Re: [ccp4bb] a challenge
Hi James, The datasets frac.80.mtz to frac.100.mtz are challenging to solve using SAD phasing. However these datasets can be easily solved using other experimental phasing method. Instead of using anomalous signal we could use isomorphous signal only. For example RIP or SIR phasing method, as there is a difference in intensity between the datasets due to scattering of S and Se. Since frac.80.mtz data contains 20% selenium that is sufficient to solve the structure against the frac.100.mtz. It seems the structure can be solved even as less as 10% selenium content (frac.90.mtz vs frac.100.mtz), and substructure can be solved easily. This is not surprising, the pair of the datasets is quite isomorphous, . We phase all reflections (centric and non-centric) where as anomalous phasing we could phase non-centric reflections only. In fact, Single Isomorphous Replacement phasing method is the first phasing technique. This method has been further extended by Ravelli et al with some deviation by introduction of X-ray or UV RIP phasing. I tried RIP (SIR) phasing protocol of Auto-Rickshaw using frac.90.mtz as before and frac.100.mtz as after. Auto-Rickshaw used SHELXC/D/E and ARP/wARP/REFMAC5 to get the partially refined model (Rfree below 30%) . Cheers Santosh Santosh Panjikar, Ph.D. Scientist Australian Synchrotron 800 Blackburn Road Clayton VIC 3168 Australia Ph: +61-4-67770851 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of James Holton [jmhol...@lbl.gov] Sent: Monday, January 14, 2013 8:12 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] a challenge I am absolutely delighted at the response I have gotten to my little John Henry Challenge! Three people already have managed to do the impossible. Congratulations to George Sheldrick, Pavol Skubak and Raj Pannu for finding ways to improve the phases over the ones I originally obtained (using the default settings of mlphare and dm) and build their way out of it. This is quite useful information! At least it is to me. Nevertheless, I do think Frances Reyes has a point. This was meant to be a map interpretation challenge, and not a SAD-phasing challenge. I appreciate that the two are linked, but the reason I did not initially provide the anomalous data is because I thought it would be too much to ask people to re-do all the phasing, etc. Yes, there do appear to be ways to improve the maps beyond the particular way I phased them, but no matter how good your phasing program is, there will always be a level of anomalous signal that will lead to phases that are off enough to make building the model impossible. Basically, once the map gets bad enough that just as many wrong atoms get built in as right atoms, then there is no escape. However, I think human beings should still have an advantage when it comes to pattern recognition, and I remain curious to see if an insightful crystallographer can tip that balance in the right direction. I am also still curious to see if tweaking some setting on some automated building program will do that too. So, my original question remains: are automated building programs better than humans? Any human? I therefore declare the John Henry Challenge still open. But yes, improving the phases can tip the balance too, and the accuracy of the anomalous differences will ultimately affect the accuracy of the phases, and so on. This is a much broader challenge. And I think the best way to frame it is with the question: How low can the anomalous signal be before any conceivable approach fails? and perhaps: What is the best procedure to use for weak anomalous signal? For those who are interested in joining George, Pavol, Raj and others in this new challenge, the full spectrum of difficulty from trivial (100% Se incorporation) to a complete waste of time (0% Se, 100% S) is here: http://bl831.als.lbl.gov/~jamesh/challenge/occ_scan/ The impossible.mtz for the John Henry Map Interpretation Challenge was derived from frac0.79.mtz and possible.mtz from frac0.78.mtz. These simulated 31% and 32% Se incorporation into Met side chains (respectively). It has now been shown that both of these can be solved automatically if you do the phasing right. But what about frac0.80.mtz? Or frac0.90.mtz ? At least on this one coordinate of Se incorporation, the prowess of a particular approach can be given a score. For example, a score of 0.78 means that the indicated procedure could solve the frac0.78.mtz dataset, but not the frac0.79.mtz dataset. Based on the reports I have gotten back so far, the difficulty score lineup is: score method 0.86 xds, xscale, right sites, crank2 (Pavol Skubak) 0.78 xds, xscale, right sites, mlphare, dm, phenix.autobuild using 20 models (James Holton) 0.75 xds, xscale, right sites, mlphare, dm, buccaneer/refmac/dm (James Holton) 0.71 xds, xscale, right sites, mlphare, dm, ARP/wARP 7.3 (James Holton) 0.51 xds, xscale, right
