Re: [ccp4bb] DNA RNA annealing problem
The concentration of oligonucleotide seems much lower than you will ultimately need for biophysical work. Annealing two strands is a bimolecular reaction and it will be driven by mass action more to completion at higher total oligonucleotide concentrations. I also agree with the posts that have suggested higher salt concentrations. The charge density of double-stranded nucleic acids is much higher than for extended or even helical single-stranded nucleic acids. Higher salt will more effectively screen those charges and promote double helix formation. Tris would not be my first choice for pH control in a sample that is going to experience a 75C shift in temperature. The pKa temperature coefficient for Tris is about -0.03 pH units per degree C, so the pH will change by over two pH units during your annealing protocol. If you are starting at pH 7.5, and 95C the pH will shift to just above 5. The pKa of cytosine is about 4.5 in solution, and in a highly negatively charged oligomer, it should shift to higher values. Protonated bases will not form Watson-Crick base-pairs as expected. I think you might do better with a buffer with a lower temperature coefficient. Back in the day, we used to anneal our oligonucleotides in cacodylate buffer. Sure it is toxic but it has a nearly flat temperature dependence and very few bacteria will live on cacodylate. On Jul 3, 2015, at 10:14 AM, ChenWeiFei weife...@outlook.com wrote: Dear all, I want to get a complex of DNA-RNA-protein. But I have a big problem of annealing DNA-RNA. The length of DNA is 19nt and RNA is 17nt. Annealing protocol: 2uM DNA 2uM RNA 10mM Tris-Hcl 100mMNaCl 1mMEDTA Heated to 95 for 5min, cooling down slowly for nearly 2h to room temperature. I can just get a result of two single strand DNA/RNA. PAGE analysis. No double helix was founded. Does anyone have the same problem or know how to fix it. Thank you for your answering. Best regards, Weifei
Re: [ccp4bb] Aimless and multiple crystals
How close are the cell constants? On May 8, 2014, at 12:17 PM, Katherine Sippel katherine.sip...@gmail.com wrote: I forgot to mention the space group is P1. Sorry about that. Thanks, Katherine On Thu, May 8, 2014 at 12:02 PM, Katherine Sippel katherine.sip...@gmail.com wrote: Hi all, I have two nice, but a hair under-complete data sets that I am trying to merge together. If I run aimless on them separately everything looks beautiful. When I merge them together the CC1/2 is still good but all the various and sundry Rs sky rocket. When I go to the log file and look at the Rs in respect to batch there is a jump between the first and second data set, so clearly they are not scaling well in respect to one another. I suspect that there is some obvious scripting command that I am missing, but I couldn't find it in the Aimless documentation or the bb archives. Any help would be appreciated. Cheers, Katherine -- Nil illegitimo carborundum - Didactylos -- Nil illegitimo carborundum - Didactylos
Re: [ccp4bb] Space group problem
It is definitely P6something or P3something with a twinning about the unique axis. The differences in merging statistics between P2 P3 and P6 are not very significant in my opinion. On May 7, 2014, at 1:16 PM, Rain Field rainfiel...@163.com wrote: additional info: If I let xds go through, it will choose P6. actually pointless suggest P62/P64. The thing is the Rmeas and Rmerge are significantly higher for P2/P3/P6 than P1, especially the highest shell. That indicates those higher symmetry ones are not the choice, it that right? (Actually, this is also the reviewer's question) P6 log from xds: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 8.454512 216 239 90.4% 3.1% 3.3% 4511 95.55 3.2% 100.0*41* 1.124 182 6.277208 330 330 100.0% 4.0% 4.0% 7208 72.47 4.1% 100.0*22* 1.211 293 5.208925 401 405 99.0% 5.4% 5.4% 8925 56.60 5.5%99.9*120.965 368 4.55 10585 470 474 99.2% 6.3% 6.5% 10585 48.76 6.5% 100.0* 30.841 436 4.09 11898 529 542 97.6% 9.1% 9.0% 11898 37.80 9.3%99.9*-60.809 495 3.75 12425 550 573 96.0% 16.9% 16.7% 12425 22.09 17.3%99.7* 10.855 519 3.48 14344 638 639 99.8% 26.3% 26.1% 14344 14.57 26.9%99.3*-30.783 601 3.26 15001 668 671 99.6% 53.6% 56.1% 150017.15 54.8%96.9*-20.745 633 3.08 14177 704 725 97.1% 114.8%124.6% 141652.95117.7%87.5*-30.632 637 total 990754506 4598 98.0% 9.3% 9.5% 99062 30.73 9.5% 100.0* 10.8334164
Re: [ccp4bb] Refmac bond restraints across special positions?
