Re: [ccp4bb] Strange Diffraction pattern! Protein/DNA complex or DNA alone crystal?
Dear Xiao and Hans: Thanks for your reply. We tried to index it but failed. Joseph On Mon, Mar 12, 2018 at 11:15 PM, Xiao Lei <xiaolei...@gmail.com> wrote: > did you try to index it? the cell dimensions may give you hint. > > On Mon, Mar 12, 2018 at 4:40 AM, Joseph Ho <sbddintai...@gmail.com> wrote: >> >> Dear all: >> >> >> I would like to seek your wisdom on our latest diffraction pattern. We >> have been working on protein/DNA complex. The protein and DNA have >> similar MW. By binding assay, we know the minimal length of DNA. (The >> Kd is 0.1-1 microMolar and we can see the complex formation in size >> exclusion chromatography up to 200mM NaCl but also some unbound form) >> After trying different length of DNA, we recently obtained many >> crystal hits (the percipient is either PEG400 or MPD). The final ratio >> (prior to protein crystallization) between protein and DNA is 1:1.6 >> considering some loss of protein during concentration. The crystal is >> birefringent. Since high conc. of PEG400 (MPD), the crystals were >> directly frozen in liquid N2. However, crystals only diffract to 8-10 >> angstrom (anisotropic) and also weird striking line are present >> (please see attachment). Do you think if it is DNA alone crystal or >> protein/DNA complex crystal? >> How should I improve the diffraction quality? >> >> >> >> PS. We have done some tests. For example, set up the same conditions >> with DNA alone. I also tried to dissolve crystals in Bradford assay >> solution and I believe I saw some blueish color. But none of these >> tests are conclusive. >> >> Thanks for your suggestion. >> >> Joseph > >
[ccp4bb] Tenure-tracked faculty - Institute of Biological Chemistry, Academia Sinica
Hello everyone: I would like to draw your attention on the tenured track faculty position ((https://jobs.sciencecareers.org/job/472001/tenure-tracked-faculty) in our institute (www.ibc.sinica.edu.tw). We will soon be equipped with two state-of-art CryoEM (Krios and Arctica all with K2/K3 detector) and current have a F20 (http://proj3.sinica.edu.tw/~core/cryotem/06iEnglish.html). In addition, synchrotron (NSRRC) is only 1 hour by driving. NSRRC (http://www.nsrrc.org.tw/) currently has four protein crystallography and one SAXS beamlines. In addition, we are constructing a more power BioSAXS and a microfocus protein crystallography beamlines now (http://tpsbl.nsrrc.org.tw/bd_page.aspx?lang=en=07A=1118). Most of scientific lectures are given in English and the grant application is also in English. Meng-Chiao Joseph Ho The following is our ads and you can find it at (https://jobs.sciencecareers.org/job/472001/tenure-tracked-faculty/) The Institute of Biological Chemistry (www.ibc.sinica.edu.tw), Academia Sinica, Taiwan, is seeking outstanding candidates to fill tenure-tracked faculty positions at all levels. Applicants should possess expertise in fields that would strengthen or complement the current research programs of IBC, particularly in i) cryo-EM and/or mass spectrometry related structural biology research, and ii) integrative omics, biological chemistry and bioinformatics of proteome wide signaling network. Selected candidates will have joint appointment at the Institute of Biochemical Sciences, National Taiwan University (ibs.ntu.edu.tw), be provided with generous start-up fund followed by annual intramural support, and enjoy full access to all shared resources in Academia Sinica including the most advanced Titan Krios and Talos Arctica instruments at the newly established Cryo-EM Facility. Applicants should send their curriculum vitae (including three names of references), a description of recent research accomplishments and future research interests (in pdf files) to the Chair of IBC Recruitment Committee, c/o Ms. Yi-Hwa Huang (email: yi...@gate.sinica.edu.tw). Screening of applications will begin on Jan 1, 2018, and will continue until the position is filled
