[ccp4bb] Group Leader position in Helsinki, Finland
(Reposting this position as it didnt go to the jobs list..:) On behalf of the search committee (see links below): -- The Institute of Biotechnology is a leading European research institute within the University of Helsinki with a mission to increase knowledge in cross‐disciplinary biology and biotechnology. The Institute has state‐ofthe‐art facilities in imaging, model organisms, proteomics, genomics, crystallography and NMR. We seek outstanding candidates for GROUP LEADER positions in the areas of Molecular Cell Biology, Genomics and Quantitative Biology, Plant Biology, Developmental Biology, or Structural Biology and Biophysics. Applicants from different levels of the career path are welcomed. Successful candidates have a suitable strong track record including a PhD or an equivalent and postdoctoral experience in an international setting and are expected to develop an independent externally funded line of research. Appointments are based on BI tenuring system with external evaluations every four years. A globally competitive remuneration package and 3‐year startup funds are negotiable. Applications should include a presentation of past achievements (1 page) and research interests (max. 3 pages), curriculum vitae, list of publications (with citation numbers), and three letters of reference. Applications and letters of reference are to be submitted at www.biocenter.helsinki.fi/bi/recruit no later than on Tuesday March 31, 2015. For further information and detailed contacts, please visit www.biocenter.helsinki.fi/bi/recruit or contact bi‐direc...@helsinki.fi. www.biocenter.helsinki.fi/bi Tommi Kajander, Ph.D. Team Leader Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-50-4480991 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/
[ccp4bb] GL position available/Finland
FYI, on behalf of the search committee (see links below): The Institute of Biotechnology is a leading European research institute within the University of Helsinki with a mission to increase knowledge in cross‐disciplinary biology and biotechnology. The Institute has state‐ofthe‐art facilities in imaging, model organisms, proteomics, genomics, crystallography and NMR. We seek outstanding candidates for GROUP LEADER positions in the areas of Molecular Cell Biology, Genomics and Quantitative Biology, Plant Biology, Developmental Biology, or Structural Biology and Biophysics. Applicants from different levels of the career path are welcomed. Successful candidates have a suitable strong track record including a PhD or an equivalent and postdoctoral experience in an international setting and are expected to develop an independent externally funded line of research. Appointments are based on BI tenuring system with external evaluations every four years. A globally competitive remuneration package and 3‐year startup funds are negotiable. Applications should include a presentation of past achievements (1 page) and research interests (max. 3 pages), curriculum vitae, list of publications (with citation numbers), and three letters of reference. Applications and letters of reference are to be submitted at www.biocenter.helsinki.fi/bi/recruit no later than on Tuesday March 31, 2015. For further information and detailed contacts, please visit www.biocenter.helsinki.fi/bi/recruit or contact bi‐direc...@helsinki.fi. www.biocenter.helsinki.fi/bi
Re: [ccp4bb] modeling flexible ends on proteins
Hi, thanks for all the answers, i will try to wrap up some conclustion once i have tested things. Best Tommi On Oct 24, 2014, at 7:09 PM, Tim Gruene wrote: Hi Tommi, I used Molscript to place a dotted line in the gap. This shows clearly from where to where the gap goes and that the data don't show interpretable density in that region. I would call everything else 'tabloid science'. Cheers, Tim On 10/24/2014 04:11 PM, Michal Jamroz wrote: Dnia 2014-10-22, o godz. 15:43:18 Tommi Kajander tommi.kajan...@helsinki.fi napisał(a): Would anyone know a software to model (just with some kind of random coil) the amino acid chain for the assumed flexible disorderd regions between domains, or at one end of protein? just for illustrative purposes. Hi Tommi, check CABSflex: http://biocomp.chem.uw.edu.pl/CABSflex You can simply put there PDB code of considered structure and get info about flexibility, native-state dynamics movie, etc. Michał -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A Tommi Kajander, Ph.D. Team Leader Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-50-4480991 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/
[ccp4bb] modeling flexible ends on proteins
Hi All, Would anyone know a software to model (just with some kind of random coil) the amino acid chain for the assumed flexible disorderd regions between domains, or at one end of protein? just for illustrative purposes. Thanks! Tommi
[ccp4bb] Stereo on macs revisited
Dear All, Can someone sugegst what would be the best options for stereo viewing for macs currently? Any good experiences with Stereo-TVs? Other? I would like know the specific combination (at least which mac computer etc) if possible to find out something that works. Thanks, Tommi Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki, Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/
[ccp4bb] Post doctoral position at the protein crystallization core facility / Institute of Biotechnology, University of Helsinki
A postdoctoral position is available in the Biocenter Finland Crystallization Core Facility, in the research group of Dr. Tommi Kajander at the institute of Biotechnology at University of Helsinki, Finland. The position is funded for two years starting from September 2014. The position will include crystallization facility responsibilities, and structural biology-related research in the Kajander lab, and possibly partly on collaborators/clients projects, as agreed. The topics of research project(s) include structural aspects of neuronal protein-protein receptor complexes, and will be discussed in detail with the selected candidates. Core facility tasks include scientific support, and help in maintenance of the services of the protein crystallization facility (http://www.biocenter.helsinki.fi/bi/xray/automation/index.html), including advising clients on structural biology projects, and data collection on synchrotron sources (e.g. Diamond and ESRF). The successful candidate must be experienced in macromolecular crystallography (crystallization, synchrotrons, structure solution, refinement etc.), and have very good teamwork, communication, and social skills, and capability to guide students. Ability to work in organized fashion and fluent English language skills are also required. Knowledge of automation, high throughput techniques and/or molecular biology and protein purification and production experience are desirable/preferable. The institute has excellent infrastructure for science and the unit has two imaging systems (Rigaku Minstrel UV and Explora Nova (+4 C), mosquito LCP, and a Hamilton star dispensing robot. The unit also has a thermofluor instrument and access to a MALLS system for upstream sample analysis. We also have a Rigaku rotating anode source w/RAXIS IV+ detector. We have regular (monthly) access to synchrotron sources. Institute of Biotechnology also has state-of-the-art cryo-EM and NMR facilities, and e.g. Biacore and thermophoresis instruments are located in the next building. Applicants should send: 1) A cover letter briefly describing previous achievements, and motivation for the position; 2) Curriculum vitae with a list of publications 3) Contact information of two referees to email address: tommi.kajander(at)helsinki.fi. The deadline for applications is August 31, 2014, or until suitable candidate is found. Contact for more information: e-mail: tommi.kajander(at)helsinki.fi (phone: +358-50-4480991) http://www.biocenter.helsinki.fi/bi/kajander/ Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki, Finland
[ccp4bb] Post doctoral position at Institute of Biotechnology, Univ. Helsinki
A postdoctoral position in structural biology is available from Sept 2014 earliest in the research group lead by Tommi Kajander at Institute of Biotechnology, University of Helsinki, Finland. The position is funded by the Academy of Finland at the Finnish university salary scale 5. The goal of the project is to understand the molecular basis of mammalian adhesion protein functions and solve structures of relevant protein-protein complexes. We are utilizing X-ray crystallography as the main methods to solve protein structures, with complementary methods such as SAXS, NMR and single particle EM and protein engineering and functional in vitro and cell-based studies. The topics of research projects will be discussed in detail during the interview of the selected candidates. Host institute has excellent infrastructure for structural biology studies. Applicants with strong background in protein expression and purification and molecular biology, and experience in crystallography/structural biology techniques are encouraged to apply (in particular experience with insect and eukaryotic expression systems is a plus). The applicant should be a team player with good English and communication skills and capability to guide studends. Applicants should send 1) a cover letter briefly describing previous achievements, future career ambitions and motivation for this position; 2) Curriculum vitae with a list of publications and contact information of two referees should be sent via email to: tommi.kajander-at-helsinki.fi The deadline for applications is July 31st, 2014, or until suitable candidates are found. Please feel free to contact for more information. Tommi Kajander, Ph.D. Team Leader Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-50-4480991 tommi . kajander-at-helsinki . fi http://www.biocenter.helsinki.fi/bi/kajander/
[ccp4bb] best transient mammalian expression systems..?
Dear all, I would be interested in comments/opinions about what is your experience on which would be the best mammalian cell culture vector/cell line/medium combination for transient expression to get the best yields. Depends, of course, but anyway obviously there are differences, HEKs are suppose to be easy to transfect, but which flavour and how and what medium or would you prefer CHO? (probably more so for stable cell lines?) ...etc. vectors...? Or does it make a difference (it seems to). Good reviews/comparisons? is it worth going beyond DMEM and supplements? (what do you add in?) Stupid questions - trying to educate myself a bit here.. and i am willing to spend a bit or at least know what difference it makes (time is money anyway ...and worse). and we are not doing suspension at the moment, but could consider it if it seems like a good idea. Thanks for the tips.., Tommi
Re: [ccp4bb] How to convert file format from CNS to CCP4
Ok. you filled my mailbox the second time today - please do stop sending junk to the list. -tommi On Mar 23, 2013, at 3:59 PM, Wei Feng wrote: Dear Steffi, Thank you very much for you patient reply! I have tried to use your script to convert the map format, but no ***omit_map.coeff can be found in all outputted file. After Heavy-atom search, Heavy-atom refinement/SAD phasing and SAD Phasing - Density Modification only two map outputted , one is fourier_map, other is density_modify.map.(see the attachments). And the cell parameter of the structure is 131.598 131.598 80.788 90.00 90.00 120.00 P622(177) Can you help me to check out why these maps can not be converted by sftools? Thank you for your time! Best! Wei At 2013-03-21 04:13:41,Stefanie Becker stefanie.bec...@uni-konstanz.de wrote: Hi Wei! i am very sorry for the belated answer. I checked my notebook and found I remembered it incorrectly. Indeed you can do the conversion with SFtools. Here is a small script that should do it (you need to change the space group and cell parameters of course ) hope that helps! steffi #!/bin/csh -f rm temp.mtz sftoolsend read composite_omit_map.coeff CNS 156.773 163.390 595.757 90 90 90 22 END write temp.mtz MTZ quit end cad hklin1 temp.mtz hklout omit_map_070419.mtzeof LABI FILE 1 ALL LABO FILE 1 E1=FWT E2=PHWT end eof Am Mittwoch, 20. März 2013 02:14 CET, ccp4...@hotmail.com schrieb: Dear steffi, Can you give me a link? I did not find it in google. Thank you! Wei 在 2013-03-19 20:55:42，Stefanie Becker stefanie.bec...@uni-konstanz.de 写道： Am Dienstag, 19. März 2013 04:37 CET, Wei Feng ccp4...@hotmail.com schrieb: Hi! don't know if you already got the answer by now, but there is a small program called map2mtz that should do it (i think you can just google it) good luck! steffi Dear all, I have used CNS to calculate the experimental phase of my structure. After Heavy-atom search, Heavy-atom refinement/SAD phasing and SAD Phasing - Density Modification - Selection of Map. Some files outputted: sad_phase2.hkl sad_phase2.sdb density_modify.hkl density_modify.map density_modify.mask ... I want to use these files to do the model building, but I do not know how to do it in CNS. So I want to convert these files to CCP4 format and do the model building by ARP/Warp, but I do not know which files should be converted and which software can be used to convert the file format from CNS to CCP4. Thank you for your time! Wei
Re: [ccp4bb] Assemble Protein-DNA complex
It is not very surprising that the affinity gets higher with lower salt, right? Why dont you measure it under _physiological_ salt concentration? (or i assume maybe you did?) and of course its not as high affinity due to screening (but physiological conditions) of the electrostatic interactions. If the complex doesnt stay together in ca. 150 mM salt (e.g in TBS) in gel filtration (which you dont say if you tried) then why not just try mixing it in the drop and screen, this is what you would do if you cant purify the complex. and try different rations. There are texts on preparation of DNA complexes in case you dont have advice for it. Tommi On Nov 9, 2012, at 6:50 PM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Wei Huang, if you are lucky you can form the complex as crystal in the drop (I assume you want to crystallise the protein-DNA complex): set up drops at high salt concentration (as low as possible to keep the protein in solution at reasonable concentration) in the presence of DNA. Prepare the buffer the same way as the protein buffer, but with reduced salt concentration (btw, you can also try divalent ions, eg. MgCl2). This way water evaporates into the drop, diluting the salt concentration. - From your discription the solubility of the protein drops faster than its concentration, hence it should precipitate. And, as I said, if you are lucky, it does so as a crystal in complex with the DNA. Best, Tim On 11/09/2012 05:14 PM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQnTRFUxlJ7aRr7hoRAvscAJ9Bm/3ix2ln69wZz74LWwaPMdBhFQCfS9C6 nvrZMfMmeekAauJ26jy6jbE= =nbV+ -END PGP SIGNATURE- Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/
[ccp4bb] solubility estimates for domains/structures?