I haven’t tried this in a long time, but in the old days, we would have simply refined one strand. On Mar 12, 2014, at 4:05 PM, Oleg Tsodikov olegtsodi...@gmail.com wrote: Colleagues, We have determined a structure of a palindromic DNA molecule, in which one half of the DNA is in the asymmetric unit. Is there a way to tell REFMAC that there are covalent bonds across asymmetric units? Without such LINK records in the PDB file, REFMAC treats this as a non-covalent interaction and pushes the two DNA halfs apart. The data are at a fairly high resolution, which helps, but the repulsion is still there. Any advice would be greatly appreciated! I imagine this situation is quite rare in macromolecular crystallography. Oleg -- Oleg Tsodikov, Ph.D. Associate Professor of Pharmaceutical Sciences University of Kentucky College of Pharmacy Department of Pharmaceutical Sciences BioPharm Bldg, Room 425 789 S. Limestone Lexington, KY 40536
Re: [ccp4bb] vitrification vs freezing
On Nov 16, 2012, at 12:26 PM, Ronald E Stenkamp wrote: I'm a little confused. Petsko and others were doing low-temperature/freezing/vitrification crystal experiments in the 1970s, right? (J. Mol. Biol., 96(3) 381, 1975). Is there a big difference between what they were doing and what's done now. Ron From Greg's paper: Since the mixed solvents used are fluid at low temperatures, diffusion of substrate into the crystals should be possible if the viscosity is not too high. One of his goals in the work reported in this paper was find fluid media that could be used at very low temperatures that would allow him to diffuse in substrates and trap a Michaelis complex and solve its structure. This is a bit different than the current typical cryopreservation practices that result in a solid system at or below 100K. It is noted that some increase in crystal lifetime is observed at the cold but not cryogenic temperatures explored in his work. Everyone will surely agree that he is one of the pioneers in collecting data on macromolecular crystals at low temperature.
Re: [ccp4bb] vitrification vs freezing
cryopreserved It says that the crystals were transferred to cryogenic temperatures in an attempt to increase their lifetime in the beam, and avoids all of the other problems with all of the other language described. I was really trying to stay out of this, because I understand what everyone means with all of their other word choices. On Nov 15, 2012, at 2:07 PM, James Stroud wrote: Isn't cryo-cooled redundant? James On Nov 15, 2012, at 11:34 AM, Phil Jeffrey wrote: Perhaps it's an artisan organic locavore fruit cake. Either way, your *crystal* is not vitrified. The solvent in your crystal might be glassy but your protein better still hold crystalline order (cf. ice) or you've wasted your time. Ergo, cryo-cooled is the description to use. Phil Jeffrey Princeton On 11/15/12 1:14 PM, Nukri Sanishvili wrote: s: An alternative way to avoid the argument and discussion all together is to use cryo-cooled. Tim: You go to a restaurant, spend all that time and money and order a fruitcake? Cheers, N.
Re: [ccp4bb] cryo for high salt crystal
You can sometimes cryoprotect crystals like this with the Mitegen low viscosity cryo oil and their micro mount loops. If that doesn't work, then you can gradually raise the concentration of sucrose or ethylene glycol so that your crystals freeze well. I don't think it should take anything like 25% sucrose to cryoprotect this in a small mount with little loose solvent around the crystal. Probably less than 10% would do the trick if you can really wick away most of the liquid and get it into the liquid nitrogen as rapidly as possible. Robert Thorne's lab at Cornell has been important in exploring this cryoprotection space. (from the lab web page) Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions. V. Berejnov, N. S. Husseini, O. A., Alsaied and R . E. Thorne, J. Appl. Cryst. 39, 244-251 (2006). Hyperquenching for Protein Crystallography. M. Warkentin, V. Berejnov, and R. E. Thorne, J. Appl. Cryst. J. Appl. Cryst. 39, 805-811 (2006). Cryocrystallography in capillaries: critical glycerol concentrations and cooling rates. M. Warkentin, V. Stanislavskaia, K. Hammes and R. E. Thorne, J. Appl. Cryst. 41, 791-797 (2008). On Jul 10, 2012, at 11:50 AM, Christian Roth wrote: Hi, I have used pure malonate (above 3M) for a similar condition. Sometimes the cryoprotectant alone, without reservoir works quite well Cheers Christian Am Dienstag 10 Juli 2012 18:28:30 schrieb m zhang: regaentDear All, I am sure this question was discussed before. But I am wondering if anyone got the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo plus original reagents in the reservoir precipitate the salts out. And more serious problem is because of high salt in the condition, while I am trying to loop the crystal, both the drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) significantly and very quickly, that cause my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar case? any suggestion will be appreciated. Thanks,Min
Re: [ccp4bb] Substituting zero vs. Fc for unobserved reflections
I would be concerned about the completeness of the data if adding Fcalc values has such a large effect on the appearance of this electron density.
Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
Do you mean N,N-dimethylformamide, aka dimethylformamide, aka DMF, or do I need a chemistry lesson? On Monday 22 January 2007 02:49 pm, Parthasarathy, Gopalakrishnan wrote: Hi Todd, DMF (Dimethyl Fluoride) is a good alternative to DMSO. Sarathy -- Craig A. Bingman, Ph.D. Center for Eukaryotic Structural Genomics and Department of Biochemistry University of Wisconsin--Madison 433 Babcock Drive Madison, WI 53706 (608) 263-5923
Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
On Monday 22 January 2007 04:57 pm, Maneesh Yadav wrote: I've noticed that the iodobenzene does largely disappear overnight (in hanging drop), I don't know if this is because of evaporation or the iodobenzene just falls into the reservoir. Maybe stick to sitting drop. Or it partitions through the vapor phase into the plastic crystallization tray. -- Craig A. Bingman, Ph.D. Center for Eukaryotic Structural Genomics and Department of Biochemistry University of Wisconsin--Madison 433 Babcock Drive Madison, WI 53706 (608) 263-5923
