[ccp4bb] Senior Crystallography Position
Posted on behalf of Professor John Hunt and Professor Liang Tong, North Eastern Structural Genomics Consortium at Columbia University USA. An accomplished and dynamic senior scientist is sought to join the high-throughput protein biochemistry / crystallography group of the Northeast Structural Genomics Consortium (www.nesg.org) at Columbia University. The successful applicant will be appointed as an Associate Research Scientist and will participate in cutting-edge projects directed at using structural information to elucidate the function of uncharacterized protein families as well as engineering proteins for improved crystallization. A track-record in successfully crystallizing proteins is essential, and experience solving structures is a plus. Preference will be given to candidates with expertise in crystallizing protein complexes with nucleic acids. A PhD in a related field and at least three years of post-doctoral experience in protein crystallization / crystallography are required. Salary commensurate with experience. Please respond or enquire to Phillip Manor pm2...@columbia.edu- regards
Re: [ccp4bb] more on lattice translocation defects
There is also another paper on this that may be useful... Acta Cryst. (2005). D61, 67-74. Correction of X-ray intensities from single crystals containing lattice-translocation defects. J Wang et al. Regards -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: Monday, January 26, 2009 6:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] more on lattice translocation defects Dear Laurie, For copyright reasons, I would suggest that you ask the authors for a reprint or pdf. They can legally do it. Concerning lattice translocation defects, as I see it, it means that neighboring layers in a crystal are sometimes translated in one direction, sometimes in another direction. These maybe an integral part of the unit cell (1/2 or 1/3 etc.) as Zhu et al. seem to have, or random values (e.g. 0.32, 0.49). This can lead to several bizarre effects in the diffraction pattern: -if the defects occur frequently, this leads to small coherent domains, which leads to smeared spots. However, spots for which these domains cancel out, stay sharp. As a result, the diffraction pattern shows a mixture of sharp and smeared spots. -The self patterson shows strong peaks for both translations. However, since the translations occur in different unit cells, there are no corresponding cross peaks. -If the translation is almost an integral part of the unit cell, one sees a modulation. E.g. if the translation is 0.45, one sees extinctions for odd h, k or l indexes if the index is around 0, extinctions for even indexes if the index is around 10, extinctions for odd indexes if the index around 20 etc. Best regards, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Laurie Betts Sent: Sunday, January 25, 2009 4:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] more on lattice translocation defects May I inquire if someone has a copy of Zhu et. al. (Acta Cryst. D. 2008 D64, 843-850) that might be posted somewhere free? I don't have subscription to Acta Cryst D and this one is possibly a problem I have in a structure and I don't understand what it means exactly. Or if someone can give a definition in terms that is simple (for my simple mind). Thanks -- Laurie Betts X-ray Crystallography Facility Manager Department of Structural Biology University of Pittsburgh 1050 BST3 3501 Fifth Ave Pittsburgh, PA 15260 412-383-5839
[ccp4bb] Off topic question
Hi CCP4ers Apologies for the off topic... I have been trying to get hold of L-ribulose-5-phosphate (D- is readily available). Does anyone know where I can get such a compound? Thanks in advance for any suggestions and Merry Xmas! Gina
Re: [ccp4bb] helix generator
I am not sure exactly if this is what you are asking but can't you just generate a alpha helix, in moleman, of the desired length, read that helix out and then change the ALA residues in that PDB to the ones you want? Gina On Jul 31, 2008, at 11:10 AM, Jacob Keller wrote: Sorry for being unclear--I meant a protein sequence. Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Kristof Van Hecke To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, July 31, 2008 10:00 AM Subject: Re: [ccp4bb] helix generator 3DNA..?! http://rutchem.rutgers.edu/~xiangjun/3DNA/ Kristof On 31 Jul 2008, at 16:55, Jacob Keller wrote: Dear crystallographers, is there a program around, ccp4 or otherwise, into which one can input a sequence and get a .pdb of an ideal helix of the input sequence? Thanks, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information.