Hi all, Does anyone a program/paper that would give some quantitative estimate for protein solubility based on surface property analysis? (excluding obvious things such as integral membrane / TM regions) Best Tommi
Re: [ccp4bb] solubility estimates for domains/structures?
Hi, Thanks, i was thinking of a situation where one can model the sequence to a structure based on homologous structures, ie assuming i know the fold - i guess PISA / ASA based estimates are the first thing. (mainly indeed the likelyhood to aggregate ie the surface property estimates are what i am looking for.) Tommi On Oct 12, 2012, at 8:38 PM, Das, Debanu wrote: Hi, Using a surface property analysis (3D structure is available), I think you can get a quantitative estimate by using PISA to find the solvent-accessible area and salvation energy and then comparing that to corresponding values obtained by using in your lab another protein for calibration (maybe ones that you label as highly, medium or minimal soluble with known mg/ml). If you just want an estimate from sequence analysis for solubility on heterologous overexpression, you can try: SOLpro in http://scratch.proteomics.ics.uci.edu/ Or http://mips.helmholtz-muenchen.de/proso/proso.seam http://www.biotech.ou.edu/ Thanks, Debanu -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tommi Kajander Sent: Friday, October 12, 2012 10:23 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] solubility estimates for domains/structures? Hi all, Does anyone a program/paper that would give some quantitative estimate for protein solubility based on surface property analysis? (excluding obvious things such as integral membrane / TM regions) Best Tommi Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/Kajander_Lab/Home.html
Re: [ccp4bb] @Ian:Death of Rmerge
well, actually i recommend having a look at the old but good scalepack manual for why Rmerge is inferior.. (i thought this was clear long ago.. so i am bit amazed that this discussio is still alive and kicking..) question of where to cut, is a different one and thats where the recent papers and developments start to come in. short quote...(scalepack manual): From a statistical point of view, I/σ is a superior criterion, for two reasons. First, it defines a resolution “limit” since by definition I/σ is the signal to noise of your measurements. In contrast, Rmerge is not directly related to signal to noise. Second, the σ assigned to each intensity derives its validity from the χ2’s, which represent the weighted ratio of the difference between the observed and average value of I, 〈I〉, squared, divided by the square of the error model, the whole thing times a factor correcting for the correlation between I and 〈I〉. Since it depends on an explicit declaration of the expected error in the measurement, the user of the program is part of the Bayesian reasoning process behind the error estimation. ..In short, I/σ is the preferred way of assessing the quality of diffraction data because it derives its validity from the χ2 (likelihood) analysis. credits to Otwinowski et al. end of story, i believe. so R-merge died long back. -tommi On Jun 4, 2012, at 9:00 AM, aaleshin wrote: Wow, it is quite a lecture here! It is very appreciated. I admit some (most?) of my statements were questionable. Thus, I did not know how sigI would be calculated in case of multiple observations, and, indeed, its proper handling should make sigI/I similar to Rmerge. Consequently, I/sigI substitutes Rmerge fairly well. Now, where the metric Rmerge=0.5 came from? If I remember correctly, It was proposed here at ccp4bb. Also, one reviewer suggested to use it. I admit that this is quite an arbitrary value, but when everyone follows it, structures become comparable by this metric. If there is a better approach to estimate the resolution, lets use it, but the common rule should be enforced, otherwise the resolution becomes another venue for cheating. Once again, I was talking about metric for the resolution, it does not need to be equal to metric for the data cutoff. Alex On Jun 3, 2012, at 2:55 PM, Ian Tickle wrote: Hi Alex On 3 June 2012 07:00, aaleshin aales...@burnham.org wrote: I was also taught that under normal conditions this would occur when the data are collected up to the shell, in which Rmerge = 0.5. Do you have a reference for that? I have not seen a demonstration of such an exact relationship between Rmerge and resolution, even for 'normal' data, and I don't think everyone uses 0.5 as the cut-off anyway (e.g. some people use 0.4, some 0.8 etc - though I agree with Phil that we shouldn't get too hung up about the exact number!). Certainly having used the other suggested criteria for resolution cut-off (I/sigma(I) CC(1/2)), the corresponding Rmerge (and Rpim etc) seems to vary a lot (or maybe my data weren't 'normal'). One can collect more data (up to Rmerge=1.0 or even 100) but the resolution of the electron density map will not change significantly. I think we are all at least agreed that beyond some resolution cut-off, adding further higher resolution 'data' will not result in any further improvement in the map (because the weights will become negligible). So it would appear prudent at least to err on the high resolution side! I solved several structures of my own, and this simple rule worked every time. In what sense do you mean it 'worked'? Do you mean you tried different cut-offs in Rmerge (e.g. 0.25, 0.50, 0.75, 1.00 ...) and then used some metric to judge when there was no further significant change in the map and you noted that the optimal value of your chosen metric always occurs around Rmerge 0.5?; and if so how did you judge a 'significant change'? Personally I go along with Dale's suggestion to use the optical resolution of the map to judge when no further improvement occurs. This would need to be done with the completely refined structure because presumably optical resolution will be reduced by phase errors. Note that it wouldn't be necessary to actually quote the optical resolution in place of the X-ray resolution (that would confuse everyone!), you just need to know the value of the X-ray resolution cut-off where the optical resolution no longer changes (it should be clear from a plot of X-ray vs. optical resolution). I is measured as a number of detector counts in the reflection minus background counts. sigI is measured as sq. root of I plus standard deviation (SD) for the background plus various deviations from ideal experiment (like noise from satellite crystals). The most important contribution to the sigma(I)'s, except maybe for the weak reflections, actually comes
Re: [ccp4bb] Akta vs HPLC
if you have peek surface or titanium parts (if i recall right) there are no problems with salt solutions. tommi On May 30, 2012, at 3:39 AM, aaleshin wrote: Back in Iowa State University we used Waters HPLC for protein purification during many years without noticeable damage to the stainless steel tubings. But Dan was right about the pumps, someone in the lab forgot to flush the high salt pump with water after its use and damaged the pump... Alex On May 29, 2012, at 5:14 PM, Daniel Anderson wrote: Hi, Ho, Your question has a lot of variables. HPLC columns should not be used on the Akta within my field of view because the Akta within my field of view does not have gradual pump acceleration and deceleration. HPLC columns can be damaged by sudden changes in pressure or composition. The HPLC within my field of view has wetted stainless steel surfaces and the mobile phase should not contain chloride ion or reductant. Chloride ion would accelerate corrosion of the stainless steel (and result in metal ions in the protein). Reductant would strip off the passivation (during maintenance I soak the stainless parts in nitric acid to keep them stainless) later resulting in corrosion. The Waters sales representative once told me that the pumps have to be salt-free and methanol-flushed at the end of every working day. Good luck implementing that policy. -Dan Ho Leung Ng wrote: Hello, My Akta Purifier is being repaired, and I'm thinking about borrowing a colleague's HPLC in the interim. What makes the Aktas different from HPLCs? I've used HPLCs for purifying small molecules and peptides but not proteins. Anything I should be careful about regarding keeping the machines, columns, and proteins happy? Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu mailto:h...@hawaii.edu
[ccp4bb] nanodrop vs larger vol measurenment of absorbance?
Dear All, I would like to make small survey on experiences with using nanodrop for low concentration protein samples vs say the small 60 ul cuvette spectrometers - my experience is that around 1-2 mg/ml (well, i mean 1 AU) or in particular less than 1AU nanodrop gives underestimated values, but i havent done a systemic test. (which now seems like a good idea.. as the samples are _quite precious_ and i certainly want to keep a track of them all the way through) --Observations? (and yes we do 2 ul samples with nanodrop.) Thanks in advance, Best, T. Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] Freezing crystal
if you use oil do direct dry Paratone-N, with paraffin oil is not as good. Li-salts should work also - i would almos imgaine you can freeze directly from so high (NH4)2SO4 conc. but perhaps not. little bit (10%) glycerol probably does it also.. Tommi On Feb 6, 2012, at 12:55 AM, Vineet Gaur wrote: Hi Theresa, Once I had crystals in 3.5 M Amm. Sulfate. I used Paraffin oil and Paraton-N-oil (in 1:1 ratio). I also used 30% Xylatol. Best, Vineet On Sun, Feb 5, 2012 at 5:49 PM, Theresa H. Hsu theresah...@live.com wrote: Hi all Is there a list of conditions to be tried *first* for cryoprotectant? My crystals diffract at room temperature capillary but no in 30% PEG 400. Crystals are from 2 M ammonium sulfate. Thank you. Theresa
[ccp4bb] mask effects in solvent flattening / NCS
Dear All, Could someone explain me why i would see density outside both solvent and NCS masked regions (which are input essentially the same in this case into DM) --- what i see is basicly density that is actually just noise - but outside both masks (and which certainly is not there in non-averaged maps?? is just because the mask is too large or too small (NCS or solvent mask) - i just would have thought things outside solvent mask at least get flattened? something that is just beyond my understanding. (or then my input solvent mask is somehow different from what it looks like.) --would like to be able to correct this. Thanks for comments. Cheers, Tommi Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] density mod., NSC and software..