Re: [ccp4bb] a challenge
Dear James I actually chose 3dko because it is a kinase (with a ligand), and therefore an interesting candidate for a molecular replacement score. I have not set this up yet, but I think if you look for PDB entries that contain the word kinase and try to molecular-replace all of them into the 3dko dataset, what fraction of them will work? I think that fraction would make a good score for a given molecular replacement pipeline. At the recent CCP4 SW in Nottingham Giovanna Scapin from Merck gave a talk on MR during which she reflected upon their attempts from some time ago to troubleshoot a recalcitrant MR case of a kinase by searching with hunderds of models derived from all kinase structures known at that time. However, I am not quite sure if they published these results anywhere (at least I could not fish out a relevant reference). Along these lines, 'Wide Search MR' (Stokes-Rees and Sliz (2010) PNAS 107: 21476-21481) and (www.sbgrid.org) may also provide some options to establish such benchmarking or MR 'scores'. Best regards Savvas On Mon, Jan 14, 2013 at 2:31 PM, Nat Echols nathaniel.ech...@gmail.com wrote: On Mon, Jan 14, 2013 at 11:18 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: I admit not having read all contributions to this thread. I understand the John Henry Challenge as whether there is an 'automated way of producing a model from impossible.mtz'. From looking at it and without having gone all the way to a PDB-file my feeling is one could without too much effort from the baton mode in e.g. coot. This should be even more possible if one also uses existing knowledge about the expected structure of the protein: a kinase domain is quite distinctive. So, James, how much external information from homologous structures are we allowed to use? Running Phaser would certainly be cheating, but if I take (for instance) a 25% identical kinase structure, manually align it to the map and/or a partial model, and use that as a guide to manually rebuild the target model, does that meet the terms of the challenge? -Nat
Re: [ccp4bb] a challenge
Dear Santosh, I think that it is a bit more complicated. SIR generally provides a stronger phasing signal than SAD, and can be better for phasing, provided that: (a) the native and derivative are sufficiently isomorphous, AND (b) the heavy atom substructure is itself chiral. For some space groups one site is enough to generate a chiral substructure but for others, e.g.P21, more than one site is necessary. Otherwise the first map will be a double image consisting of two overlapping positive images, and density modification will not in general be able to untangle them. SAD also gives a double image in such cases, but then instead of two positive images we have one negative and one positive image, and the simplest form of density modification - setting negative density to zero - will break the pseudosymmetry. One can also break such pseudosymmetry by using SIRAS or RIPAS instead of SIR or RIP, even if the anomalous signal alone is not sufficient to phase the structure. If MAD doesn't work and one happens to have a native (Met) dataset as well as SeMet, one should always consider analyzing the data as SIRAS. Whether this is better than SAD on the SeMet data alone will depend primarily on how isomorphous the two datasets are. Best wishes, George On 01/15/2013 11:06 AM, Santosh Panjikar wrote: Hi James, The datasets frac.80.mtz to frac.100.mtz are challenging to solve using SAD phasing. However these datasets can be easily solved using other experimental phasing method. Instead of using anomalous signal we could use isomorphous signal only. For example RIP or SIR phasing method, as there is a difference in intensity between the datasets due to scattering of S and Se. Since frac.80.mtz data contains 20% selenium that is sufficient to solve the structure against the frac.100.mtz. It seems the structure can be solved even as less as 10% selenium content (frac.90.mtz vs frac.100.mtz), and substructure can be solved easily. This is not surprising, the pair of the datasets is quite isomorphous, . We phase all reflections (centric and non-centric) where as anomalous phasing we could phase non-centric reflections only. In fact, Single Isomorphous Replacement phasing method is the first phasing technique. This method has been further extended by Ravelli et al with some deviation by introduction of X-ray or UV RIP phasing. I tried RIP (SIR) phasing protocol of Auto-Rickshaw using frac.90.mtz as before and frac.100.mtz as after. Auto-Rickshaw used SHELXC/D/E and ARP/wARP/REFMAC5 to get the partially refined model (Rfree below 30%) . Cheers Santosh Santosh Panjikar, Ph.D. Scientist Australian Synchrotron 800 Blackburn Road Clayton VIC 3168 Australia Ph: +61-4-67770851 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of James Holton [jmhol...