[ccp4bb] Postdoctoral Position at the University of Georgia Center for Vaccines and Immunology
Postdoctoral Research Position in Antibody-Antigen Interactions The Mousa Laboratory at the University of Georgia (UGA) Center for Vaccines and Immunology (CVI) https://vet.uga.edu/cvi in Athens, GA is seeking a creative and enthusiastic postdoctoral fellow to drive forward a research project focused on antibody-antigen interactions. This is a multidisciplinary project with enormous training opportunity in human immunology, virology, and structural biology, including X-ray crystallography and electron microscopy. The Mousa Laboratory studies monoclonal antibodies from humans, mice, and nonhuman primates as therapeutics and to inform vaccine design. The project will focus on the isolation of antibodies, and the immunological and structural characterization of antibody interactions with Pneumovirus and Paramyxoviruses. The postdoctoral fellow also has the opportunity to design protein vaccine constructs informed by structural data and to examine the experimental vaccines in mice and nonhuman primates directly or collaboratively. The Mousa Laboratory uses a number of techniques that offer high potential for training including: X-ray crystallography, electron microscopy, flow cytometry analysis and sorting, protein biochemistry, biolayer interferometry, virus growth and neutralization analysis, enzyme-linked immunosorbent assays, and monoclonal antibody isolation from human, mouse, and nonhuman primate B cells, among many other techniques. The postdoctoral fellow will be expected to design and conduct research within a specified field while receiving advanced training from a designated Principal Investigator to enhance professional skills and research independence needed for pursuit of a career. The postdoctoral fellow will be expected to design and evaluate experiments, develop new ideas and promote current research, and prepare and publish scientific manuscripts under the direction of the Principal Investigator. The postdoctoral fellow may be responsible for operation of scientific equipment. The postdoctora l fellow may be responsible for teaching techniques to others, including training of students and supervision of research staff. Positions are temporary appointments as a research trainee. Funding for this position is available for at least two years. The initial appointment is for one year, and renewal is expected if progress is satisfactory and funds are available. Appointments cannot exceed five years. MINIMUM QUALIFICATIONS: A doctoral degree or equivalent (Ph.D., M.D., ScD., D.V.M., etc.) in an appropriate field is required. Excellent scientific writing ability and strong oral communication skills are required, as is the ability to work effectively and collegially with colleagues. ADDITIONAL PREFERRED QUALIFICATIONS: Previous experience working with protein expression and crystallization of viral proteins is preferred, but not required. Previous experience with X-ray crystallography and/or electron microscopy is preferred. SCIENTIFIC ENVIRONMENT: The CVI at UGA is a unique and collaborative research environment currently housing four research laboratories focusing primarily on respiratory pathogens and emerging infectious diseases. The Mousa Lab is housed in the CVI and trainees have generous bench space, and access to state of the art instrumentation including an OctetRED384 system. UGA houses a Mosquito drop-setting robot, a state of the art in-house X-ray beamline, and negative-stain electron microscopes. UGA is a member of the Southeast Regional Access Collaborative Access Team (SER-CAT) at Argonne National Laboratory, and abundant beamtime is available. We also have access to the Electron Microscopy facility at Emory University, which includes a FEI Talos Arctica 200 KV cryo-electron microscope. The University of Georgia (UGA), a land-grant and sea-grant university with statewide commitments and responsibilities is the state's oldest, most comprehensive, and most diversified institution of higher education (http://www.uga.edu/). UGA is currently ranked among the top 20 public universities in U.S. News & World Report. The University's main campus is located in Athens, approximately 65 miles northeast of Atlanta. UGA employs approximately 1,800 full-time instructional faculty and more than 7,600 full-time staff. The University's enrollment exceeds 36,000 students including over 27,500 undergraduates and over 8,500 graduate and professional students. Interested applicants should send their CV and list of 2-3 references to Jarrod Mousa, Ph.D. at jarrod.mo...@uga.edu. Publications of interest are below: Wen, X., Mousa, J.J., Bates, J. Lamb, R.A., Crowe, J.E., Jardetzky, T.S. Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus. Nature Microbiology 2, 2017, 16272. Mousa, J.J., Kose, N., Matta, P., Gilchuk, P., Crowe, J.E. A novel pre-fu