Re: [ccp4bb] Spooky, moving crystals
Hi Mark we also had a problem like this but in one case it was due to an instability in the goniometer head system so that the device still moved even after being "homed". Another few cases were due to an unstable magnetic base that was wobbling about due to the screw, on the gonimeter head, not holding the magnetic base in place correctly. Hope that helps! Gina On Jul 21, 2008, at 4:25 PM, Rizkallah, PJ (Pierre) wrote: Hi Mark, Cryocooled loops stored in tubes inside dewars do flex a little when the loops are put on the goniometer. This is natural, as in the dewar, the whole pin was in liquid nitrogen, but on the goniometer, it is only the loop that is in the cryo stream. Having said that, the degree of movement is small enough not to worry about, but will stabilise within a couple of minutes. Different makes of loops move to different extents, due to the different amount of metal in them. I agree that the Mitegen loops move least, as they are made of more plastic than others, which doesn't change dimensions upon temperature cycling as much as metal pins. When it comes to loops disappearing out of the field of view, then that is almost certainly due to a pin not glued to the base. It works for a little while, but when you start rotating it, gravity sets in, and it jumps randomly. The solution is quite simple. Just a tiny drop of super glue between pin and base, wait long enough for it to set, then rotate. As for empty loops, what Eddy Snell described is very likely if your loop is taken to the cryo stream first, with the stream obstructed. Eddy gave an accurate description of the problem. It might be better to put the loops in a tube in a small open dewar to avoid exactly this problem, then take the tube to the goniometer. Many different ways of achieving the same result. Isn't research wonderful! Pierre ** * Pierre Rizkallah, Daresbury Laboratory, Warrington, Cheshire WA4 4AD, U.K. Phone: (+)44 1925 603808 Fax: (+)44 1925 603124 e-mail: [EMAIL PROTECTED] html: http://www.srs.ac.uk/px/pjr/ -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mark J. van Raaij Sent: 21 July 2008 18:47 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Spooky, moving crystals Dear all, in a recent synchrotron trip we had a problem with our crystals moving after mounting them onto the goniometer, in some cases they moved out of the beam and even out of the zoomed camera picture - it seemed the pins, upon equilibrating to room temperature, extended. It happened with pre-mounted litho-loops only, not with pre-mounted mitegen loops on the same trip, so one possible cause is different metal allows used in the pins, somehow the mitegen ones being more suitable. We used two-component glue to stick the pins into the metal bases (Spine), so that might be another possible culprit. Perhaps we did not allow sufficient time for the glue to react before freezing into liquid N2 and it continued its reaction upon thawing, somehow pushing the pin a bit out of the base. In this case the difference between litholoops and mitegen loops may have been the thickness of the pins, the latter somehow allowing expansion of the glue along the sides, the former not. In any case, I am wondering if any of you has seen this before, so we know how to avoid it in the future. In some cases, it took 10-20 min. for the crystal to stop moving, which, with the current data collection speed and robotic mounting, is significant. Fortunately, it did not affect our trip too much, as we has sufficient time in the end. Greetings, Mark Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/
Re: [ccp4bb] Concentrating protein
Hi there I actually just consulted, about your question, with one of the longer term members of the department about this. And we came to the conclusion that 4- 500ml was probably the maximum size. Gina On Jun 30, 2008, at 11:15 AM, Radisky, Evette S., Ph.D. wrote: Another question re: Amicon stirred cells... I also seem to recall seeing 1L size stirred cells in older labs of my youth. My current lab has acquired one of 400 mL, but looking to purchase a bigger one, I can't find any. Any ideas about where we might find one? Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Gina Clayton Sent: Friday, June 27, 2008 7:37 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Concentrating protein Hi there I quite like the Amicon stirred ultra concentration cell systems. You can put large volumes in, maximum 1 litre size, I think. As well you can attach an inert gas such as Argon or Nitrogen, for the gaseous pressure, this reduces oxidation of your sample while it concentrates. My experience has been that, depending on the filter, the filters are very resistant to various salts even GuHCl, and you get good recovery. I used to concentrate large volumes of protein down to say 50-25ml then switch to the same system, in a much smaller cell i.e. 10ml, to get down to 1-2ml. And they are fairly fast too. I get the impression, perhaps incorrectly, they are not as fashionable as they used to be, but perhaps "older labs" tend to have them milling about somewhere in the back of a cupboard. So most