Thanks judit, well one problem here would be that if you cant feed in operators, that you got say from the map correlations with something like GETAX, and you only have two sites (one per monomer) thats not enough to define a two-fold... same goes of course if you dont have a model (because you would need to get the averaging to work first... ) Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi On Dec 21, 2011, at 12:56 PM, Debreczeni, Judit wrote: DM is kind of past tense (unless you are dealing with multicrystal averaging) -- I'd use parrot or shelxe instead (both pretty fast and automated). NCS operators as input do not seem to be in fashion these days, so you might have to put up with heavy atoms or partial MR models… I would not necessarily expect NCS related bits of the density to be fully identical. JED. AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tommi Kajander Sent: 20 December 2011 16:19 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] density mod., NSC and software.. Dear all, Stupid question: if i do density modification (flattening and extension say from 6 to 4Å) w/o averaging on a symmetrical, say dimer, would you expect the thing stays symmetric nevertheless? (in particular if there is a strong peak in the NCS??) (seems to me it doenst necessarily...) favorite programs for NCS averiging?? DM obviously, and solomon doesnt do it. Others that do it (with out a PDB or atoms to derive it from, but rather operators) Thanks, Tommi Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
[ccp4bb] density mod., NSC and software..
Dear all, Stupid question: if i do density modification (flattening and extension say from 6 to 4Å) w/o averaging on a symmetrical, say dimer, would you expect the thing stays symmetric nevertheless? (in particular if there is a strong peak in the NCS??) (seems to me it doenst necessarily...) favorite programs for NCS averiging?? DM obviously, and solomon doesnt do it. Others that do it (with out a PDB or atoms to derive it from, but rather operators) Thanks, Tommi Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] symmetry for ages 6 and up
we have a whole street made of them... welcome to Helsinki... t. On Dec 14, 2011, at 1:10 AM, Jacob Keller wrote: Does anyone know where one can acquire some Penrose tiles? I think they'd be great toys as well, and drive you a little bonkers. Maybe a kitchen/bathroom floor made from them? Jacob On Tue, Dec 13, 2011 at 5:03 PM, VAN RAAIJ , MARK JOHAN mjvanra...@cnb.csic.es wrote: reminds me of these symmetric 2D P3 lizards: http://www.worldofescher.com/store/Z51.html I bought the RGB-coloured set/puzzle after visiting an Escher exhibition and sometimes use them in crystallography/symmetry teaching. Nice to make the students assemble them and then decide on the symmetry operator, unit cell and asymmetric unit. Quoting Phoebe Rice: Hi all, For those who teach xtallography - we found some plastic turtles that can be snapped together in an amazing variety of space groups. Worked well in a workshop for our students, so I thought I'd share the shopping tip. They're called Reptangles, and we got them from Amazon. http://www.amazon.com/Fat-Brain-Toys-FA042-1-Reptangles/dp/B00392NSQ4 Have fun! Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
[ccp4bb] ncs axis translation
Hi, anybody have a script to to find a translation for 2-fold NCS (rotaion) axis based on the center of NCS symmetry..?? (ie to the midpoint of line between to heavy atoms) This search option doenst seem to be endoed anywhere (why??) ...stupid me... Thanks, tommi
[ccp4bb] sftools bug
Hi, if you change crystal infro (SG P2 -- P21) and column labels at the same time F col label (like FP) cant be changed to anything else at the same time for whatever reason... at least i had this prob. ccp4 vs 6.2 tommi
Re: [ccp4bb] small 3ml Superdex column performance
Hi, if you really want it SMART was replaced be Äkta Ettan and nowaays Äkta micro. The nice thing is the easy of use (same software). However i other think HPLCs might be more flexible and cheaper (e.g. we have schimadzu - cant complain about anything, interface is more complex, but you dont really need to care about all that... you use just a few options - and there are several other manufacturers..) --- just as a comment on the analytical systems. you can inject few microliters... And the ca. 20 ml S-200/S-75 10/300 works fine for small volumes by the way. at least on our HPLC. why would you need to go smaller?? 20-50 ug. in 20 ul should be fine did you try? you could try just chaning the tubing on your current Äktä purifier and injecting via Hamilto syringe?? Connected to HPLC certainly will work. Of couse if you want faster runs, thats another thing (i think these smaller columns are mainly good for fast screening of quality) HTH, tommi On Oct 16, 2011, at 4:14 AM, Artem Evdokimov wrote: Hi, You're probably referring to the Superdex 5/150 Tricorn column, with working volume of 3ml, and not the 15/150? Those columns work quite nicely for small sample volumes. For analytical runs 15-25ul injection is pretty nice. The PC columns are originally designed to be used with the SMART system, which by the way used to be one of the best analytical products for macromolecules -- optimized path length, one-volume pumps suitable for directly running typical protocols, etc. etc. and for its time the OS was also damn good (OS/2). Sadly, GE did not come up with any direct replacement for this machine, as far as I can tell. AKTA is not optimal for analytical runs - tubing is too long, etc. If you have an old HPLC system moldering in a corner I recommend either of these columns mounted directly in front of the detector. In our current setup the entire portion of the HPLC that is responsible for column selection and heating and so on is bypassed, so there are literally ~4-5 mm of (the thinnest available PPEK) tubing in between the injection valve and the column, and in between the column and the detector. Autosampler is a very helpful feature, esp. when analyzing fractions output from previous step, and as long as the column isn't clogged the run is 8-12 minutes (depending on buffer composition). Now, Agilent software for HPLC is absolutely horrible for this kind of work but it suffices. Artem P.S. for lower protein quantities don't forget to record the A210, in addition to A280 and A260. On Sat, Oct 15, 2011 at 5:04 PM, Alexandra Deaconescu deac...@brandeis.edu wrote: Hi everyone: I was hoping I could get your opinion on the performance of the small approx. 3ml gel filtration columns from GE Healthcare. We currently have an Akta fplc and we would like to do small runs using small volumes of sample. As far as I know we have two options: 1. use the Superdex 15/150 columns these appear to have a slightly lower resolution (the no. of theoretical plates is about 25000 m-1) these apparently can be directly hooked up to our Akta fplc 2. use Superdex PC 3.2 these have somewhat higher resolution (the no of theoretical plates is 3 m-1) these can only be hooked up using a special Precision Holder equipped with titanium end fittings (which I have heard clog easily?) What is your experience with these columns? Which option of the two do you recommend (cost is not a huge issue, but resolution and overall performance is)? Have you seen significant band broadening when using these small columns with a regular fplc (rather than Akta's microFPLC)? I would greatly appreciate your comments! Many thanks... Bests, Alex . Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] detect dsDNA
i wouldn't recoommend that. here is the info from somebody forwarded from our genetics department with regards to safety of that while back Sybr is just as toxic/poisonous/harmful as ethidium bromide, only far more expensive. There have been very few tests concerning it's use up until now and therefore it should be treated with even more caution than ethidium bromide. Waste must be separated and processed by the company and use of Sybr in the lab must involve safety and care levels at least as stringent as with Ethidium bromide. (not sure thow if it was SYBR-safe... safe must mean safe, right... in particular with chemical companies...) Feel free to handle your gels as sloppy as you like (or eat them or whatever), but mind the other people's health in the lab. There are people who actually have to work with this stuff for decades. I would advice following the safety instructions. Tommi On Oct 2, 2011, at 9:10 PM, Jacob Keller wrote: There exists a less toxic chemical than EtBr to stain DNA: SYBR safe DNA stain (a fluorescence dye sold by a certain vendor). SYBR Safe is about 10X less sensitive though. Can you do the toothbrush test with SYBR Safe? JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** Tommi Kajander, Ph.D. Junior Group Leader Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] question regarding secondary-structure restraints
-Just to make a note, there has actually been some discussion in the published literature recently (ok maybe past ten years) about what terms; simply steric (as originally) or hydrogen bonding etc might be needed to explain observed backbone angular values. Tommi On Sep 26, 2011, at 5:44 PM, Dirk Kostrewa wrote: Dear Nat, yes, I fully agree - all these restraints that improve the geometry either by restraining to high-resolution structures or by introducing H-bond restraints for secondary structures are very useful for low-resolution structures! I see your argument with the Ramachandran plot. But imagine a set of very strong non-bonding/bonding restraints that would result in an absolutely clean Ramachandran plot for any structure, then the Ramachandran plot would become useless even in the absence of any phi/psi-restraints. So, I prefer to err a bit on the safe side here by saying not a truly independent measure. Personally, I think, that ALL refinement programs, including the real-space refinement in Coot, would benefit from inclusion of proper H-bonding terms (something, that for instance the very old X- Plor version did), since this would automatically restrain secondary structures and other hydrophilic interactions to some reasonable geometry, even at very low resolution. Best regards, Dirk. Am 26.09.11 16:17, schrieb Nat Echols: On Mon, Sep 26, 2011 at 1:53 AM, Dirk Kostrewa kostr...@genzentrum.lmu.de wrote: when I played with H-bond restraints for secondary structures for the refinement of a 4.3 A structure (only a few weeks before they were introduced in phenix), I've made the following observation: at low resolution without H-bond restraints for secondary structures, the carbonyl groups of these secondary structures take the liberty within their globbish electron densities to deviate from their ideal H-bond conformation, resulting in a tight belt of outliers around the preferred Ramachandran regions, with typical deviations of only a few degrees. Introducing the additional H-bond restraints for maintaining secondary structures pulls these outlier carbonyl groups back into the preferred Ramachandran regions. In my case, the number of Ramachandran outliers was reduced to less than one half! Although, these H-bond restraints do not directly include information about allowed Ramachandran regions, the Ramachandran plot is actually affected by these restraints. Thus, at least in my opinion, the Ramachandran plot is then not a truly independent measure for model quality, anymore. The same holds true for all geometrical restraints, of course. It depends on how strictly you assess the independence of validation criteria. The Ramachandran plot is considered valid in most cases because refinement programs traditionally do not restrain phi and psi angles, so we need to rely on the accuracy of the data (and our placement of atoms) and various complementary geometry restraints (especially nonbonded) to keep residues in the favorable regions of the plot. There are a variety of ways to make the plot better by modification of the model and/or restraints (adding hydrogens, increasing the weight on the nonbonded restraints, secondary structure restraints, etc.), none of which are as drastic as directly restraining the model to the plot. I don't really view this as biasing the plot, for two reasons: a) the quantity being measured is independent of the quantity restrained, and b) at least in my hands, these modifications never completely fix the problem of Ramachandran outliers. (It's the loop regions that are really awful.) Anyway, I don't think anyone should feel bad about using this kind of restraint at low resolution. The caveat is that of all the specialized restraints that we (Jeff Headd and I) have been testing for low-resolution refinement (in Phenix), nothing works nearly as well in preserving good geometry, and usually improving the R- factors, as restraining model parameters to a related high- resolution structure, when one is available. Fortunately, every modern refinement program has this ability in some form, and I expect that this is going to have the most impact in improving the overall quality of low-resolution structures. -Nat -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de *** Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] low res. SAD phasing