Re: [ccp4bb] Questions about antibody in crystallization
Yes, the hinge region between Fab and Fc is highly flexible. If you look at IgG by EM you will see many different conformations. Best to use Fab or scFv. Jarrod Mousa On Thu, Jun 22, 2017 at 1:39 PM, Cheng Zhang <chengzh1...@gmail.com> wrote: > Hi all, > > I have a naive question about antibodies. Many people used Fab fragments > in crystallization. I am wondering if it is possible to use the whole IgG > molecule with Fc fragment as well. Or it would be too flexible and bad for > crystallization? > > Thanks, > > Cheng > > > -- > - > Cheng Zhang >
[ccp4bb] Lipid molecule
Hi all, Picture attached. I solved the structure of a membrane protein using LCP. When trying to identify the cation-binding site, I co-crystallized with Rb+. I see strong density corresponding to Rb+, and it was confirmed in an isomorphous difference map. One problem: in the native structure I see strong density for a lipid molecule from the LCP, in which I can model in very well. In the Rb+ structure, I see more of a blob of density at low rmsd, but when at higher the spherical Rb begins to show. Based on the isomorphous differnece map showing nice spherical denstiy for Rb+, it seems the crystal I shot contains protein molecules with the lipid and others with Rb+. I currently have Rb+ modeled in, but there is lots of extra density surrounding it, corresponding to the lipid molecule. Should I put both molecules in the structure, even though it doesn't make much sense and would results in odd lipid-cation clashes? Or should I just leave the Rb+ without lipid since this makes sense chemistry-wise? Thanks, Jarrod - Jarrod J. Mousa, Ph.D. Bruner Laboratory Department of Chemistry University of Florida email: mo...@chem.ufl.edu
[ccp4bb] Best screens for Fabs and antibody-antigen complexes
Hi all, A little off-topic: I am beginning a post-doc in a non-crystallography lab in a month, and I am working to get a list together of screens to purchase before I arrive. Can anyone recommend screens for crystallizing antibodies (Fab fragments), and Fab-antigen complexes? Thanks, -Jarrod
[ccp4bb] RfreeRwork
Hi all, I am wondering what the conditions are when Rfree is less than Rwork. I have seen this when trying certain models to solve my structure, but I am not sure why this occurs, or if this is valid or not. Thanks, Jarrod Mousa U. of Florida
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
Hi Thomas, I have been doing LCP for a while and I am using the teflon ferrules that come with the 250 uL RN syringes instead. So on one side you would have two teflon ferrules and the other side you would have the ferrule that is attached to the needle and another plastic ferrule. I have also had luck using a 100 uL teflon ferrule (you have to hollow it out a bit) for this purpose. We have a machine shop here that can do the ferrule removal. I didn't have much luck doing this on my own. Hope that helps, Jarrod Mousa Graduate Research Assistant University of Florida -Jarrod Mousa On Mon, Dec 15, 2014 at 2:09 PM, Thomas Cleveland thomas.clevel...@gmail.com wrote: Hi all, I'm trying to put together some homemade syringe couplers following the published instructions from the Caffrey group. I'm having a bit of trouble with this part: The stainless steel ferrule of the second needle is removed and placed on the free end of the coupling needle such that the double thumb nut is held symmetrically between the two steel ferrules Has anyone done this, and if so, how did you remove the stainless steel ferrule from the second needle in order to place it over the first? The stainless steel ferrule appears to be firmly attached and I'm having trouble removing it. Thanks, Thomas Cleveland
[ccp4bb] Merging Data from Multiple Crystals
Hi, I am trying to solve the structure of a membrane protein. The protein has 12 helices and I have a good molecular replacement model that seems to work for about half of the structure. I used chainsaw to convert the amino acid residues to that of my protein sequence, and the density fits the structure well on one side of the protein, but on the other side (about 5 helices), there doesn't seem to be any density for the side chains. Has anyone had experience with this? The completeness is high ~99% for 3.2 angstroms. The data was collected from fairly small crystals ~ 20um. Thanks.
