Re: [ccp4bb] 3D
I'm still using passive, interleaved 3D, Zalman-style... Hopefully coot etc will retain support for that...! To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] Crystallization with no precipitant
We have seen a similar thing once, where we obtained proteins crystals simply by dehydration of the drop: Sharpe, M. L., Baker, E. N., and Lott, J. S. (2005) Crystallization of a protein using dehydration without a precipitant. Acta Crystallogr Sect F Struct Biol Cryst Commun 61, 565–568. They diffracted, but weren't much use to us in the end, as they were hard to reproduce or optimise. Hopefully you have more luck than we did!
Re: [ccp4bb] Stereo monitors for use with Pymol and Coot
Thanks to everyone who responded, on and off the list. I thought I'd post a quick summary. To clarify my question slightly (if belatedly) I'm specifically looking for a passive 3D solution to plug into my MacBook Pro whilst on sabbatical in the USA. I'll be using it for a mixture of 2D and 3D viewing, not for many hours of dedicated hand building of my 3.5A maps. Well, I hope not! ;) The LG D2343PB-BN gets honourably mentioned, and is available in several places: http://www.adorama.com/LOCD2343PBBN.html $273.50 including shipping http://www.bhphotovideo.com/c/product/1002116-REG/lg_electronics_d2343pb_bn_23_mon_led_lcd.html $273.50 + shipping Another candidate is this from AOC: http://www.amazon.com/AOC-E2352PHZ-23-Widescreen-Flicker/dp/B005LORQGG $250 including shipping The Asus VG27AH also got good reports, but is listed as either 'out of stock' or 'unavailable'. However, its kid brother, the VG23AH is available: http://www.pinnaclemicro.com/computer/dsku.php?g=VG23AH&m=ASUS $251 including shipping (you need to enter the promo code SAS10 to get $10 off!) This monitor got a poor review on cnet (http://reviews.cnet.com/lcd-monitors/asus-vg23ah/4505-3174_7-35306085.html) but much better and more thoughtful reviews elsewhere (http://3dvision-blog.com/7861-review-of-the-23-inch-asus-vg23ah-passive-3d-ips-display) so I'm going to go with the Asus for the following very scientific reasons: 1) It's $20 cheaper than the LG 2) The colours felt a bit washed out on the only AOC monitor I've used previously 3) It has built in speakers and I don't have any on my desk My feeling is that these will all perform in a very similar fashion - I'll report back once I've plugged it in! cheers Shaun
Re: [ccp4bb] Stereo monitors for use with Pymol and Coot
A rather US-centric question on passive 3D monitors... I'm just getting set up in the US, and I'm surprised on how few passive 3D monitors seem to be around - many models seem listed as 'out of stock' when looking in the usual places (Amazon, NewEgg, BestBuys, Walmart etc.) The best deal I have found is for an LG D2343PB-BN (http://www.lg.com/us/commercial/lcd-computer-monitors/lg-D2343PB-BN) at US$274 Does anyone have any experience with this model, or any suggestion about where best to buy 3D monitors in the US? many thanks in advance Shaun
Re: [ccp4bb] OSX and stereo
Yep - works fine on Coot and Chimera. Others may correct me, but I don't think Schrodinger have re-instated support in PyMol? I use it daily - the Zalman is the primary monitor on my desk. The great advantage of this type of monitor (IMHO) is that it is hardware independent.