@lbl.gov] Sent: Monday, January 14, 2013 8:12 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] a challenge I am absolutely delighted at the response I have gotten to my little John Henry Challenge! Three people already have managed to do the impossible. Congratulations to George Sheldrick, Pavol Skubak and Raj Pannu for finding ways to improve the phases over the ones I originally obtained (using the default settings of mlphare and dm) and build their way out of it. This is quite useful information! At least it is to me. Nevertheless, I do think Frances Reyes has a point. This was meant to be a map interpretation challenge, and not a SAD-phasing challenge. I appreciate that the two are linked, but the reason I did not initially provide the anomalous data is because I thought it would be too much to ask people to re-do all the phasing, etc. Yes, there do appear to be ways to improve the maps beyond the particular way I phased them, but no matter how good your phasing program is, there will always be a level of anomalous signal that will lead to phases that are off enough to make building the model impossible. Basically, once the map gets bad enough that just as many wrong atoms get built in as right atoms, then there is no escape. However, I think human beings should still have an advantage when it comes to pattern recognition, and I remain curious to see if an insightful crystallographer can tip that balance in the right direction. I am also still curious to see if tweaking some setting on some automated building program will do that too. So, my original question remains: are automated building programs better than humans? Any human? I therefore declare the John Henry Challenge still open. But yes, improving the phases can tip the balance too, and the accuracy of the anomalous differences will ultimately affect the accuracy of the phases, and so on. This is a much broader challenge. And I think the best way to frame it is with the question: How low can the anomalous signal be before any conceivable approach
[ccp4bb] Problems in scaling up expression
Dear All. A question on protein expression. We have been doing small scale test expressions in 15ml of terrific broth+kanamycin using E.coli KRX cells in falcon tubes. On reaching OD600 of ~0.6 we induced with rhamnose+IPTG and expressed for 4 hours at 37 C. There is a big band corresponding to our protein in the soluble fraction and not much in the insoluble fraction. However, on scaling up to 1 L TB cultures (with same concentrations of kanamycin and inducing with same concs of rhamnose+IPTG) we don't get strong over expression. Has anyone else experienced problems when scaling up expression? (and more importantly, solved them?) best wishes James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895
[ccp4bb] crystal dehydration
Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith
Re: [ccp4bb] Problems in scaling up expression
Hi James, Just a thought or two after reading your notes. I don't exactly know what KRX cells are but I suspect (based on your mention of rhamnose) that they are cells in which T7 RNAP levels are under the control of a rhamnose promoter. If that is so, it would be important to add rhamnose during cell growth to prevent an leaky expression of your protein, which may be toxic to E. coli, as opposed to adding it during the stage of induction along with IPTG like you describe. Just to rephrase, add inoculum, antibiotics and rhamnose to TB, grow to the OD you desire, then add IPTG to induce protein expression. Often, toxicity effects are felt in larger cultures and not seen in 15mL or 40mL cultures. Also, I have found that there are certain cases when it is better to use Luria Broth as opposed to TB. But that is not my primary concern in your case. My two cents! Raji On Tue, Jan 15, 2013 at 9:48 AM, Murray, James W j.w.mur...@imperial.ac.ukwrote: Dear All. A question on protein expression. We have been doing small scale test expressions in 15ml of terrific broth+kanamycin using E.coli KRX cells in falcon tubes. On reaching OD600 of ~0.6 we induced with rhamnose+IPTG and expressed for 4 hours at 37 C. There is a big band corresponding to our protein in the soluble fraction and not much in the insoluble fraction. However, on scaling up to 1 L TB cultures (with same concentrations of kanamycin and inducing with same concs of rhamnose+IPTG) we don't get strong over expression. Has anyone else experienced problems when scaling up expression? (and more importantly, solved them?) best wishes James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] crystal dehydration