Hi, Thanks for all the comments, i was more wondering what the state of the art might be here, we have done similar thing at 4 Å (with 3 Å data though) 10 years or so back with SnB. Maybe we need to get a heavier atom derivative indeed the break the phases, but i'll keep banging for now.. I have tried quite a lot, and was mainly wondering what was the world record nowadays in desperate solutions by SHELX/other direct methods... playing around with XDS, data is finally quite ok to 6 Å low res anom CC 100-90%, SigAno about 3... goes down obviously but not terribly fast there is signal all the way to 6Å. Inverse beam data collection would probably be a good idea indeed next time, native or second wavelength or rahter both might help i assume.. thanks to Tim, Clemens, Poul and others for comments, Best, Tommi On 15.6.2011, at 17.16, Tim Gruene wrote: Hi Tommi, Give it a try? You give very little information, e.g. - the data quality, - possible radiation damage, - isomorphism with a native data set, and - what you have tried so far. If you have a native data set with an isomorphous unit cell, try SIRAS instead of SAD. Tim On Wed, Jun 15, 2011 at 04:36:59PM +0300, tommi kajander wrote: Dear all, Does anyone have suggestions for 6 Å resolution phasing with large number (40-50) Se sites (SAD so far)?? Thanks a bunch, Tommi Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940 -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
I would add that there are some issues with air, you have to be careful with nanodrop that the path is ok, and also if concentrations are low, 1 mg/ml for instance, i am not sure one can trust it - compare 50 ul 1 cm path results with nano at 0.5-1 mg/ml... i get inconsistency there.. its good for concentrated samples and fast. and handy, we use it a lot, but for dilute samples, i always use 1 cm light path. just my feel on it. Tommi On Jun 16, 2011, at 11:20 PM, aaleshin wrote: Filip, 25% accuracy is observed only for very diluted (OD280 0.1) or concentrated samples. But those sample a rarely used for ITC or CD. The concentrated samples require dilution but a regular spec does it too. Since the light passway is very short in Nanodrop it is accurate with more concentrated samples, which we crystallographers use, so Nanodrop is ideal instrument for our trade. If the drop is within recommended volume like 1-2 ul for our model, its size has a very small influence on the measurement. Cuvettes will give a better accuracy provided you clean them properly. I hated those times when I had to measure a concentration because of a need to wash a cuvette. In a biological lab they are always dirty. We switched to plastic disposable cuvettes for that reason... Alex On Jun 16, 2011, at 1:06 PM, Filip Van Petegem wrote: 25% is not acceptable for ITC or CD experiments though... I was just sharing our bad experience with a demo nanodrop we had. Even if evaporation is not an issue, one has to take pipetting errors into account when dealing with small volumes. The relative error on 1-2ul is a lot bigger than on 50ul. Unless you want to pre-mix 50ul and use a small quantity of that, which defeats the purpose of miniaturization... It all depends on your applications and sample availability, but if you want a very accurate measurement, miniaturized volumes just won't get you the same accuracy. Cuvettes will give a better accuracy provided you clean them properly. Just some water or EtOH is *not* enough... Filip Van Petegem On Thu, Jun 16, 2011 at 12:52 PM, aaleshin aales...@burnham.org wrote: I also like our Nanodrop, but I do not recommend using it for Bradford measurements. The 25% accuracy mentioned by Flip is pretty good for biological samples. Using 50 ul cuvette in a traditional spectrophotometer will not give this accuracy because cleanness of the cuvette will be a big issue... Alex On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote: I completely disagree with Filip’s assessment. I’ve been using nanodrop nearly 5 years and never had inconsistency issues. If you work at reasonable speed (if you put a drop there then lower the lever and click measure before you do anything else) there will be no issues. At very high concentrations the accuracy and therefore consistency may become lower. Concentrations between 5 and 10 mg/ml should be fine. The instrument is pricey though. Vaheh From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip Van Petegem Sent: Thursday, June 16, 2011 3:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford. Dear Arnon, the Bradford method is not recommended for accurate measurements. The readings are strongly dependent on the amino acid composition. A much better method is using the absorption at 280nm under denaturing conditions (6M Guanidine), and using calculated extinction coefficients based on the composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds). This method is also old (Edelhoch, 1967), but very reliable. One thing about the nanodrop: smaller volume = more evaporation. On the demo we've had, I was so unimpressed with the precision (25% variability between two consecutive measurement) that we didn't consider this instrument at all. So unless you just want a 'rough' estimate, I wouldn't recommend it at all. But most respectable spectrophotometers will take cuvettes with 50ul volumes - a big step up from 1ml volumes... Filip Van Petegem On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote: Dear fellow crystallographers - a question about spectrophotometers for protein concentration determination. We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein conc. determination. We have been considering buying a Nanodrop machine (small volume, no dilution needed, fast, easy). However, while testing our samples using a colleague's machine, we have gotten readings up to 100% different to our Bradford assay (all fully purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So while it is fun/easy to use the Nanodrop, I am not sure how reliable are the measurements (your thoughts?). So QUESTION 1: What are people's experience regarding the
[ccp4bb] low res. SAD phasing
Dear all, Does anyone have suggestions for 6 Å resolution phasing with large number (40-50) Se sites (SAD so far)?? Thanks a bunch, Tommi Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] off topic: problematic protein
I would think thing here is that this protein actually associates to those lipid nanodiscs...(around the disc) and Na cholate CMC is around 10 mM. so, yes you can solubilise proteins that bind lipids, the question is does this protein bind lipids or not? or is it just scrambled or whatever, doesnt like to be overexpressed etc??? or sticky because it is part of a larger complex naturally and not stable alone, for instance. which i wouldn't know of course. regards, Tommi On Apr 20, 2011, at 1:08 AM, Arthur Glasfeld wrote: I recently followed a protocol from Stephen Sligar's lab for the purification of his nanodisc protein, which has strong hydrophobic character as it associates with phospholipids. His protocol includes washes with 1% Triton X-100 and then with 50 mM cholate (both at pH 8 in the presence of 300 mM NaCl). Worked great, and I saw stuff coming off the column in both washes. The reference is: Bayburt et al. (2002) Nano Letters, vol. 2, pp 853-856. http://pubs.acs.org/doi/abs/10.1021/nl025623k Good luck, Arthur Arthur Glasfeld Department of Chemistry Reed College 3203 SE Woodstock Blvd. Portland, OR 97202 USA On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non- micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/ tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] what to do with disordered side chains
you queried, however. A somewhat key question might be: across the various molecular visualization programs, what is the default way to handle atoms with occ=0? Perhaps those programs might be the best place to fix the problem... JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
Hi, they buy it from some small company, idea being the applicability to SLS, optimized for minimized bleeding/sheding or material from the column, which will show up only in the light scattering detector. but i have to say some of our proteins stick to their columns (happened during the demo), whereas they dontt to TOSOH, on the other hand some proteins, as mentioned dont like silica at all --- so you have to try, or if you know GE columns work for you (i have some proteins that like to stick to sacharide based things...) can go with them quite ok. Few tens of micrograms is certainly enough on a 10/300 S200 for instance... narrow bore silica columns (eg 4.8 mm TOSOH) appear to be bit limiting since the flow rate would be better to be bit faster than what you can get there (0.4 ml/min vs 0.5-1ml/min on S200 superdex) Tommi On Mar 8, 2011, at 6:56 PM, Jacob Keller wrote: So from where does Wyatt outsource their columns? JPK On Tue, Mar 8, 2011 at 10:49 AM, Sally Pham Thanh Van sally.pha...@gmail.com wrote: I should say clearer that I did a comparison between GE (Superdex 200 10/30) and the Wyatt column of the same separation range on AKTA connected with MALS. The Wyatt column gave better resolution for size exclusion chromatography as well as signal-noise ratio for light scattering. So I think it is worth for you to try it. They offer money-back guarantee. Best wishes, Sally. On Tue, Mar 8, 2011 at 3:36 PM, Sally Pham Thanh Van sally.pha...@gmail.com wrote: Dear Sebastiano, From my experiences, Wyatt columns are the best. Best wishes, Sally. On Tue, Mar 8, 2011 at 2:03 PM, Sebastiano Pasqualato sebastiano.pasqual...@gmail.com wrote: Dear all, I was wondering if somebody could help me out by suggesting the best column to be used in a Static Light Scattering (I guess it would be the same for a Multi Angle Light Scattering) instrument. We were suggested using a silica-based column, with very high separation properties, but it seems that these columns are highly sensitive to (even slightly) basic pH's. Even running the column in PBS, it looks like injecting samples at pH 8.0 ruins the column resin, making it unusable. On the other hand, GE Healthcare columns would require a huge amount of material to be loaded. What are you guys using in your instruments? Thanks a lot in advance for the feedback, best, ciao Sebastiano -- Sebastiano Pasqualato, PhD Crystallography Unit IFOM-IEO Campus Cogentech - Consortium for Genomic Technologies via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5172 fax +39 02 9437 5990 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] CCP4 for iphones
why, If we are lucky they will all just vaporize and disappear... (...sorry couldn't resist...) tommi On Feb 25, 2011, at 10:51 PM, Xiaoguang Xue wrote: But I don't know what will happen if we make a cluster by hundreds and thousands of iphones. On Fri, Feb 25, 2011 at 9:25 PM, Xiaoguang Xue bio...@gmail.com wrote: Hi, Your idea is very cool, but i think it's difficult to run CCP4, Coot or Phenix on iphone/itouch. Because the CPU of iphone/itouch is very slow and the graphics engine is also not good enough for these programs. But I think we can install an SSH client app on iphone/itouch/ipad, such as iSSH, then we can control our server or workstation by iphone/itouch, and we can run these 'big' software remotely. If this can be considered working on an iphone, I think we can do that. Nice weekend! Xiaoguang On Fri, Feb 25, 2011 at 8:54 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Hello All, is there a ccp4 app for iphones yet? Has any structure been solved on an iphone? I know BR uses an iphone to drive his robots, but what about structures? (A question for Friday afternoon...) JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- Xiaoguang Xue, PhD student Utrecht University Crystal Structural Chemistry Padualaan 8. Room N807 3584 CH Utrecht The Netherlands Tel. +31-30-253-2383 -- Xiaoguang Xue, PhD student Utrecht University Crystal Structural Chemistry Padualaan 8. Room N807 3584 CH Utrecht The Netherlands Tel. +31-30-253-2383 Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
[ccp4bb] MALLS analysis question
Hi, if you happen to have Wyatt's light scattering detector + their RI detector rEX, together with analytical HPLC/SEC system what are your experiences on the stability of the aligment? the digital connections dont seem to work for us. ie. apparently synchronizing the signals from different detectors (computers) to the analysis software computer dont work, even if the alignment (ie delay volumes between detectors) has been done. Should work with analog cables to one detector and then LAN connection from there on to the analysis PC. testing it.quit certain it will. but i would be happy to hear if anyone had similar problems Thanks! tommi Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] The 2nd Advanced Protein Characterisation and Crystallisation Course in Helsinki, Finland
