Re: [ccp4bb] Off topic: Flourescence anisotropy measurement
With this type of behavior, one suspicion is that you didn’t let the reaction come to equilibrium prior to taking the measurement and the variability between time of mixing and measuring between individual replicates is introducing extra variability. Take a concentration at which you know there is a significant signal change and measure the polarization every few minutes for an hour. You might find that you need to pre-incubate the samples for an extended time prior to taking measurements. With that said, my first suspicion is that you have lousy binding. Eric From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad Khan Sent: Friday, July 21, 2017 9:33 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement Dear all, I am trying to measure the difference in polarization upon the binding of the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in difference of polarization with decrease in protein concentration. However, the results are difficult to reproduce and also vary greatly within triplicates of an experiment. Similar observations have been observed by my colleagues with their proteins. Are there any tips or precautions to keep in mind while setting up these reactions? Looking forward for suggestions. Thank you.
[ccp4bb] Postdoctoral Position at the Institute for Bioscience and Biotechnology Research
Dear crystallographic community The Institute for Bioscience and Biotechnology Research (IBBR) at the University of Maryland is seeking a highly motivated postdoctoral fellow. We are seeking an individual with an interest in protein engineering and structural biology to participate in a new IBBR initiative in conjunction with the Center for Biomolecular Therapeutics (CBT). IBBR is a joint research institute, which brings together partner institutions including the University of Maryland College Park (UMCP), University of Maryland Baltimore (UMB) and the National Institute of Standards and Technology (NIST), and is geographically located at the nexus of these institutions and in the hub of Maryland's biotechnology corridor (www.ibbr.umd.edu). The mission of IBBR is to conduct groundbreaking biomolecular and measurement science research to generate innovative technologies and solutions for medical and public health applications. IBBR is committed to providing an exceptional environment for specialized training and to mentoring tomorrow's biotechnology workforce. Interested applicants can apply at the following link: https://ejobs.umd.edu/postings/30999 Regards, Eric _ Eric A. Toth, Ph.D. Assistant Professor University of Maryland School of Medicine Department of Biochemistry and Molecular Biology Marlene and Stewart Greenebaum Cancer Center Section Leader, Structural Biology Center for Biomolecular Therapeutics 9600 Gudelsky Drive Rockville, MD 20850 Email: et...@som.umaryland.edu Phone: x-240-314-6516 Faculty Profilehttp://medschool.umaryland.edu/FACULTYRESEARCHPROFILE/viewprofile.aspx?id=8032 _ Eric A. Toth, Ph.D. Assistant Professor University of Maryland School of Medicine Department of Biochemistry and Molecular Biology Marlene and Stewart Greenebaum Cancer Center Section Leader, Structural Biology Center for Biomolecular Therapeutics 9600 Gudelsky Drive Rockville, MD 20850 Email: et...@som.umaryland.edu Phone: x-240-314-6516 Faculty Profilehttp://medschool.umaryland.edu/FACULTYRESEARCHPROFILE/viewprofile.aspx?id=8032
Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
Have you tried fixing the molecule that looks correct and searching for others? You might have greater than one but less than 9 molecules per ASU. When you do this, try imposing severe restraints on the packing function. This worked for me in Phaser with a difficult case. My anecdotal experience is that, when you have lots of molecules per asu, the correct solution gets swamped by poorly-packed solutions if the default packing penalties are used. Good luck. Sent from my iPhone On May 15, 2014, at 6:50 PM, Matthew Bratkowski mab...