Dear Judith, in Acta Cryst D61 (2005), 1173-1180 different handling methods for dehydration are described besides other post-crystallization treatments to improve diffraction quality. You can also try Dehydration Salts. A screen with ready made salt solutions is available from Jena Bioscience. Useful might also be the MicroRT System from Mitegen. Ulrike
[ccp4bb] changing resolution bins statistics in CORRECT.LP
Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of CORRECT.LP. Please, note that I am not referring to the table output by XSCALE, in which you can change the resolution bins with the keyword RESOLUTION_SHELLS=, but rather the table output by the CORRECT job of XDS. Thanks in advance, ciao, Sebastiano -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990 sebastiano.pasqual...@ieo.eu
Re: [ccp4bb] crystal dehydration
Dear Judith, Indeed we do. Please see our website for further details. http://www.diamond.ac.uk/Home/Beamlines/MX/I02/Humidifier.html Let me know if you have any questions or need any further information. Hope it helps Juan Juan Sanchez-Weatherby, PhD Beamline Scientist - I02 Tel: +44 (0)1235 778661 Mob:+44 (0)7795 641259 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Judith A Ronau Sent: 15 January 2013 14:47 To: ccp4bb Subject: [ccp4bb] crystal dehydration Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith
Re: [ccp4bb] crystal dehydration
Hi Judith, a very effective method is the use of a humidity control device - this was actually the reason we developed the equations that predict the RH in equilibrium with precipitant solutions. It has the great advantage that you can characterize changes that occur and also move straight to data collection. There are several HC1 devices (developed here at the EMBL) in Europe and at least 1 in the USA - there is also the FMS. Below are sole links that might help, best wishes, Matt. Website for experiments: http://www.esrf.eu/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b Calculation server for mother liquor RH equilibria: http://go.esrf.eu/RH Paper describing above: http://scripts.iucr.org/cgi-bin/paper?S1744309111054029 Papers describing device and methods: http://journals.iucr.org/d/issues/2009/12/00/gm5010/index.html and http://www.sciencedirect.com/science/article/pii/S1047847711000499 Interface in MXCuBE that allows the design of dehydration gradients, data collection and analysis of the images http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/workflows/dehydration-workflow. On 2013-01-15 15:46, Judith A Ronau wrote: Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ ===
Re: [ccp4bb] Problems in scaling up expression
Hi James, The first place I'd look is oxygen conc, as you'll get different aeration from different vessel shapes/sizes. You could play around with shaking speed and/or flask geometry to try to get more or less aeration. Shane Caldwell McGill University On Tue, Jan 15, 2013 at 9:48 AM, Murray, James W j.w.mur...@imperial.ac.ukwrote: Dear All. A question on protein expression. We have been doing small scale test expressions in 15ml of terrific broth+kanamycin using E.coli KRX cells in falcon tubes. On reaching OD600 of ~0.6 we induced with rhamnose+IPTG and expressed for 4 hours at 37 C. There is a big band corresponding to our protein in the soluble fraction and not much in the insoluble fraction. However, on scaling up to 1 L TB cultures (with same concentrations of kanamycin and inducing with same concs of rhamnose+IPTG) we don't get strong over expression. Has anyone else experienced problems when scaling up expression? (and more importantly, solved them?) best wishes James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895
Re: [ccp4bb] a challenge
Santosh, Although I appreciate your ingenuity and I agree that SIRAS is an excellent idea in the real world if you have only partial Se occupancy, I'm afraid I think it is cheating to use more than one of the challenge datasets at a time. The scenario I wanted to test is the all-too-common we only had that one good crystal situation. Then again, I do think it is interesting to ask how low the Se incorporation can go before SIRAS fails. Even if it is under the idyllic perfect isomorphism situation here. I have now put up 1% increments between frac0.90.mtz and frac1.00.mtz. Do you think you/Autorickshaw can solve it with frac0.99.mtz vs frac0.1.00.mtz ? If you'd like to test in the presence of non-isomorphism, I'd recommend using the radiation damaged simulated dataset here: http://bl831.als.lbl.gov/~jamesh/workshop2/decaying.mtz as the derivative. It is about 18% different from frac0.00.mtz (100% Se, but badly decayed). Thanks for all the great ideas! -James Holton MAD Scientist On 1/15/2013 2:06 AM, Santosh Panjikar wrote: Hi James, The datasets frac.80.mtz to frac.100.mtz are challenging to solve using SAD phasing. However these datasets can be easily solved using other experimental phasing method. Instead of using anomalous signal