Dear all, I would like to draw your attention to the following protein crystallization and characterization course we are offering: 2nd Advanced Protein Characterisation and Crystallisation Course: May 2.- 6. 2011 The Biocenter Finland Protein crystallisation unit is again organising an advanced workshop in Protein Characterisation and Crystallisation. The dates are May 2nd to May 6th, 2011. The course will start at 9 am on Monday and end at 2 pm on Friday. The course will be held at the Institute of Biotechnology in Helsinki. We have again assembled a world-class team of international experts in protein crystallisation and characterisation, and the course will be appropriate for graduate students and also for postdoctoral fellows wishing to acquire new skills. for details: http://www.biocenter.helsinki.fi/bi/xray/automation/kurssi.htm Best regards, Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Se-Met Protein Purification
I think the likely reason here are the points where you did the mutations having adverse effects - how do the mutants behave without labeling? this would be of course an important control... -Tommi On 25.1.2011, at 12.52, Paula Salgado wrote: Dear Abhilash You could try an alternative labelling option: if your protein has cysteines, try labelling those with selenium. We succeeded in doing this using non-auxotrophic strains, meaning you deviate less from your native expression protocol and are therefore more likely to obtain well folded and soluble sample, in the correct dimeric form. For details of our protocol, see: http://journals.iucr.org/d/issues/2011/01/00/dz5216/index.html Good luck. Paula On 25 January 2011 10:34, Abhilash Padavannil g0800...@nus.edu.sg wrote: Dear All, I am working on structure determination of a bacterial protein. The protein was expressed as a GST fusion protein in E. coli. I got crystals of the native protein. I am trying to label my protein with Se-Met. The purification protocol involves GST affinity purification, cleavage of the fusion protein followed by gel-filtration. The native protein behaves well and on gel filtration majority of the protein elutes as a dimer with very little eluting in the void volume. However, using the same protocol with the Se-Met protein, most of the protein elutes in the void volume with very little or nothing as a dimer. I am struggling with this for quite some time. I also tried by adding 10mm DTT in the buffers only to see that the fusion protein does not bind to the beads at this concentration of DTT. Could someone suggest me on how to increase the proportion of dimeric protein? I have one more question, does the position of methionines in the protein effect its hydrophobicity? For the sake of labeling I have mutated a few leucines in the native protein to Met and they all lie very close to one another. Could this be the reason for heavy aggregation? Thanks in advance, Abhilash
Re: [ccp4bb] Space group and R/Rfree value
Wouldnt it be easier (in case anyone didnt suggest it yet) to look at the parity analysis from e.g. dataman or probably phenix.xtriage to tell you if you have pseudosymmetry (if you processed it in P212121) -- in C1 you obvioulsy already assume _real_ centering. visually inspecting maybe bit tedious unless you are really hard core on it... Tommi On Dec 1, 2010, at 9:25 PM, harry powell wrote: Hi I'm not sure if anyone has mentioned this yet, but if you use hklview to view the mtz file (assuming you're working in ccp4- land), you can probably get a better handle on the systematic absences than by looking at the original images. I'm told there are other tools available to examine your reflection file graphically, but I haven't used them so I can't comment on them. On 1 Dec 2010, at 17:58, Phoebe Rice wrote: Its may be an interesting question of pseudosymmetry. The best thing would be to find a local mentor who could have a good hands- on look, but here are some thoughts: From the space group tables you will see that P212121 has half as many asymmetric units as C2221, and C2221 has crystallographic twofold axes that your dimer could lie on. So both space groups are perfectly consistent with the protein being a dimer. (You must be able to see the symmetric dimer when you rebuild your model in C2221 during the refinement process?) The R-factor might be higher in P212121 because you're refining two molecules instead of one, and thus there are more degrees of freedom. If the space group does turn out to be P212121, you should probably find a local expert for detailed refinement advice. The centering in C2221 should give a different pattern of systematically missing spots, so you should have a carefull look at the original images and the output of the scaling in C2221, and see if the additional missing spots are really missing or not. (Don't forget the axes may be permuted between the two indexing systems). If they're present but weak, its P212121. In that case, you should note that the systematically weak spots will distort the standard twinning statistics. Hope that helps! Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 1 Dec 2010 16:17:58 +0800 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Xiaopeng Hu huxp...@mail.sysu.edu.cn) Subject: [ccp4bb] Space group and R/Rfree value To: CCP4BB@JISCMAIL.AC.UK Dear all, I am working on a data-set (2.3A) and the space group problem bothers me a lot.The space group of the data-set could be C2221 or P212121, since our protein functions as a dimer, and P212121 gives two molecular in the asym-uint, I think P212121 is more reasonable than C2221.However with C2221, I can refine the R/Rfree to 20/24 or lower, while with P212121 only to 26/30. Also Phenix points out that the crystal is probably a twin with P212121 but is OK with C2221. I am not a real crystallographer, perhaps this problem is stupid, any help will be appreciated!! Best wishes, Xiaopeng Hu Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
[ccp4bb] met auxotroph S.cerevisiae strain?
Hello all, Would anyone happen to have a met auxotroph S. cerevisaea strain at hand that you could send some of to us for testing Semet labelling? Thanks very much! Regards, Tommi Tommi Kajander, Ph.D., Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] LS / RI detector systems
Dear All, I would like to ask people's comments on usage/preferences on different providers MALLS/RALS detectors and RI detectors (to combine with HPLC SEC) Mainly we are looking at Wyatt vs Viscotek (or perhaps Varian/Agilent now also) tetradetektors(or triple) detectors at the moment, i am not aware of many other options.. In particular what is your take on Wyatt MALSS accuracy *(why bother with several angles although i kind of like the software and instrument...) vs RALS for proteins (which end up being angle independent in scattering anyhow) --or what do you think overall of the different manufacturers equipments accuracy, etc... e.g. viscometer seems rather unnessary to me, as does online DLs.. Thanks for comments, Tommi -- Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] insect cell media
Hi, well, actually this is availalbe as powder (HYClone), as it says on the page.. (we have it made in our media kitchen on a regular basis, but not for huge scale... so i dont know about the prices..) https://www.thermoscientific.com/wps/portal/ts/products/detail?navigationId=LA11074__10347categoryId=82051productId=11960716 HTH, Tommi On Jun 7, 2010, at 5:52 PM, Dima Klenchin wrote: We have happily made a transition last year from using Invitrogen's SFM medium and cellfectin to Insect-Xpress (Lonza) and polyethyleneimine for transfection. We are moving several protein targets to large-scale cultures and would consider cost-cutting alternatives. For example, Invitrogen and Thermo Sci both offer media in powder form at an attractive price. Has anyone made a systematic comparison of these media? Any particular recommendations regarding powder media? Last I checked, no manufacturer offered serum-free powedered medium. Fetal calf serum is very expensive - once you count in its cost, the overall price of the media becomes comparable. If your protein is secreted, serum-containing media are not even a realistic choice. If it is intracellular, buying dry medium and adding FBS work well: is a lot more hassle, is about 20% cheaper, and usually gives slightly higher titers and expression levels. Of all the common media we compared, TNM-FH works best for us with Sf9 cells. Switching to dry medium also means some initial investment: bottles, filtration equipment and filters. So it all pays off only of you need large scale cultures long-term. If you are aware of a dry serum-free insect cells medium, please let me know - I'd love to try it. Dima Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] SCA
yes, other people can comment probably but i think entropy based estimates are better (as i remember less dependent on sample set size). and indeed yale has a server. which may or may not do what you want. secondly its not proper to distributed ohter people's software w/o their permission (actuaally its abs wrong, if not illegal, availibility is anohter question of course...). i would advise to look at papers on entropy based measures of coupling and write to the authors. my two cents..., tommi Quoting Jacob Keller j-kell...@md.northwestern.edu: Hi Azadeh, I looked into this and other related methods extensively once, and came out with the understanding that SCA is not really the best of this type of analysis (you can read some of the papers out there which analyze the several methods). I found that the java package from Mark Gerstein's group at Yale does any/all of the analyses in parallel (if you want) and is relatively easy to set up. The best, as I recall, was the one based on: Gobel,U. et al. (1994) Correlated mutations and residue contacts in proteins. Proteins: Struct. Funct.Genet., 18, 309-317. I think you need an MTA for the actual SCA software, as well. 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: j-kell...@northwestern.edu *** - Original Message - From: Azadeh Shahsavar To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, January 18, 2010 4:25 AM Subject: [ccp4bb] SCA Dear all, Does anyone have the current (or old) version of SCA? (SCA: statistical coupling analysis) It should be as a toolbox of Matlab software. Thank you in advance, Azadeh -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] FW: pdb-l: Retraction of 12 Structures....
Would the exact analysis of how each of these things were wrong and fabricated be somewhere available Would be fair (apart from the known case of C3b) to have the whole analysis available instead of just this kind of news feed. I suspect its not obvious by five minute check in all cases. Perhaps there needs to be ways within PDB in form of automated tools that would raise those red flags in suspicious cases (e.g. some data analysis --such as the contribution by solvent etc now that data beyond 8Å is by default used in refinement) - as it appears peer review/editing by journals isn't/cant always be(?) stringent enough. In any case, some type of automated analysis of the whole data base might be a good idea, as there can be other cases (with another couple of thousand papers citing them..). tommi On Dec 10, 2009, at 4:16 PM, Ibrahim Moustafa wrote: After a thorough examination of the available data, which included a re-analysis of each structure alleged to have been fabricated, the committee found a preponderance of evidence that structures 1BEF, 1CMW, 1DF9/2QID, 1G40, 1G44, 1L6L, 2OU1, 1RID, 1Y8E, 2A01, and 2HR0 were more likely than not falsified and/or fabricated and recommended that they be removed from the public record, the university said in its statement this week.
[ccp4bb] UV detection of crystals/survey
Hello, I would be interested in people experiences and opinions about what types of fluorescence microscope setups have worked adequately to distinguish small protein crystals from background/ autofluorescence etc. ...general comments? (regular dichroic mirros etc dont allow Trp peak to pass through for instance etc and quartz becomes qute expensive i think?) i am looking for the cheapest but effective system (ie i want to see 10 um size things) regular sterefluor. microscope with filters + fiber UV light from the side? anybody has found something like that to work? thank you very much for comments, Tommi Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] phenix rigid body ref.
Apologies, i know this is not phenix bb but since i am here (and not subscr. there), does anyone know how to control rigid body ref positions in phenix.refine, i am trying to do very low res check (6 Å) with quite many molecules, and they start landing on each other while the MR solution from molrep has perfect packing isnt there a packing constraint somwhere saying atoms cant be on top of each other... there was something about clashes but didnt seem to do anything.. the parameter definitions in .eff are bit cryptic to me in places... (would it be possible to step back a bit (sorry if i am wrong) to the ancient world of x-plor, still like that manual a lot..). thanks Tommi K.