@cornell.edu wrote: Hello all, I am working on the structure of a small protein in space group P212121. The protein is monomeric in solution based on gel filtration analysis. The Matthews Coefficeint program indicates that 9-10 molecules per asymmetric unit results in ~50% solvent content, while 1 molecule per asymmetric unit results in ~95% solvent. I tried molecular replacement with a search model which is essentially identical in sequence to my protein, and searched for 9 or 10 molecules/asu. Using MolRep with 9 or 10 molecules/asu, I get poor contrast scores around 1-1.5. However, when using Phaser, I get a solution with one molecules/asu. Likewise, when I went back and tried MolRep with 1 molecule/asu, I got a contrast score of 3.12. This model still has some issues, but looks more correct compaired to models created with 9 or 10 molecules/asu. It seems highly unlikely that a crystal would contain 95% solvent, but is there any possiblility that this could be the case? Assuming that the Matthews coefficient is correct, does anyone have an idea why MR seems to work better for 1 molecule/asu with 95% solvent content compared to 9-10 molecules with 50% solvent content? Alternatively, is there any reason why the Matthews coefficient could be calculating incorrectly? Any suggestions would be helpful. Thanks, Matt
Re: [ccp4bb] Spots not getting indexed properly in protein-DNA complex
That looks like a garden variety mosaic/kinda-crappy crystal. Without being there, it's impossible to tell if it was morphology (e.g. cracks and divots, etc.), crystal handling, or just bad luck. The bottom line is you need better data. The good news is that those crystals diffract to a reasonably good resolution on your home source. The bad news is that the data you have in hand are probably not that useful. It might be a case of just trying 50 crystals before you get a good one or you might have to modify your cryo, crystal growth, etc. Good luck. _ Eric A. Toth, Ph.D. Assistant Professor Department of Biochemistry and Molecular Biology Marlene and Stewart Greenebaum Cancer Center University of Maryland School of Medicine 108 North Greene St. Baltimore, MD 21201 Email: et...@som.umaryland.edumailto:etoth...@umaryland.edu Phone: x-410-706-5345 Fax: x-410-706-8297 http://medschool.umaryland.edu/FACULTYRESEARCHPROFILE/viewprofile.aspx?id=8032 http://crystal.umaryland.eduhttp://crystal.umaryland.edu/ [Description: Description: Description: UMSOM New logo] A Third Century Where Discovery Transforms Medicine From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Appu kumar Sent: Thursday, August 01, 2013 9:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Spots not getting indexed properly in protein-DNA complex Dear all ccp4 user, We have collected a data set of 370 frames with 0.5 OSC for a protein-DNA complex on RAXIS IV detector. The spots look mostly clustered from 50A to 6A region but the whole data is completely spreaded to 2.8A. There are many spots which are very near to each other as if they were merged but closely placed. When we are trying to index the data, its not picking all the spots correctly and giving a unit cell dimension variable from 150 to 500A in one of the axis. Rest two axis axes of unit cell are almost similar ~ 55A, 111A. We tried processing with HKL2000 but not able to index it., I am attaching four 4 images for every 90 degree frames collected. Please look at these images and give your valuable input regarding indexing problem. your suggestions and support will be highly appreciated. Please guide me and help me sorting out the problem. Thank you inline: image001.jpg
Re: [ccp4bb] completeness in scala
In sports, maximal effort is considered to be 110%, so you're actually 9.9% short of getting everything you could out of that crystal at its resolution limit. All things considered, that's not bad. _ Eric A. Toth, Ph.D. Assistant Professor Department of Biochemistry and Molecular Biology Marlene and Stewart Greenebaum Cancer Center University of Maryland School of Medicine 108 North Greene St. Baltimore, MD 21201 Email: et...@som.umaryland.edu Phone: x-410-706-5345 Fax: x-410-706-8297 http://medschool.umaryland.edu/FACULTYRESEARCHPROFILE/viewprofile.aspx?id=8032 http://crystal.umaryland.edu A Third Century Where Discovery Transforms Medicine -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Tuesday, May 15, 2012 1:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] completeness in scala Just a curiosity - I have a dataset at 1.45A for which SCALA reports the highest resolution shell completeness at 100.1%. I am impressed :-) -- Hurry up before we all come back to our senses! Julian, King of Lemurs