likely you would only have to buy membranes -PM or YM it think depending on you sample. Hope that helps Gina On Jun 27, 2008, at 9:19 AM, Roger Rowlett wrote: Guenter Fritz wrote: A mild and quick method is to use dry Sephadex G-25. The material will swell and take up all the liquid except molecules larger than ca. 5 kDa. Dear All, we have GCSF protein produced in inclusion bodies. we solubilise it refold it and then concentrate it using proflux system. still the concentration of the protein we get is less and volume is more for us to load in Ion exchange chromatography. is there any simple technique that can be performed in lab without using any hi-fi instrument to concentrate the protein in small volume of buffer. the protein we obtain is about 0.7 mg/ml and we get 450 ml solution. our column is 110ml lab scale and we have to work in that only. i have heard of NH4SO4 precipitation. but it requires protein conc more than 1 mg/ml. kindly help me to progress in my experiment. One of the beauties of ion-exchange chromatography is that it is an excellent concentration step as well as a purification methodology. It may take less time and involve less protein loss to pass all the solution through the IEX column and bind the protein, assuming you have the protein in a low ionic strength buffer at the appropriate pH. Elution in a smaller volume can be accomplished by increasing the NaCl concentration to an appropriate level. In the bad old days before bacterial overexpression, we used to to this routinely to concentrate a liter or more of protein extract to 50-100 mL after elution from a small, high-capacity IEX column. Cheers, -- - - -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
[ccp4bb] Disordered domains in crystal strutures
Dear CCP4ers can anyone recommend papers describing crystal structures of proteins with a large functionally important disordered domain or domains. Thanks in advance Gina
Re: [ccp4bb] Concentrating protein
Hi there I quite like the Amicon stirred ultra concentration cell systems. You can put large volumes in, maximum 1 litre size, I think. As well you can attach an inert gas such as Argon or Nitrogen, for the gaseous pressure, this reduces oxidation of your sample while it concentrates. My experience has been that, depending on the filter, the filters are very resistant to various salts even GuHCl, and you get good recovery. I used to concentrate large volumes of protein down to say 50-25ml then switch to the same system, in a much smaller cell i.e. 10ml, to get down to 1-2ml. And they are fairly fast too. I get the impression, perhaps incorrectly, they are not as fashionable as they used to be, but perhaps "older labs" tend to have them milling about somewhere in the back of a cupboard. So most likely you would only have to buy membranes -PM or YM it think depending on you sample. Hope that helps Gina On Jun 27, 2008, at 9:19 AM, Roger Rowlett wrote: Guenter Fritz wrote: A mild and quick method is to use dry Sephadex G-25. The material will swell and take up all the liquid except molecules larger than ca. 5 kDa. Dear All, we have GCSF protein produced in inclusion bodies. we solubilise it refold it and then concentrate it using proflux system. still the concentration of the protein we get is less and volume is more for us to load in Ion exchange chromatography. is there any simple technique that can be performed in lab without using any hi-fi instrument to concentrate the protein in small volume of buffer. the protein we obtain is about 0.7 mg/ml and we get 450 ml solution. our column is 110ml lab scale and we have to work in that only. i have heard of NH4SO4 precipitation. but it requires protein conc more than 1 mg/ml. kindly help me to progress in my experiment. One of the beauties of ion-exchange chromatography is that it is an excellent concentration step as well as a purification methodology. It may take less time and involve less protein loss to pass all the solution through the IEX column and bind the protein, assuming you have the protein in a low ionic strength buffer at the appropriate pH. Elution in a smaller volume can be accomplished by increasing the NaCl concentration to an appropriate level. In the bad old days before bacterial overexpression, we used to to this routinely to concentrate a liter or more of protein extract to 50-100 mL after elution from a small, high-capacity IEX column. Cheers, -- -- -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
Re: [ccp4bb] Bacterial induction at 18C
Raji aside from the possibilites of toxic protein as already mentioned.. we had great results with overnight induction at 20oC for a protein that was somewhat insoluble at 37oC. One thing that might work for you is to grow the cells to OD 0.5 then lower the temperature to say 18 or 20. After an hour or so (retake OD) induce for overnight growth. Alternate is higher starting OD prior to temperature reduction (i.e greater mass of cells) and shorter growth time say 5-6 hours as you are thinking (we had protein that we only induced for 1 and a half hours too). Hope that is useful! Gina -- Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks.