Re: [ccp4bb] phenix rigid body ref.
this doesnt exactly go to the detail i was looking for... but thanks, i do know where the pages are. tommi On Nov 26, 2009, at 9:52 PM, Jacques-Philippe Colletier wrote: Hi Tommi You will find all the information you need on the following web page: http://www.phenix-online.org/documentation/refinement.htm as per your question, you could try : phenix.refine yourdataset.mtz yourmodel.pdb strategy=rigid_body rigid_body.number_of_zones=1 sites.rigid_body=chain A sites.rigid_body=chain B sites.rigid_body=chain C sites.rigid_body=chain D etc... (I suggest you take only one zone for the rigid body refinement because at 6A you only have so many reflections) Best Jacques Le Nov 26, 2009 à 8:32 PM, Tommi Kajander a écrit : Apologies, i know this is not phenix bb but since i am here (and not subscr. there), does anyone know how to control rigid body ref positions in phenix.refine, i am trying to do very low res check (6 Å) with quite many molecules, and they start landing on each other while the MR solution from molrep has perfect packing isnt there a packing constraint somwhere saying atoms cant be on top of each other... there was something about clashes but didnt seem to do anything.. the parameter definitions in .eff are bit cryptic to me in places... (would it be possible to step back a bit (sorry if i am wrong) to the ancient world of x-plor, still like that manual a lot..). thanks Tommi K. Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
[ccp4bb] arpwarp improve maps
Hi, the latest arpwarp via ccp4 6.1.2 GUI on Mac OS X 10.5 something does not work, it wants a sequence file which isnt an option in the GUI... (you put in the coordinates and update them thats all right?) i just want to improve the maps if possible. also the model if possible.. flex-warp didnt quite do what i wanted it to do.. thanks for suggestions tommi
[ccp4bb] loop modelling?
Dear all I was wondering if anyone knows a simple way to generate a missing loop (strech of amino acids) really in just some simple manner (no fancy minization necessary, of course some constraints probably dont hurt), just for visualization purposes of further simulations etc thanks! tommi
Re: [ccp4bb] Anomalous map creating
to the original poster: please do not attache several MB files... Tommi On Oct 27, 2009, at 11:24 AM, Eleanor Dodson wrote: Well - doing an mtzdump on testXXX.mtz shows most of the DANO are missing.. and also most of the F_gp5-gp27(+)_nat so the problem is farther back.. OVERALL FILE STATISTICS for resolution range 0.000 - 0.119 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. Low High label 1 ASC 0 41 0 100.00 19.7 19.7 127.21 2.89 H H 2 NONE 0 24 0 100.00 6.9 6.9 127.21 2.89 H K 3 NONE -131 131 0 100.00 1.5 49.7 127.21 2.89 H L 4 NONE0.019.0 5 99.98 9.48 9.48 64.49 2.89 I FreeR_flag . 16 NONE8.5 4494.8 294998.34 895.29 895.29 74.82 2.89 G F_gp5-gp27(+)_nat 17 NONE6.0 162.8 294998.3437.8337.83 74.82 2.89 L SIGF_gp5-gp27(+)_nat 18 NONE8.5 4681.4 353 98.90 812.18 812.18 74.82 2.89 G F_gp5-gp27(-)_nat 19 NONE5.8 162.8 353 98.9025.8925.89 74.82 2.89 L SIGF_gp5-gp27(-)_nat 20 BOTH0.0 0.0 294998.34 0.00 0.00 74.82 2.89 D DANO_gp5-gp27_nat 21 BOTH0.0 0.0 294998.34 0.00 0.00 74.82 2.89 Q SIGDANO_gp5-gp27_nat The cad log file looks OK so you need to go back to the scala step and see how this happened.Maybe you didnt measure enough equivalent observations to get a DANO set? Eleanor Sergii Buth wrote: Hello everybody! I am faced with a problem of calculating an anomalous map from a Se- Met dataset, and I cannot interpret the error message. So, detailed problem description: I was given a Se-Met dataset of my protein. I scaled it in Scala and made .mtz file, but I do not phases. And I cannot do a MR, but I have a coordinate file. This is my situation So, what I did. I made a copy of .mtz and did a refinement in refmac - to generate phases. During that I lost all anomalous data. After I did CAD procedure - I took from original .mtz anomalous data (F(+), F(-), DANO, IMEAN, I(+), I(-), with sigmas) and from refined mtz - H K L FreeR_flag, F, SIGF, FC, PHIC, FC_ALL, PHIC_ALL, FWT, PHWT, DELFWT, PHDELWT, FOM. And then I did anomalous FFT in the fields I put: PHI - PHIC Weight - FOM DANO - DANO Sigma - SIGDANO I tried with and without excluding of R-free, but result was the same - FAILED... And error message was FFTBIG: No reflexions pass acceptance criteria! Check RESOLUTION, EXCLUDE, missing data. And I cannot find how to fix this. It have also one more warning message - * Missing value set to NaN in input mtz file but as I read it is not a problem - mtz is still readable. I would be glad for any help or advice. Thanks. Sergii P.S. Please, find attached mtz and logs. Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] How to compare the binding affinity between two domains structurally?
this will only give an estimate of *electrostatic* contributions via poisson boltzmann calculations NOT *binding* energy. certainly one way to try to estimate those (and hyrdophobics via buried surface area?). perhaps FEP/MD or something that actually takes into account hydrogen bonding properly might be better, but perhaps also rather time consuming (no suggestions). tommi Quoting Chandra Verma chan...@bii.a-star.edu.sg: if the link between the domains is not part of the interface then perhaps a good first approximation may be to cut the linker and then use a program such as APBS to compute the binding energy.. Hi, Recently we determined two structures of the same protein in complex with different molecules. The protein contains two domains (called domain A and B here). In the two structures, domain A and B have different arrangements relative to each other, resulting different interaction interface. I want to know which inter-domain interaction is stronger. Is there a way to quantatively compare the interaction energy or intensity between the two domains? I have calculated the buried surface area. However, just comparing the buried surface does not provide definitive answer, given that the interacting residues on the interface are also different. BTW, we were not able to purify individual domains, so we cannot measure the binding affinity by wet lab approaches (so far). Thank you in advance for your inputs. -- Best regards, Joe -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] MOLREP
Hi, I have been using a dimer as a search model in MOLREP (there will be several in AU), for some reason the program tends to break the dimer into monomers wihtout asking me.. how is this determined in the program... a more detailed manual would be nice, also on the output as the different contrasts and their meaning appear bit cryptic to me. (i am beginning to get the hang of it but its still bit fuzzy..) Also if i am searching for number of say these dimers and there is a speudo-translation vector should it be used all the time? (i would assume not all the searched unit are related necessarily by the translational NCS) (to make it even merrier, there is both proper speudo centering and just translational NCS .. and you can only specify one vector... if we are lucky its also twinned... thanks for comments, Tommi
Re: [ccp4bb] MSA formatting...
thanks for all who replied, got the answers already in several flavours i believe! tommi On 14.9.2009, at 2.46, Tim Gruene wrote: Hello Tommi, could you please paste one or two lines of what you've got and one or two lines of what you want. That might make the problem description a bit easier. Tim Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] MSA formatting
Hi, off-crystal topic: i was wondering if anyone has a simple solution to combining e.g. CLUSTAL etc ouput files to format (ie idcodes then 60 residues strech and start again below the same...) ...that has only one line per sequence. hopefully that was clear enough... basic problem is simply combining lines which start with same set of characters (ID) but removing the IDs codes from everyelse but the beginning. i am not much of a script writer obivously Thanks a bunch! Tommi Tommi Kajander Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] MSA formatting...
...actually now i remember the problem was that i want it in excel with 800 column and each amino acid needs to be separated to a column (ie comma between each position) excel puts words into one column. bit tedious to define columns by hand maybe wrong forum, but thanks anyway.. tommi On Sep 13, 2009, at 3:52 PM, Tommi Kajander wrote: Hi, off-crystal topic: i was wondering if anyone has a simple solution to combining e.g. CLUSTAL etc ouput files to format (ie idcodes then 60 residues strech and start again below the same...) ...that has only one line per sequence. hopefully that was clear enough... basic problem is simply combining lines which start with same set of characters (ID) but removing the IDs codes from everyelse but the beginning. i am not much of a script writer obivously Thanks a bunch! Tommi Tommi Kajander Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] Problems with phasing a protein (1300aa)
i was just refering to the mentioned ideal case with 43 se and 1300 aa in AU, and some real (successful) examples of same size range. -tommi Quoting Bernhard Rupp b...@ruppweb.org: What is not true? Also in your case the applet estimates an expected 4-5% signal which is quite doable with decent data. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tommi Kajander Sent: Friday, March 20, 2009 3:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) this cant be true, in the idea case (not Rmerge 15%, then again one can apply a resolution cutoff, perhaps, while this sounds like a very desperate case) the answer must be yes. didnt do the calculation right now (but it _should_ back this up) we for instance have solved a structure long time ago -- and this probably wasnt on the limit (well it was at the time but not anymore), 365 res x 8 in AU and 80 Se. at least looking at the SnB success list (very old list ) http://www.hwi.buffalo.edu/SnB/SnBSuccesses.htm there are plenty of others. -tommi On Mar 20, 2009, at 10:53 PM, Bernhard Rupp wrote: One can estimate this from http://www.ruppweb.org/new_comp/anomalous_scattering.htm and the answer as Jim says is no. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Friday, March 20, 2009 1:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) Well, what do you expect the anomalous signal contributed from your 45 seleniums in a perfect world to be when the asymmetric unit contains 1300 aa? Do you think a dataset with Rmerge of ~15% is good enough to detect a signal of even 2%? (Note: I did not do the calculation, so I just made up the number of 2%.) Jim On Fri, 20 Mar 2009, Kumar wrote: Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. ... Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Problems with phasing a protein (1300aa)
this cant be true, in the idea case (not Rmerge 15%, then again one can apply a resolution cutoff, perhaps, while this sounds like a very desperate case) the answer must be yes. didnt do the calculation right now (but it _should_ back this up) we for instance have solved a structure long time ago -- and this probably wasnt on the limit (well it was at the time but not anymore), 365 res x 8 in AU and 80 Se. at least looking at the SnB success list (very old list ) http://www.hwi.buffalo.edu/SnB/SnBSuccesses.htm there are plenty of others. -tommi On Mar 20, 2009, at 10:53 PM, Bernhard Rupp wrote: One can estimate this from http://www.ruppweb.org/new_comp/anomalous_scattering.htm and the answer as Jim says is no. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Friday, March 20, 2009 1:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) Well, what do you expect the anomalous signal contributed from your 45 seleniums in a perfect world to be when the asymmetric unit contains 1300 aa? Do you think a dataset with Rmerge of ~15% is good enough to detect a signal of even 2%? (Note: I did not do the calculation, so I just made up the number of 2%.) Jim On Fri, 20 Mar 2009, Kumar wrote: Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. ... Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] Small lines in diffraction pattern (more info)