[ccp4bb] Merging Data from Multiple Crystals
I am also trying to merge data from multiple crystals that I collected from. I have tried to reindex the data in CCP4 after indexing in iimosflm, and then put these .mtz files through sortmtz before scala, but I keep getting an error in sort mtz: From ccp4_lwbat: warning: attempt to add new batch with existing batch number 1! SORTMTZ: LWBAT: error in ccp4_lwbat, see messages above Times: User: 0.3s System:0.0s Elapsed: 0:00 *** * Information from CCP4Interface script *** The program run with command: /Applications/ccp4-6.4.0/bin/sortmtz HKLOUT /Users/bruner/Desktop/combinedfiles.mtz has failed with error message SORTMTZ: LWBAT: error in ccp4_lwbat, see messages above *** #CCP4I TERMINATION STATUS 0 SORTMTZ: LWBAT: error in ccp4_lwbat, see messages above #CCP4I TERMINATION TIME 26 Mar 2014 18:50:58 #CCP4I MESSAGE Task failed Any help would be greatly appreciated!
[ccp4bb] cTruncate problem
I can index my data through imosflm with no problems, but when I try to scale using aimless, the program will not run if I also run truncate (or old truncate). Here is the error message I get below. Could anyone elaborate on what this means? Thanks. P.S. I can take the scaled .mtz file from aimless that was not truncated and get an ok MR solution. COMPLETENESS ANALYSIS (using intensities): Using I/sigI 3 with completeness above 0.85, the estimated useful Resolution Range of this data is 17.964A to 8.982A The high resolution cut-off will be used in gathering the statistics for the dataset, however the full dataset will be output TRANSLATIONAL NCS: No translational NCS detected (with resolution limited to 8.98 A) ANISOTROPY ANALYSIS (using intensities): Eigenvalues: 0. 0. 0. Eigenvalue ratios: nan nan nan ctruncate: Anisotropy correction failed - negative eigenvalue. Times: User: 0.5s System:0.0s Elapsed: 0:00 *** * Information from CCP4Interface script *** The program run with command: /Applications/ccp4-6.4.0/bin/ctruncate -hklin /tmp/bruner/ClbM_29_3_mtz.tmp -hklout /tmp/bruner/ClbM_29_5_mtz_New.tmp -colin /*/*/\[IMEAN,SIGIMEAN\] -colano /*/*/\[I(+),SIGI(+),I(-),SIGI(-)\] -colout New has failed with error message ctruncate: Anisotropy correction failed - negative eigenvalue. ObjectCache: Leaked 0005 refs to *** #CCP4I TERMINATION STATUS 0 ctruncate: Anisotropy correction failed - negative eigenvalue. ObjectCache: Leaked 0005 refs to #CCP4I TERMINATION TIME 25 Mar 2014 18:32:11 #CCP4I TERMINATION OUTPUT_FILES /tmp/bruner/ClbM_29_2_mtz.tmp ClbM /Users/bruner/Desktop/Jarrod/ClbM_29.scales ClbM /Users/bruner/Desktop/Jarrod/ClbM_29_rogues.log ClbM /Users/bruner/Desktop/Jarrod/ClbM_29_normplot.xmgr ClbM /Users/bruner/Desktop/Jarrod/ClbM_29_anomplot.xmgr ClbM /Users/bruner/Desktop/Jarrod/ClbM_29_rogueplot.xmgr ClbM /Users/bruner/Desktop/Jarrod/ClbM_29_correlplot.xmgr ClbM #CCP4I MESSAGE Task failed