we could use isomorphous signal only. For example RIP or SIR phasing method, as there is a difference in intensity between the datasets due to scattering of S and Se. Since frac.80.mtz data contains 20% selenium that is sufficient to solve the structure against the frac.100.mtz. It seems the structure can be solved even as less as 10% selenium content (frac.90.mtz vs frac.100.mtz), and substructure can be solved easily. This is not surprising, the pair of the datasets is quite isomorphous, . We phase all reflections (centric and non-centric) where as anomalous phasing we could phase non-centric reflections only. In fact, Single Isomorphous Replacement phasing method is the first phasing technique. This method has been further extended by Ravelli et al with some deviation by introduction of X-ray or UV RIP phasing. I tried RIP (SIR) phasing protocol of Auto-Rickshaw using frac.90.mtz as before and frac.100.mtz as after. Auto-Rickshaw used SHELXC/D/E and ARP/wARP/REFMAC5 to get the partially refined model (Rfree below 30%) . Cheers Santosh Santosh Panjikar, Ph.D. Scientist Australian Synchrotron 800 Blackburn Road Clayton VIC 3168 Australia Ph: +61-4-67770851 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of James Holton [jmhol...@lbl.gov] Sent: Monday, January 14, 2013 8:12 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] a challenge I am absolutely delighted at the response I have gotten to my little John Henry Challenge! Three people already have managed to do the impossible. Congratulations to George Sheldrick, Pavol Skubak and Raj Pannu for finding ways to improve the phases over the ones I originally obtained (using the default settings of mlphare and dm) and build their way out of it. This is quite useful information! At least it is to me. Nevertheless, I do think Frances Reyes has a point. This was meant to be a map interpretation challenge, and not a SAD-phasing challenge. I appreciate that the two are linked, but the reason I did not initially provide the anomalous data is because I thought it would be too much to ask people to re-do all the phasing, etc. Yes, there do appear to be ways to improve the maps beyond the particular way I phased them, but no matter how good your phasing program is, there will always be a level of anomalous signal that will lead to phases that are off enough to make building the model impossible. Basically, once the map gets bad enough that just as many wrong atoms get built in as right atoms, then there is no escape. However, I think human beings should still have an advantage when it comes to pattern recognition, and I remain curious to see if an insightful crystallographer can tip that balance in the right direction. I am also still curious to see if tweaking some setting on some automated building program will do that too. So, my original question remains: are automated building programs better than humans? Any human? I therefore declare the John Henry Challenge still open. But yes, improving the phases can tip the balance too, and the accuracy of the anomalous differences will ultimately affect the accuracy of the phases, and so on. This is a much broader challenge. And I think the best way to frame it is with the question: How low can the anomalous signal be before any conceivable approach fails? and perhaps: What is the best procedure to use for weak anomalous signal? For those who are interested in joining George, Pavol, Raj and others in this new challenge, the full spectrum of difficulty from trivial (100% Se incorporation) to a complete waste of time (0% Se, 100% S) is here:
Re: [ccp4bb] crystal dehydration
In case if dehydration needs to be done slowly and under tight control of all parameters, one possibility is to use micro-dialysis buttons. We used it for a large membrane protein complex and diffraction improved from ~7 to 2.7 A. The crystal is fished out and put into mother liquor solution in the button, sealed with dialysis membrane and the button is then placed into about 5 mls of mother liquor with slightly higher PEG concentration. Then you just exchange outside buffer every day or so for solutions containing higher concentrations of PEG. We went from ~9 to 30 % PEG4000 in about a week. You can easily observe crystal under microscope and if it cracks - you went too far/too quickly with PEG and need to use a bit less next time. Also, this method allows you to control all other components of the dehydrating solution - we needed to decrease salt concentration at the same time as increasing PEG. You can also introduce/increase cryo-protectant concentration at the same time. With these crystals, otherwise excellent dehydration machines already mentioned did not work, possibly because the process had to be really slow. The reference is here: http://www.ncbi.nlm.nih.gov/pubmed/21822288 Best wishes.