For this discussion another relevant reference might be: The 1.8 A crystal structure of a statically disordered 17 base-pair RNA duplex: principles of RNA crystal packing and its effect on nucleic acid structure. Shah SA, Brunger AT. J Mol Biol. 1999 Jan 29;285(4):1577-88. -tommi On Jan 29, 2009, at 1:53 PM, Ian Tickle wrote: Hi Herman Aren't detwinning methods appropriate only in the case of true twin domains which are larger than the X-ray photon correlation length in order for the assumption to be valid that |F|^2 from each domain can be summed? This wouldn't give rise to the apparent 'diffuse scatter' phenomenon. However if what you are describing is rather static disorder of unit cells, which would give rise to diffuse scatter, where A B type cells are randomly mixed (so a domain is only one or at most a few unit cells), as opposed to being confined to A B type domains, then detwinning would not be appropriate. Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: 29 January 2009 11:19 To: margriet.ova...@chem.kuleuven.be; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Small lines in diffraction pattern (more info) Dear Margriet, From your description and what James Holton wrote, it seems that you have 2 types of unit cells: A: with the sense strand in position 1 and the antisense strand in position 2 B: with the antisense strand in position 1 and the sense strand in position 2 If the crystal contacts are mainly via the backbone, your crystal may contain a random distribution of both and the electron density you see is a superposition of both and for the crystal packing, both chains are identical. This situation is similar to the situation when an asymmetric inhibitor is bound to a dimeric, symmetric molecule like e.g. HIV protease. In this case, both orientations are deconvoluted using detwinning methods for perfect twinning (see e.g. Proc Natl Acad Sci U S A. 2002 November 12; 99(23): 14664-14669). The 34,34,34 cell is definitively too small, so I would process in the 34,34,170 cell and detwin. You molecular replacement solutions should tell you which twinning operator to use. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: Thursday, January 29, 2009 10:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Small lines in diffraction pattern (more info) Dear all, There were some comments about detector issues, but these can be ruled out, to my opinion, since the lines appeared on different beamlines. Default settings of mosflm (spot picking) finds the cell 34 34 34 90 90 90 (pointless indicating P41212) Structure was solved by SAD phasing on the phosphates in this space group. Double helices stack in continuous helices, the backbone is well defined in the (refined) density maps but the individual bases are messy (purines and pyrimidines seemed to overlap) + obviously not all spots were covered and the duplex does not fit in the A.U. For this reason the integration was repeated in the higher cell 34 34 170 Space group most probably P212121, but solutions can be found in P41212 as well (still disordered bases) There are also indications that the 41 screw axis is rather a pseudo axis than a pure crystallographic one, also in the small cell Reindexing the cell to 34 34 340 also gives a solution, which supports the theory of Holton Rmerg is around 5% for the small cell, about 8% for the 170Å cell (both in P41212) Which refinement procedure would be best to follow? kind regards Margriet Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information. Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
the tandem KH domain of FMRP crystallized as a very convincing dimer (valverde et al 2007), but is a monomer in solution, although it is not the whole protein but just two domains of it.. anyway, i would think these ar much more common than the other way around. Tommi Quoting Poul Nissen [EMAIL PROTECTED]: I would say that all crystals represent hyper-oligomeric structures, but never mind, I know what you mean ;-) the E. coli EF-Tu:EF-Ts complex is a good example - the structure clearly indicates an (EF-Tu:EF-Ts)2 dimer, and the T. thermophilus EF-Tu:EF-Ts is even a disulphide-linked dimer. However, all solution studies indicate that the E.coli EF-Tu:EF-Ts complex is in fact a monomeric complex. Poul On 11/12/2008, at 17.09, Santarsiero, Bernard D. wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Protein folding pattern schematic
ALSCRIPT used to be popular, bit old now i guess and not so simple. but does the job. tommi On Nov 10, 2008, at 8:39 PM, Partha Chakrabarti wrote: Hi Charu, Indonesia is one option, system independent but you need Java for that. http://xray.bmc.uu.se/dennis/ Cheers, Partha On Mon, Nov 10, 2008 at 6:53 PM, Charu Chaudhry [EMAIL PROTECTED] wrote: Hello, Does anyone know of a program that can automatically generate a folding pattern schematic diagram showing the arrangement of secondary structure elements for a protein ? Presumably one would have to feed it a PDB file with secondary structure assigned from DSSP. Thanks! Charu Mayer lab/NIH -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: [EMAIL PROTECTED] Phone: + 44 208 816 2515 Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [EMAIL PROTECTED]
[ccp4bb] recipees for prep of test derivatives
Hi, Could anyone give a more or less exact recipee for preparing a derivate for lysozyme or proteinase K? like what used, concentration and soak time. we'd need some data sets for a lab course only thing that really worked so far was lysozyem KI SIRAS (which however doesnt seem to be good enough for demonstration purposes, so we need more) Thanks very much! best, Tommi Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [EMAIL PROTECTED]
[ccp4bb] test data sets part II...
Also, i think that would be nice if this type of info could be put on the web, part of the wiki for instance.. if there is some consensus to what works + the typical proteins easily available. Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [EMAIL PROTECTED]
Re: [ccp4bb] Progresss with Stereo 3D under Mac OS X Leopard
you use 3D stereo visualization (i.e. stereo glasses)? ALWAYS OFTEN SOMETIMES RARELY NEVER Answer: 8) Is there any software you would like to have available in the computing environment to assist you in molecular model building and/or visualization that is not currently available? Answer: Thank you for your time. Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [EMAIL PROTECTED]
[ccp4bb] baculovirus GFP vectors
Hi, bit off topic, but would anyone have baculovirus vectors that co-express GFP for checking infection (i dont want a fusion protenin) ??? such as the pUltraBac-1 by Philipps B, Rotmann D, Wicki M, Mayr LM, Forstner M. Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells. Protein Expr Purif. 2005 Jul;42(1):211-8 or if i can reach one of the authors is reachable this way and willing to send some material i would be greatful. thank you, tommi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Concentrating protein
couldnt agree more.. just pump the dilute solution through the ion exhange column.. or was there salt in it to prevent binding? or what was wrong with just using 80 ml centripreps or equivalent? not that high-tech, all you need is a regular 250 ml centrifugre tube rotor... (well the centrifuge also). tommi Quoting Phoebe Rice [EMAIL PROTECTED]: A layer of dry PEG on the outside of the dialysis tubing works too, without changing the ionic strength. But you'll probably get small molecular weight contaminants from the PEG entering the bag, so you'll have to dialyze against real buffer afterwards. The whole procedure in the end might be more annoying than just loading a very large volume onto an ion exchange column. Phoebe Original message Date: Fri, 27 Jun 2008 10:16:53 -0500 From: First, Eric [EMAIL PROTECTED] Subject: Re: [ccp4bb] Concentrating protein To: CCP4BB@JISCMAIL.AC.UK Back in the old days, we used to put the protein in dialysis tubing and surround it with solid NaCl (a 2 liter graduated cylinder works well for this). After several hours, rinse off the outside of the dialysis tubing, readjust the dialysis clips and remove excess tubing, and dialyze against your favorite buffer. I suggest wrapping parafilm around the ends of the dialysis clips, as the dialysis tubing will swell when you dialyze out the NaCl. You lose some protein due to adhesion to the dialysis tubing, but it works fairly well. Eric First Dep't of Biochemsitry and Molecular Biology LSU Health Sciences Center in Shreveport -Original Message- From: CCP4 bulletin board on behalf of Exec Sent: Thu 6/26/2008 10:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Concentrating protein 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. Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Concentrating protein
yes, but you have to check fisrt the protein doesnt crash out in 1 M (NH4)2SO4 or whatver concentration needed.. problem with amm. sulfate is that its good at salting-out proteins also, which we of course know... (which is of course another way to concetrate your protein, but there are more gently ways.), -just to add the cautinary note there. i would go with e.g. the large vol centripreps (amicon/vivaspin whatever) /IEX and dialysis or desalting columns if needed. in whatever combination needed. Best, Tommi Quoting Roger Rowlett [EMAIL PROTECTED]: There are actually two very good ways to concentrate dilute protein while carrying out a purification step. If the solution is low ionic strength, then IEX is the way to go. If the solution is too high in ionic strength to do IEX directly, then add ammonium sulfate (usually to about 1.0 M) and bind protein to a hydrophobic interaction chromatography medium. We usually use butylsepharose for HIC purification. It will be necessary to work out conditions for both binding and elution before committing the entire sample, of course. 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] Tommi Kajander wrote: couldnt agree more.. just pump the dilute solution through the ion exhange column.. or was there salt in it to prevent binding? or what was wrong with just using 80 ml centripreps or equivalent? not that high-tech, all you need is a regular 250 ml centrifugre tube rotor... (well the centrifuge also). tommi Quoting Phoebe Rice [EMAIL PROTECTED]: A layer of dry PEG on the outside of the dialysis tubing works too, without changing the ionic strength. But you'll probably get small molecular weight contaminants from the PEG entering the bag, so you'll have to dialyze against real buffer afterwards. The whole procedure in the end might be more annoying than just loading a very large volume onto an ion exchange column. Phoebe Original message Date: Fri, 27 Jun 2008 10:16:53 -0500 From: First, Eric [EMAIL PROTECTED] Subject: Re: [ccp4bb] Concentrating protein To: CCP4BB@JISCMAIL.AC.UK Back in the old days, we used to put the protein in dialysis tubing and surround it with solid NaCl (a 2 liter graduated cylinder works well for this). After several hours, rinse off the outside of the dialysis tubing, readjust the dialysis clips and remove excess tubing, and dialyze against your favorite buffer. I suggest wrapping parafilm around the ends of the dialysis clips, as the dialysis tubing will swell when you dialyze out the NaCl. You lose some protein due to adhesion to the dialysis tubing, but it works fairly well. Eric First Dep't of Biochemsitry and Molecular Biology LSU Health Sciences Center in Shreveport -Original Message- From: CCP4 bulletin board on behalf of Exec Sent: Thu 6/26/2008 10:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Concentrating protein 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. Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940 - -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O
Re: [ccp4bb] about anisotrophic diffraction
You could use salts such as LiSO4 for cryo-protection (also amm sulphate with small crystals has worked to a degree with us, when nothing else worked, also 1.6-1.7 M amm. sulphate was exchangable to 40% PEG 400 (very quick), although with not so great results...). which oil did you try? that would make a lot of difference.. (see also the recent discussion on LN2/propane etc and how to freeze) hth, Tommi Quoting Ji lee [EMAIL PROTECTED]: Dear, I have a crystal diffracted anisotrophically. I tested with a few different cryo conditions like oil, glycerol in different concentration to get a better data but these conditions didn't help any. Using capillary method improved the diffraction (isotrophic) but the crystal couldn't survive during the data collection. Is there any methods or cryo conditions I can use to improve my diffraction data? This crystal grew in 2.5M Ammonium Sulfate. Thank you so much. Ji -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation and mosaicity
according to literature,see below and references http://www.px.nsls.bnl.gov/courses/papers/ZD_EG_papers.html, it is not clear that liq. propane plunged item would cool faster. (whilst i havent tested this)... Would anyone have actual experimental data with protein crystals on the hyperquenching suggested by Warkentin, V. Berejnov, and R. E. Thorne, J. Appl. Cryst. (2006) 39, 805-811. (no diffraction data in the paper). in particular with small samples. thanks, Tommi Quoting Petr Leiman [EMAIL PROTECTED]: yes you are right, but I assumed if people see a cloud of condensed fog over their LN2 bath they should remove that by a) filling up the bowl completely e.g. some LN2 drips out of the bowl b) blow the fog away before you dip I think the original poster meant the relatively low heat conduction of liquid N2, which causes boiling around the crystal immediately after plunging. The best way to freeze things is to put a small container of liquid ethane or propane into a liquid N2 bowl, and plunge into the ethane/propane (this methods was suggested earlier). Petr -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] mosaicity,was:crystallisation