[ccp4bb] Protein or DNA crystals
Dear all: I would like to seek your opinion on our crystal hits. We are working on protein/dsDNA complex. By changing different protein and DNA (14-22bp) constructs, we recently got some hits from commercial screens using sitting drop vapor diffusion (very small xtals). The precipitant is PEG and the picture of crystals are attached. In this particular condition, it is 30%PEG3350, sodium succinate pH5.5 and 100mM NaCl. The crystal seems floating and sit in the bottom. We do some test shot from other conditions and it is not salt crystals. The crystals can suck in izit dye. I do some google and it seems izit dye also turns dsDNA crystal into blue. We also do UV/Vis microscope but no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp. This is our first time to work on protein/DNA complex crystals and we are not certain if this is just DNA or protein/DNA crystals. Can you provide your comments on our hits? Thank you for your help Joseph
[ccp4bb] suggestion on protein crystallization optimization from phase separation
Dear all: I would like to seek your suggestion on protein crystallization from phase separation. We recently observed many small round droplets shown in our protein/DNA crystallization. Condition are 0.8M-1.6M LiSO4; 20mM MgCl2; pH 5-8; protein conc. ~15mg/ml). The UV microscope confirms those are protein-rich phase separation. We have tried to change conc. of LiSO4 and pH. Still we got different size and amount of small round droplets. At 20 degree, those droplets appear within one day and at 4 degree, it takes two-three days. We also tried additive and silver bullet screen. So far, we have not found a condition to have protein crystals. The protein is already truncated. Several DNA constructs are on-going. At this point, I would like to seek your advice on the method to optimize the condition. Based on PS. Any people have luck with protein crystallization by streaking the Gelationous protein to new drop as shown in http://xray.bmc.uu.se/terese/tutorial3.html . Can you please share your experience with us? Thanks for your help. Joseph Ho
[ccp4bb] postdoc/RA positions available at Academia Sinica
Dear All, The Tsai and Ho labs at the Institute of Biological Chemistry of Academia Sinica in Taiwan seeks several motivated structural biologists/protein chemists at post-doctoral/RA level with interest in structural and functional of phospho-protein protein interaction related to cell signaling. The position is available from now on for initially up to 12/31/2016 with highly possible extension to 12/31/2017. Applicant should have a strong background in protein expression and purification. Experiences in native chemical ligation, un-natural amino acids incorporation using amber codons and structural biology are plus. This position provides the opportunity or broad training in protein crystallography, small angle X-ray scattering, NMR, enzymology and protein-protein interaction. The IBC is equipped with many state of art instruments (http://bcf.assic.sinica.edu.tw/ ; http://www.ibc.sinica.edu.tw/Facility_Protein_E.asp http://www.ibc.sinica.edu.tw/Facility_Biophysics_E.asp ). We also have access to Taiwan synchrotron beamline, ALS, SSRL, Spring-8 and high-field NMR facility (http://www.nmr.sinica.edu.tw/en/). He/She will also closely work with cell biologists in this projects and the language of the application will be English/Chinese. Applicants should submit the curriculum vitae and the names and contact information of three professional references via email to: sbddintai...@gmail.com For more information about the lab, please visit http://www.ibc.sinica.edu.tw/MDTsai/ and http://www.ibc.sinica.edu.tw/ho We are looking forward to receiving your application! Joseph Ho
[ccp4bb] Have everyone had a Scorpion Screen Builder or a Dragonfly screen optimizer?