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
Just to add my 2c worth... The department here has a couple of nanodrops as a shared facility, one for DNA/RNA and one for protein. It has been noticeable that over time people has been getting decreased reliability of measurements on the latter machine cf cuvette measurements, presumably due to the build-up of protein deposits over time - so I would say that although it's easier to clean than a cuvette, the nanodrop is not immune to the problem. The biggest issue I see with the nanodrop is evaporation of sample. Even here in moist Auckland, where RH is very often 80%+, taking a series of measurements with the nanodrop over a period of just a minute or two shows increasing concentration in the sample. So, for consistent results, one has to be careful to measure quickly. It's probably fine for comparative measurements, but as has been observed above, not great for super-accurate values for biophysics, and I think rather operator dependent. But all our students are super-careful, right? ;) Worth to note also that ProtParam calculates extinction coefficients based on Gill & von Hippel, (Gill, S.C. and von Hippel, P.H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319-326) who claim accuracy of ~5% for 'normal globular' proteins without extra chromophores. Whilst on this subject, I would put in a plug for the good old BCA (aka Pierce) assay for protein concentration. It's a little slow, but gets away from sequence dependency somewhat as it is primarily dependent on the peptide backbone rather than sidechains and works well in micro-titre plates etc. It is certainly very superior to Bradford. (Smith, P.K., et al. (1985). "Measurement of protein using bicinchoninic acid". Anal. Biochem. 150 (1): 76–85. doi:10.1016/0003-2697(85)90442-7). cheers Shaun
Re: [ccp4bb] Using chaperones to boost expression in E. coli
As with all these things, YMMV. In our experience, chaperone co-expression can help not at all, can produce more folded protein or can produce more soluble protein, with co-purifying chaperone. With GroEL/S at least, chaperone release is easy (add ATP) and sometimes results in the liberation of folded protein, sometimes not. One case where for us chaperones made an enabling difference: Acta Cryst. (2005). F61, 403–406 Like most things, worth trying! cheers Shaun
[ccp4bb] Chair in Structural Biology at The University of Auckland
The School of Biological Sciences at the University of Auckland, New Zealand invites applications for a Chair in Structural Biology. The ideal candidate will combine X-ray crystallography with a broad range of biophysical, biochemical and cell technologies to solve relevant biological problems of fundamental and applied nature. The successful candidate will establish his/her own research portfolio and actively contribute to teaching and graduate supervision. Commensurate with the seniority of the position, the appointee will also provide leadership for the Structural Biology Section within the School of Biological Sciences, which currently comprises five research groups and around 40 staff and students. See http://www.bioscienceresearch.co.nz/research/structural-biology for details. Facilities currently available in the Structural Biology Laboratory include a Rigaku Micromax 007HF rotating anode generator and two Mar345 image plate detectors on a Dtb with mounting robot, with Osmic mirror optics and cryocooling; a Cartesian nanolitre dispensing robot for crystallization; numerous FPLC systems, and access to DLS, ITC, CD, high-field NMR (Bruker 600 MHz with cryoprobe), excellent cryo-EM (FEI Tecnai 12 and forthcoming Tecnai 20) and advanced mass spectrometry facilities. The Laboratory is situated in the School of Biological Sciences, which has close to 200 staff and supervises over 150 graduate students. It also has strong links with the University of Auckland Medical School and forms part of the Maurice Wilkins Centre, a national Centre of Research Excellence. It is located on the University’s city campus which is in a beautiful park setting close to Auckland’s picturesque harbour. For general information on the position and informal enquiries, please contact Prof. Ted Baker (ted.ba...@auckland.ac.nz) Applications close on 15 October 2010. To apply for this position and for further information and other conditions go to http://www.auckland.ac.nz/opportunities and search for position number 12305. The School of Biological Sciences at the University of Auckland, New Zealand invites applications for a Chair in Structural Biology.
Re: [ccp4bb] macbook pro 13"
I have the previous model with NVIDIA GeForce 9400M and it's been just fine - on the desktop I use a Zalman stereo and it's great.
Re: [ccp4bb] Deglycosylation enzymes
Or you could just bug me to find the PNGaseF expression plasmid that I should have in the freezer somewhere! LOO T., PATCHETT M. L., NORRIS G. E. & LOTT J. S. “Using Secretion to Solve a Solubility Problem: High-yield Expression in E. coli and Purification of the Bacterial Glycoamidase PNGase F.” Protein Expression and Purification, 24, 90-98 (2002)
Re: [ccp4bb] domain boundary, new fold, structure-based sequence alignment
In my experience, DALI can be better than SSM at detecting distant structural similarities. You might also want to try CE. To decide if a fold is 'new' rather than 'old but decorated' often boils down to a somewhat subjective call, but asking Alexei Murzin is as good a way as any to decide :-) For domain definition, try http://pdomains.sdsc.edu/ which offers a handy interface for comparing a number of different methods for automatic domain calling. hope this helps Shaun
Re: [ccp4bb] heavy atom derivative choice
>For gel-shift assay, do people normally use a special gel tank for heavy >metal work? We used to use a Pharmacia Phast system, as this is bufferless and made it very easy to contain heavy metal contamination. Ours caught fire and died a couple of years ago, and I have yet to find a good cheap replacement - does anyone know of a native gel equivalent of the Invitrogen E-PAGE for example? In the absence of such niceties, just treat the gels and electrophoresis solutions as heavy metal waste.