[ccp4bb] off-topic: citations for GST dragging gunk into solution?
Does anybody have a nice, cite-able reference for the observation that GST tags can sometimes drag otherwise-aggregated/unfolding/generally unhappy proteins into solution? We need to politely explain why a His-tagged version of our protein (purified in my lab) has activity but a GST-tagged version (purified in a different lab) doesn't. thanks, Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] crystal dehydration
Hi Partha, as far as I know the HC1 is available at the CLS and soon at the APS and ALS, best wishes, Matthew. On 2013-01-15 22:27, Parthasarathy Sampathkumar wrote: Dear Dr. Bowler, Which beamline in USA has this Humidity Control Device? Best Wishes, Partha On Tue, Jan 15, 2013 at 11:02 AM, Matthew BOWLER mbow...@embl.fr [10] wrote: Hi Judith, a very effective method is the use of a humidity control device - this was actually the reason we developed the equations that predict the RH in equilibrium with precipitant solutions. It has the great advantage that you can characterize changes that occur and also move straight to data collection. There are several HC1 devices (developed here at the EMBL) in Europe and at least 1 in the USA - there is also the FMS. Below are sole links that might help, best wishes, Matt. Website for experiments: http://www.esrf.eu/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b [1] Calculation server for mother liquor RH equilibria: http://go.esrf.eu/RH [2] Paper describing above: http://scripts.iucr.org/cgi-bin/paper?S1744309111054029 [3] Papers describing device and methods: http://journals.iucr.org/d/issues/2009/12/00/gm5010/index.html [4] and http://www.sciencedirect.com/science/article/pii/S1047847711000499 [5] Interface in MXCuBE that allows the design of dehydration gradients, data collection and analysis of the images http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/workflows/dehydration-workflow [6]. On 2013-01-15 15:46, Judith A Ronau wrote: Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 [7] Fax: +33 (0) 4.76.88.29.04 [8] http://www.embl.fr/ [9] === Links: -- [1] http://www.esrf.eu/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b [2] http://go.esrf.eu/RH [3] http://scripts.iucr.org/cgi-bin/paper?S1744309111054029 [4] http://journals.iucr.org/d/issues/2009/12/00/gm5010/index.html [5] http://www.sciencedirect.com/science/article/pii/S1047847711000499 [6] http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/workflows/dehydration-workflow [7] http://embl.fr/tel:%2B33%20%280%29%204.76.20.76.37 [8] http://embl.fr/tel:%2B33%20%280%29%204.76.88.29.04 [9] http://www.embl.fr/ [10] mailto:mbow...@embl.fr -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ ===
[ccp4bb] advices
Hello, all, My crystal grows very fast even though the protein conc. is now 1.5 mg/ml. The shape of the crystal likes a ruler plate with one side very thin. The crystal quality has diffraction to about 3.5A in a home source of 1.2 KW sealed tube Angilent equipment. But the data can not be indexed due to one direction has poor diffraction and the crystal quality. Seeking advices on improving the crystal quality. Thank you very much Mike