Re: [ccp4bb] The importance of USING our validation tools
I thought that when a structure is deposited the databank does run its own refinement validation and geometry checks and gives you back what it finds i.e distance problems etc and rfactor? Quoting Eleanor Dodson <[EMAIL PROTECTED]>: The weighting in REFMAC is a function of SigmA ( plotted in log file). For this example it will be nearly 1 for all resolutions ranges so the weights are pretty constant. There is also a contribution from the "experimental" sigma, which in this case seems to be proportional to |F| Yesterday I attached the wrong TRUNCATE log file - here is the correct one, and if you look at the plot "Amplitude Analysis against resolution" it also includes a plot of Eleanor Dominika Borek wrote: There are many more interesting things about this structure - obvious fake - refined against fabricated data. After running refmac I have noticed discrepancies between R and weighted R-factors. However, I do not know how the weights are calculated and applied - it could maybe help to find out how these data were created. Could you help? M(4SSQ/LL) NR_used %_obs M(Fo_used) M(Fc_used) Rf_used WR_used NR_free M(Fo_free) M(Fc_free) Rf_free WR_free $$ $$ 0.0052205 98.77 3800.5 3687.2 0.12 0.30 121 4133.9 4042.7 0.12 0.28 0.0153952 99.90 1932.9 1858.7 0.20 0.60 197 2010.5 1880.5 0.21 0.40 0.0255026 99.81 1577.9 1512.3 0.23 0.62 283 1565.0 1484.6 0.26 0.54 0.0345988 99.76 1598.0 1541.5 0.23 0.61 307 1625.7 1555.6 0.23 0.42 0.0446751 99.79 1521.2 1481.6 0.18 0.41 338 1550.3 1523.8 0.18 0.61 0.0547469 99.81 1314.5 1291.2 0.14 0.29 391 1348.3 1337.7 0.15 0.27 0.0648078 99.87 .5 1089.1 0.16 0.36 465 1096.1 1077.9 0.18 0.42 0.0738642 99.84 976.7 959.2 0.15 0.32 488 995.3 988.4 0.16 0.50 0.0839255 99.88 866.4 848.0 0.16 0.36 490 856.8 846.0 0.17 0.38 0.0939778 99.88 747.6 731.4 0.16 0.36 515 772.8 747.3 0.18 0.38 0.103 10225 99.86 662.6 649.1 0.17 0.38 547 658.9 643.6 0.20 0.36 0.113 10768 99.83 597.2 584.7 0.18 0.42 538 593.4 590.0 0.20 0.49 0.122 11121 99.86 535.5 521.9 0.19 0.48 607 556.2 542.0 0.20 0.47 0.132 11692 99.85 489.3 479.2 0.19 0.46 607 476.4 467.3 0.23 0.42 0.142 11999 99.83 453.9 443.1 0.19 0.48 621 455.3 440.6 0.22 0.55 0.152 12463 99.79 419.2 407.3 0.19 0.44 655 435.3 424.3 0.22 0.53 0.162 12885 99.78 384.0 373.9 0.20 0.53 632 384.1 376.1 0.22 0.43 0.171 12698 95.96 357.2 348.5 0.21 0.57 686 353.9 338.6 0.24 0.51 0.181 11926 87.78 332.0 323.3 0.21 0.66 590 333.4 322.6 0.24 0.57 0.191 11204 80.39 309.9 299.6 0.22 0.59 600 302.1 296.3 0.26 0.77 $$ Eleanor Dodson wrote: There is a correspondence in last weeks Nature commenting on the disparities between three C3B structures. These are: 2icf solved at 4.0A resolution, 2i07 at 4.1A resolution, and 2hr0 at 2.26A resolution. The A chains of all 3 structures agree closely, with each other and other deposited structures. The B chains of 2icf and 2i07 are in reasonable agreement, but there are enormous differences to the B chain of 2hr0. This structure is surprisingly out of step, and by many criteria likely to be wrong. There has been many articles written on validation and it seems worth reminding crystallographers of some of tests which make 2hr0 suspect. 1) The cell content analysis suggests there is 80% solvent in the asymmetric unit. Such crystals have been observed but they rarely diffract to 2.26A. 2) Data Analysis: The reflection data has been deposited so it can be analysed. The plots provided by TRUNCATE showing intensity statistic features are not compatible with such a high solvent ratio. They are too perfect; the moments are perfectly linear, unlikely with such large volumes of the crystal containing solvent, and there is absolutely no evidence of anisotropy, again unlikely with high solvent content. 3) Structure analysis a) The Ramachandran plot is very poor ( 84% allowed) with many residues in disallowed regions. b) The distribution of residue B values is quite unrealistic. There is a very low spread, which is most unusual for a structure with long stretches of exposed chain. The baverage log file is attached. c) There does not seem to be enough contacts to maintain the crystalline state.
[ccp4bb] density modification
Hi CCP4ers firstly apologies that this is slighty off ccp4 topic but does anyone know of a programme whereby I can use solvent flipping with multi domain masks in addition to a solvent mask for my phases i.e. not based on a model? I wanted to use CNS density_modify (my maps are currently from DM multi -uses solvent flattening) but DM in CNS accepts only one mask. So I guess my real question,which is why it is off topic, does anyone know of a way of successfully modifying density_modify to get it to accept multi masks or an alternative (other than resolve)?" thanks for any advice in advance! Gina