[ccp4bb] Applications invited for PhD Projects in Structural Biology at the University of Auckland, New Zealand
PhD Scholarships available in Structural Biology in the School of Biological Sciences at the University of Auckland, New Zealand Applications are invited for PhD scholarships in the following areas: The structure of insecticidal toxins from Yersinia entomophaga. (Dr. Shaun Lott, Dr Mark Hurst, Biocontrol and Biosecurity, AgResearch) The insecticidal tc toxins produced by a number of bacteria form large (~2.5MDa) complexes, where the assembly of up to seven proteins are required to show full insecticidal activity. The general architecture of these complexes has previously been established using single- particle EM analysis, but the structural details of the complexes, and their mode of action, remain obscure. This project aims to elucidate the structures and functions of components of the tc toxin complex from the bacterium Yersinia entomophaga. Drug targets from M. tuberculosis. (Dr. Shaun Lott, Professor Ted Baker) We have recently solved the structure of several enzymes known to be essential for the bacterium to cause disease, including anthranilate phosphoribosyl transferase (AnPRT; TrpD), the enzyme which catalyses the second committed step in tryptophan biosynthesis, isopropylmalate synthase (IPMS; LeuA), the enzyme which catalyses the first committed step in leucine biosynthesis, salicylate synthase (MbtI) which catalyses the production of salicylate, essential for the production of the siderophore mycobactin, and others. Through a combination of in silico modelling and in vitro assay, we have identified a set of weak AnPRT inhibitors, and are embarking on the structure-guided synthesis of more potent versions. We plan to use a similar approach with the other enzymes also, with the intention of producing useful anti- mycobacterial agents for the future. Host lipid-induced transcriptional regulation in M. tuberculosis. (Dr. Shaun Lott, Dr Sharon Kendall, Royal Veterinary College, London) We have recently showed that the essential transcriptional regulator KstR, which has previously been implicated in pathogenesis, directly controls the expression of many lipid metabolism genes in M. tuberculosis. Additionally, a similar transcriptional regulator, KstR2, has also been identified to control a smaller regulon. KstR and KstR2 both belong to the TetR family of transcriptional regulators, and our hypothesis is that the activation of KstR and/or KstR2 is triggered by lipid ligands derived from the human host, triggering bacterial adaptation to the intracellular environment. We are aiming to structurally and functionally characterise these transcription factors to discover more about their mode of action. Novel proteins from Orf virus. (Dr. Chris Squire, Professor Ted Baker, Prof. Andrew Mercer, Virus Research Unit, Otago University) Orf virus is an animal virus, and a member of the poxvirus family. Its genome sequence shows that in addition to the genes required for replication and assembly, it encodes genes for a large number of novel proteins that appear to have no counterparts in other organisms. We believe that many of these proteins are involved in infection, or in immune avoidance (by mimicking components of the host immune system). Some of these have potential therapeutic applications. We have carried out a preliminary bioinformatic survey, and identified a subset of these proteins that we predict to have important functions, and well defined structures. The aim is to express and purify the proteins, test their predicted functions and determine their 3D structures by X- ray crystallography. These scholarships are funded by research grants from the NZ Foundation for Research, Science & Technology, and include University Fees, working expenses and a stipend of NZ$25,000 p/a. Candidates should have a First or Upper Second Class Honours or Masters Degree or equivalent, and have a strong interest in using protein structure to elucidate biological function. Applicants should write to Dr Shaun Lott (s.l...@auckland.ac.nz) or Prof. Ted Baker (ted.ba...@auckland.ac.nz) in the first instance, enclosing a CV and an academic transcript, citing reference AGNRF1, by May 15th 2009. Informal inquiries are welcomed at the same addresses. Auckland was recently ranked joint 4th in the world in the Mercer Quality of Living Survey: http://www.mercer.com/qualityofliving#Top_5_ranking_cities_by_region More information about living & studying in Auckland can be found here: http://www.aucklandnz.com/ More information about the School of Biological Sciences at The University of Auckland can be found here: http://www.sbs.auckland.ac.nz/uoa/science/about/departments/sbs/about-us.cfm More information about Dr Shaun Lott’s Research Group can be found here: http://shaunlott.blogspot.com More information about Professor Ted Baker’s Research Group can be found here