Dear all: We are interested in purchasing either a Scorpion Screen Builder from ARI or a Dragonfly from TTP labtech for setting up the grid screen. I am wondering if anyone can share their personal experience or opinions with me. Your help/comments are highly appreciated. Joseph
[ccp4bb] positions for PhD students
Dear all: I would like to ask your kind help to distribute TIGP application announcement for 2014 on your network. The Chemical Biology and Molecular Biophysics program of Taiwan International Graduate Program is a Ph.D. program, founded by Academia Sinica, the foremost research institution of Taiwan in 2002. In cooperation with top universities in Taiwan, our CBMB program focuses on synthetic chemistry, structural biology, bioinformatics, kinetics and spectrometry and biotechnology and translational research. The TIGP-CBMB students will learn in all-English teaching and research environments, and enjoy accessing to world-class faculty and state-of-the-art research facilities at Academia Sinica and partner universities. All applicants who are admitted to TIGP will receive a fellowship from Academia Sinica. The application deadline is 31st March 2014. No application fee is required. For more information, please visit TIGP CBMB website. TIGP-CBMB: http://proj1.sinica.edu.tw/~tigpcbmb/ TIGP website: http://tigp.sinica.edu.tw/applying.html. If you have any further inquiries, please contact Ms. Elve Lin. Email: elve...@gate.sinica.edu.tw Thank you in advance for advertising TIGP program. Joseph Ho
Re: [ccp4bb] Removing a tight binding ligand
We often dialysis protein against charcoal to remove small molecules that tightly bind to protein. Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
Re: [ccp4bb] Removing a tight binding ligand
It is just like the regular dialysis but the deserved buffer contains charcol powder. Meng-Chiao Joseph Ho How do you do this ? I have not heard of this, but I also never had to deal with getting rid of a ligand. However I would be interested to learn more about this method. Thanks, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 8, 2010, at 11:01 AM, Joseph Ho wrote: We often dialysis protein against charcoal to remove small molecules that tightly bind to protein. Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
Re: [ccp4bb] Crystal rescue
Hi, Zhiyi: You can always put a layer of oil on the top of your drop when you open the coverslip. It will get you more time to mount the crystals. Good luck Meng-Chiao Ho On Tue, 2010-01-26 at 11:48 -0500, Zhiyi Wei wrote: I forgot to mention a phenomenon when I did crystal mounting. I found that if open a coverslip, phase separation will appear in drops in minutes and damage to the crystals. And I have tried additive screen. The optimized crystals are really big (a few hundred microns) with sharp edge but diffraction pattern is similar. On Tue, Jan 26, 2010 at 10:42 AM, Zhiyi Wei ustcwebri...@gmail.com wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while 8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] phaser troubles
Hi, Sylvia: Did you also install Phenix in your machine? Phenix contains another version of phaser and will alias phaser to the phaser in Phenix. Try to type which phaser. If your command is not aliased to the phaser in your ccp4 version. Just use runview com file in the ccp4 and type in the phaser with the ccp4 directory (for example, /your directory/ccp4-6.1.1/ccp4-6.1.1/ccp4i/bin/phaser/). Meng-Chiao Joseph Ho Postdoc Dept. of Biochemistry Albert Einstein College of Medicine On Mon, 2009-08-24 at 18:03 +0200, Sylvia Fanucchi wrote: Hi This is probably an obvious one but I’m having trouble getting Phaser to run. It simply says the status is “starting” and will not give me more information in the log file. I suspect the problem has to do with the F and Sigma F values but I am not sure what to assign them as? Any help would be appreciated Sylvia This communication is intended for the addressee only. It is confidential. If you have received this communication in error, please notify us immediately and destroy the original message. You may not copy or disseminate this communication without the permission of the University. Only authorized signatories are competent to enter into agreements on behalf of the University and recipients are thus advised that the content of this message may not be legally binding on the University and may contain the personal views and opinions of the author, which are not necessarily the views and opinions of The University of the Witwatersrand, Johannesburg. All agreements between the University and outsiders are subject to South African Law unless the University agrees in writing to the contrary.
[ccp4bb] refining anisotropy B factors on only a subset of atoms
Hello, I have a refined 1.8A structure that I wonder if I could squeeze out some anisotropy information. I did TLS refinement on the protein, and it helped my Rfree. But I would like to ask a biological question based on the thermal movement of only a few waters (7 total). In theory, that is not adding a lot of parameters for anisotropy refinement because I am not refining anisotropy of the whole protein. Correct me if I am wrong. So, 1) How do I use Refmac to refine anisotropic B's on only 7 waters? There is an option to use mixed for B factor refinement, but I don't know how to define the atoms. 2) If this can be done, given my resolution, how much confidence can I put in my anisotropy refinement of those waters? In another word, if I do see thermal ellipsoids of the waters, are they believable? And if I don't, is it because my resolution isn't high enough to see it anyways? I have a total of ~1700 atoms, and ~24000 unique reflections at 1.8A resolution. Thanks! Joseph
[ccp4bb] rastep r3d file for pymol
Hello, I would like to display thermal ellipsoids in pymol. What I did was using rastep to make a r3d file and open it in pymol. If I generate the r3d file with no extra options: rastep infile.pdb outfile.r3d, pymol can open it. But if I generate the file with rastep -auto -fancy1 infile.pdb outfile.r3d, pymol cannot open it. I know I can use render to make a picture, but I would like to be able to view the picture in 3-D in pymol, so I need to make a r3d file (with the -auto -fancy1 options in rastep) that pymol can read. Is it possible? Any suggestions for other ways of doing the same thing? Joe
