[ccp4bb] Assistant Professor Position in Biochemistry & Cell Biology at Dartmouth
Dartmouth is searching for a new tenure-track junior faculty member to join the Department of Biochemistry & Cell Biology. A description is appended below - please feel free to share. Questions about the position can be addressed to biochemis...@dartmouth.edu TENURE-TRACK FACULTY POSITION The Department of Biochemistry & Cell Biology at Dartmouth’s Geisel School of Medicine invites applications for a tenure-track faculty position at the Assistant Professor level. We seek outstanding scientists working in broadly defined areas of biochemistry, biophysics, cell, chemical, or structural biology. The department offers a dynamic and interactive environment, with a commitment to research excellence and a thriving multidisciplinary graduate program. Candidates may also benefit from synergies with the Norris Cotton Cancer Center and the new COBRE-funded Institute for Biomolecular Targeting. A generous start-up package as well as access to state-of-the-art research facilities will be provided. Successful candidates are expected to develop an independent research program and to participate in graduate level teaching. Applicants should have a PhD, MD or equivalent degree with relevant postdoctoral training. Application materials should include a cover letter, curriculum vitae, statements of research accomplishments and goals, and three letters of recommendation. Please upload application materials electronically to: apply.interfolio.com/38066. Review of applications will begin on November 14, and continue until the position is filled. Dartmouth is an Equal Opportunity/Affirmative Action Employer with a strong commitment to diversity and inclusion. We prohibit discrimination on the basis of race, color, religion, sex, age, national origin, disability, veteran status, marital status, or any other legally protected status. Applications by members of all underrepresented groups are encouraged. -- Dean R. Madden, Ph.D. Professor of Biochemistry & Cell Biology Director, COBRE Institute for Biomolecular Targeting Geisel School of Medicine at Dartmouth 7200 Vail Building, Rm 408A Hanover, NH 03755-3844 USA tel: +1 (603) 650-1164 fax: +1 (603) 650-1128 e-mail: dean.mad...@dartmouth.edu
Re: [ccp4bb] ultrafiltration/buffer exchange device for insect cell cultures
As an alternative to the Pellicon system, Millipore also makes PrepScale TFF cartridges (http://www.millipore.com/techpublications/tech1/ef063). Unlike the Pellicon, they are sealed units, but like the Pellicon membranes, they are true tangential-flow devices and can be reused many times (20preps or ~1year in our hands). The time required to concentrate insect-cell culture medium from 10-20L to 1L, is also on the order of hours, depending on the MWCO and the size of the cartridge. With 10L, the 2.5ft^2 3MWCO cartridge should take 1-2 hours. For 10L at 3MWCO, use the 6ft^2 cartridge. When we compared prices a number of years ago, the upfront cost was lower with the TFF. If you're going to be a long-term heavy user, it may be worth it to invest in the Pellicon. Prices may have changed... Another point: if you use a peristaltic pump like the Masterflex, keep a close eye on the tubing in the head. The back pressure can be significant at the required flow rates and can cause tubing failure. We replace the section within the pump head after each use. Dean Michael Hothorn wrote: Dear all, sorry for the non-CCP4 question. I am expressing a protein in insect cells that is targeted for secretion into the medium. After expression, I want to concentrate the medium containing my protein of interest (from 10 l to 0.3 L) and at the same time change the buffer to something that is compatible with my first ion exchange chromatography step. I would be interested to know what kind of ultrafiltration devices can be used for this purpose, and whether some of you could suggest a quick, reliable and efficient system that we could purchase for our lab. Thanks! Michael -- Dean R. Madden, Ph.D. Department of Biochemistry Dartmouth Medical School 7200 Vail Building, Rm 408A Hanover, NH 03755-3844 USA tel: +1 (603) 650-1164 fax: +1 (603) 650-1128 e-mail: dean.mad...@dartmouth.edu
Re: [ccp4bb] an over refined structure
Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. The same effect applies to a large extent even if the NCS is not crystallographic. Bottom line: thin shells are not a perfect solution, but if NCS is present, choosing the free set randomly is *never* a better choice, and almost always significantly worse. Together with multicopy refinement, randomly chosen test sets were almost certainly a major contributor to the spuriously good Rfree values associated with the retracted MsbA and EmrE structures. Best wishes, Dean Dirk Kostrewa wrote: Dear CCP4ers, I'm not convinced, that thin shells are sufficient: I think, in principle, one should omit thick shells (greater than the diameter of the G-function of the molecule/assembly that is used to describe NCS-interactions in reciprocal space), and use the inner thin layer of these thick shells, because only those should be completely independent of any working set reflections. But this would be too expensive given the low number of observed reflections that one usually has ... However, if you don't apply NCS restraints/constraints, there is no need for any such precautions. Best regards, Dirk. Am 07.02.2008 um 16:35 schrieb Doug Ohlendorf: It is important when using NCS that the Rfree reflections be selected is distributed thin resolution shells. That way application of NCS should not mix Rwork and Rfree sets. Normal random selection or Rfree + NCS (especially 4x or higher) will drive Rfree down unfairly. Doug Ohlendorf -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eleanor Dodson Sent: Tuesday, February 05, 2008 3:38 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] an over refined structure I agree that the difference in Rwork to Rfree is quite acceptable at your resolution. You cannot/ should not use Rfactors as a criteria for structure correctness. As Ian points out - choosing a different Rfree set of reflections can change Rfree a good deal. certain NCS operators can relate reflections exactly making it hard to get a truly independent Free R set, and