment. > __ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com
[ccp4bb] Ligand insertion and orientation...
Good day all, I have what may be a very simple question. I am trying to insert a substrate/ligand into the active site of my enzyme using COOT, into electron density that I have already utilized in "O" for this purpose. I've created the substrate library file (*.cif) using a pdb file from PRODRG in the ccp4 sketcher. In COOT, I went to File->Get Monomer..., but when I type the 3 letter code, I get nothing. Further, I have imported the substrate as a separate pdb file, and can move it close to the active site, but I have no idea how to orient/manipulate the ligand into the electron density. If I can eventually get the ligand as a monomer into the screen, I still don't know how to manipulate it's orientation prior to writing it into the enzyme's pdb, so I guess that I'm just generally stuck here. Any help/suggestions/advice would be appreciated. Thank you all for your time here. Need Mail bonding? Go to the Yahoo! Mail Q&A for great tips from Yahoo! Answers users. http://answers.yahoo.com/dir/?link=list&sid=396546091