there are other reasons to make it a blunt edged tool. The map is the best validator - are there blobs still not fitted? (maybe side chains you have placed wrongly..) Are there many positive or negative peaks in the difference map? How well does the NCS match the 2 molecules? etc etc. Eleanor George M. Sheldrick wrote: Dear Sun, If we take Ian's formula for the ratio of R(free) to R(work) from his paper Acta D56 (2000) 442-450 and make some reasonable approximations, we can reformulate it as: R(free)/R(work) = sqrt[(1+Q)/(1-Q)] with Q = 0.025pd^3(1-s) where s is the fractional solvent content, d is the resolution, p is the effective number of parameters refined per atom after allowing for the restraints applied, d^3 means d cubed and sqrt means square root. The difficult number to estimate is p. It would be 4 for an isotropic refinement without any restraints. I guess that p=1.5 might be an appropriate value for a typical protein refinement (giving an R-factor ratio of about 1.4 for s=0.6 and d=2.8). In that case, your R-factor ratio of 0.277/0.215 = 1.29 is well within the allowed range! However it should be added that this formula is almost a self-fulfilling prophesy. If we relax the geometric restraints we increase p, which then leads to a larger 'allowed' R-factor ratio! Best wishes, George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-2582 *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail: [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] *** -- Dean R. Madden, Ph.D. Department of Biochemistry Dartmouth Medical School 7200 Vail Building Hanover, NH 03755-3844 USA tel: +1 (603) 650-1164 fax: +1 (603) 650-1128 e-mail: [EMAIL PROTECTED]
Re: [ccp4bb] an over refined structure
Hi Ed, This is an intriguing argument, but I know (having caught such a case as a reviewer) that even in cases of low NCS symmetry, Rfree can be significantly biased. I think the reason is that the discrepancy between pairs of NCS-related reflections (i.e. Fo-Fo') is generally significantly smaller than |Fo-Fc|. (In general, Rsym (on F) is lower than Rfree.) Thus, moving Fc closer to Fo will also move its NCS partner Fc' closer to Fo' *on average*, if they are coupled. Dean Edward Berry wrote: Actually the bottom lines below were my argument in the case that you DO apply strict NCS (although the argument runs into some questionable points if you follow it out). In the case that you DO NOT apply NCS, there is a second decoupling mechanism: Not only the error in Fo may be opposite for the two reflections, but also the change in Fc upon applying a non-symmetrical modification to the structure is likely to be opposite. So there is no way of predicting whether |Fo-Fc| will move in the same direction for the two reflections. I completely agree with Dirk (although I am willing to listen to anyone explain why I am wrong). Ed Edward Berry wrote: Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. I don't think this is right- remember Rfree is not just based on Fc but Fo-Fc. Working in your lower symmetry space group you will have separate values for the Fo at the two ncs-related reflections. Each observation will have its own random error, and like as not the error will be in the opposite direction for the two reflections. Hence a structural modification that improves Fo-Fc at one reflection is equally likely to improve or worsen the fit at the related reflection. The only way they are coupled is through the basic tenet of R-free: If it makes the structure better, it is likely to improve the fit at all reflections. For sure R-free will go down when you apply NCS- but this is because you drastically improve your data/parameters ratio. Best, Ed -- Dean R. Madden, Ph.D. Department of Biochemistry Dartmouth Medical School 7200 Vail Building Hanover, NH 03755-3844 USA tel: +1 (603) 650-1164 fax: +1 (603) 650-1128 e-mail: [EMAIL PROTECTED]
Re: [ccp4bb] an over refined structure
Hi Phil, Here I will disagree. R-free rewards you for putting in atom in density which an atom belongs in. It doesn't necessarily reward you for putting the *right* atom in that density, but it does become difficult to do that under normal circumstances unless you have approximately the right structure. However in the case of multi-copy refinement at low resolution, the refinement is perfectly capable of shoving any old atom in density corresponding to any other old atom if you give it enough leeway. ... So there's evidence, w/o simulation, that the 12-fold or 16-fold multicopy refinements are worth 7-8% in R-free, and I'm doubtful that NCS can generate that sort of gain in either crystal form. I've certainly never seen that in my own experience at low resolution. Remember that there are two things at work here: putting atoms into real density (which does reduce Rfree) and putting atoms into noise (overfitting, which shouldn't help Rfree). At low res, there's a lot of noise. If you think about it, there is an analogy to relaxing geometrical constraints, which also allows the refinement to put atoms into density. The reason it usually doesn't help Rfree is that the density is spurious. At least some of the incorrect structure determinations of the early 90's (that spurred the introduction of Rfree etc.) had high rms deviations, suggesting that this is how the overfitting occurred. Nevertheless, once hit with a bit of simulated annealing, the Rfree values of such models deteriorated significantly. I would argue that 12-fold or 16-fold multicopy refinements simply permitted overfitting of noise. In other words, it is worth 7-8% in R*work*, but not Rfree. In this case, the main reason Rfree also dropped is because the test set was coupled *by NCS* to the overfit working set. Use of a random test set in the presence of NCS could easily prevent the Rfree value from serving as a warning of overfitting. Of course, to be absolutely sure, one would have to repeat the multicopy refinements of the inverted structures with a test set chosen in thin shells, and then see if Rfree dropped as before. I think only the original authors would be in a position to do that properly. Dean -- Dean R. Madden, Ph.D. Department of Biochemistry Dartmouth Medical School 7200 Vail Building Hanover, NH 03755-3844 USA tel: +1 (603) 650-1164 fax: +1 (603) 650-1128 e-mail: [EMAIL PROTECTED]