Are you including beam divergence is the defition of mosaicity? if you are, what happens when you go to a synchrotron? i.e., we know what happens: the beam is less divergent and hence your spots sharper. in any case you will have probably quite a bit better resolution from the extra photons at a synchrotron source. + you could try oil, e.g. paratone-N (for cryo protection), this does not penetrate the crystal and hence destroy the crystal contacts either. probably someone suggested the obivous additive screens and changing the PEG/salt etc. hth, tk Quoting Christian Biertuempfel [EMAIL PROTECTED]: Hi Sajid, Just a simple test for your problem: Incubate your crystal longer in your cryo/stabilization solution. This helps sometimes to lower mosaicity. Of course, you can also try co-crystallization with glycerol (2%, 5% or 10%). Good Luck, christian sajid akthar wrote: Dear All My protein size is ~30kD and crystallizes with 19%Peg3350, 0.2M Nacl, and 0.1M Na Cacodylate buffer. Please refer the attached crystal image with this. The crystal looks like good enough for home source. These crystals appears in 4-5 days at room temp. Sometimes I'm getting crystals like this, but very few in 24 well tray. Most of the time, I found the drop contains needles. If I reduce the precipitant little bit, I wont find any change in the drop even after long time. Changing pH (or temp)of the buffer does not help me any better. The crystal appears only around 5.5pH. The problem is mosaicity. This crystal diffracted in home source upto 3.2A and the mosaicity is 2.5degree. Almost all the good crystal like this having same mosaicity. Good cryo condition so far that I found was 10%Glycerol with mother liquor. Other conditions weekens the diffraction quality or increase mosaicity. In many crystal I could see some crack in the middle of the crystal, it looks like twin crystal. Or the crystal appears with some sattelite crystals. Can anyone suggest me some good way to overcome these problems. Thankz Sajid From Chandigarh to Chennai - find friends all over India. Go to http://in.promos.yahoo.com/groups/citygroups/ ___ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___ -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] space group problem
pseudo C2 centering in P21 will result in C2 systematic absenses being weak. you can check this with e.g. dataman (parity check of even and odd reflections of that type). and you will have that native patterson peak as mentioned. this will make life bit more complicated of course since large portion of the data maybe quite weak. also if its really P21 but because of some reason you dont count the weaker reflections in indexing that will lead you to _misindex_ the data in C2 and you are missing the weak data that would tell you some details about it in realite _differs_ from C2. (it is not a matter of incompleteness in either space group/lattice per se.) -i believe this was the point..? hth, -tommi Quoting Junyu Xiao [EMAIL PROTECTED]: Dear all, Thanks for all the advices. I am especially grateful to Dr. Clemens Vonrhein, now I am clear about the relationship between this two choices. Dr. Zwart raises another interesting point, which of course is my major concern. C2 will have the advantage for phasing, since it has a smaller ASU; but incomplete data won't help. Can I get more education on this? Thanks a lot, Junyu On May 1, 2008, at 1:07 PM, Peter Zwart wrote: Clemens is right of course. If you ignore the lattice centring in C2, the cells are the same. I was however under the impression that auto-indexing goes via the primitive cell. Which makes the two solutions unique. Ignoring the possible lattice translation in P2 will show up in a Patterson function (at 1/2,1/2,0 i think) . The lattice translation might of course be a pseudo translation. In the C2 case, you would miss weak reflections if P2 would be the right answer. P 2008/5/1 Clemens Vonrhein [EMAIL PROTECTED]: Dear Junyu, it looks to me like you encounter a classical monoclinic feature: one can index monoclinic always in two ways origin | V A' -- A \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / // C C' One cell (A,B,C) has B coming towards you and the other (A',B',C') has B' pointing away from you. The two axes A and A' have identical length as have B and B'. But C' is the diagonal in the AC-plane. In your case you can just swap the A and C axis of the C2 (to follow the above picture) and then calculate the C' (diagonal) to 136.8. So to summarize: these are identical cells - just different choice of axes (and nothing to do with the C2 versus P2 choice ... I think). Cheers Clemens On Thu, May 01, 2008 at 12:03:16PM -0400, Junyu Xiao wrote: Hi all, We run into an interesting space group problem. The same diffraction image can be either indexed into space group C2, with a=145, b=44, c=67, and beta=110.5; or space group P2 (should be P21 after scaling), with a=67, b=44, c=136, and beta=96.8. Both are refined ok during index. These two must somehow be related. Can anyone give some comments on that? Thanks a lot, Junyu = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 == -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) *** -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net - = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 == -- Tommi Kajander, Ph.D
Re: [ccp4bb] isopropanol as a precipitant
Hi, if you look at papers on crystallization of the E.coli protein ROP, you might find interesting info, in particular e.g.: Papanikolau Y, Kotsifaki D, Fadouloglou VE, Gazi AD, Glykos NM, Cesareni G, Kokkinidis M. Ionic strength reducers: an efficient approach to protein purification and crystallization. Application to two Rop variants. Acta Crystallogr D Biol Crystallogr. 2004 Jul;60(Pt 7):1334-7 (sounds like a lot of work, but you asked for it ... ;-) ) volatility is obviously a big problem, but as for cryo use paratone-N (or related dry highly viscous, see previous discussions on its behaviour on ccp4bb) oil, thats probably the easiest thing to do. (only thing ever work for me with methanol and ethanol) obvioulsy you can try butanols etc and MDP as the less volatile relatives. HTH, Tommi ps sounds suspiciously close to ROP in size... not a fourhelix bundle is it??? (none of my business obviously though) (on another note i think it would be nice if people would subscribe with their institute email addresses so that we are all on the same level in public discussions. not that it should be mandatory but it would be sort of nice given its a professional forum) Quoting shivesh kumar [EMAIL PROTECTED]: Dear all Sorry for non-CCP4 query. I have crystallized a 7kDa protein in 50-60% of isopropanol,pH 4.0-4.6.Theinteresting thing is that the xtal appears within 5 hrs at 16 degree. The protein conc is 5mg/ml and drop size is 3+1 with mother liquor 300 micro lt.Numerous xtals appears and but small is size.Do anyone can share their experience with Isopropanol as a precipitant and to improve the xtal quality with other precipitants.All suggestions are welcome. Thanx in advance. Shivesh -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] rescuing crashing-out protein eluted from Nickel column
first aid panic rescue might be to quickly add more salt. (e.g. in couple of steps to 0.5 M-1 M NaCl and see if it clears up). then add EDTA/or/and dialyse. (have to get teh salt out probably...) the Ni-ions leaking out are a likely cause. among others. tommi Quoting José Trincão [EMAIL PROTECTED]: Hi Jacob, try adding a bit of edta (1mM) to remove any Ni that might come off the column. You could also add some DTT or bME to keep cysteines reduced (careful, add only after the edta!). In my experience the gradient did not work very weel because the protein with have a lot of impurities. You can also think about adding a different tag (GST is usually helpful in keeping proteins soluble). Hope this helps!. Jose Jacob Wong wrote: Dear all, I just ran into this problem and would like to see if I could get some helpful tips before my protein completely crashes out. I have a protein as 6His fusion and it remained bound to the Ni resin with 40 mM Imidazole wash (added to 1XPBS) but then was eluted off with 200 mM (added to 1XPBS). The protein seemed to be highly concentrated in the elution and began to get cloudy right away, with more and more precipitation produced over a matter of minutes. I felt so helpless, didn't know what to do, and then decided to add 5% of glycerol into one of the fractions but that made it even more cloudy (ohh no...). While the protein is dying in the tube, do you have some quick remedy for me? Thanks very much, -J.J. -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] xprep vs shelxc
Dear All, A very simple stupid question: is it worth while getting xprep instead of shelxc to do the same job? i realize xprep does a lot of other things, but also seem to get the idea from some places that it does a better job than shelxc at preparing data from shelxd, or is there any difference (getting xprep will cost some money so i would like to know where the difference lies if there is any significant difference??) Thanks for advice, Tommi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Solvent content of membrane protein crystals
Quoting Edward Berry [EMAIL PROTECTED]: Savvas Savvides wrote: Indeed, but wouldn't consideration of micelle size affect our estimation of the number of molecules in the asu, in some cases significantly? Good point- I think now that is taken into account by just saying membrane proteins tend to have a high solvent content and taking that into consideration when you guess the number of molecules. But it would be nice to account for the detergent explicitly. Say by analyzing detergent content of the crystals, or in some ideal cases neutron diffraction with perdeuterated detergent. with regard to this has anyone actually checked how the micelle properties with or without protein embedded might differ?? are we assuming empty micelle and the protein-added micelle are the same size/Mw? is this really so? --- of course this may further vary depending on the oligomeric state of the protein --suppose some neutron scattering studies on model systems might give the answer --havent looked. just wondering.. -tommi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] HT-thermal melts
Dear All, Concerning matters upstream of crystallization... (well also optimization) I would be interested in any advice on companies selling fluorimeters with temperature controlled (heatable, ie capable of thermal melts) 96-well sample holder. also opinions on 96 DSC, + price range (i guess europe most relevant) thanks very much, tommi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] refmac problem in running arp/warp
Quoting Anastassis Perrakis [EMAIL PROTECTED]: On Aug 9, 2007, at 15:02, Tommi Kajander wrote: so, a) WHAT IS GAMMA?? gamma is possibly the letter of the greek alphabet that has had most abuse from scientists. thats what i thought... http://en.wikipedia.org/wiki/Gamma_%28disambiguation%29 b) why the difference between the machines??, is this a bug, or diagnostic of some problem...? (with my refiment.. which isnt really working...) some platform dependency is not shocking for minimizers ... you always see that. i have not really observed serious differences in the end result though, except once that i had serious trouble with 64-bit machines and code compilation. That was ages ago though and all these problems must be solved by ccp4. well this gamma issue seems to be there for refmac 5..(???) havent tested regular refinement with a complete model and refmac. probably that works ok. I would absolutely stand by my suggestion to always use HL restraints, in all cases that well estimated HL coefficients (eg Sharp) are available. i should probably recalculate phases with sharp (as soon as we have it up and running..) or some other, at the moment there's only density modified phases from shelxe (would be nice if shelx actually automatically output phases prior to density modification..(this is again probably my lazyness because i am using the _very nice_ hkl2map gui, ...and from other sources, but i use a blurring factor.. thanks again for everyone for their replies, tommi
[ccp4bb] refmac problem in running arp/warp
dear all (well experts i suppose), can someone explain what this means (REFMAC): Resolution limits= 19.944 2.600 Number of used reflections = 12652 Percentage observed = 100. Percentage of free reflections = . Overall R factor = .3922 Overall weighted R factor= .3737 Overall correlation coefficient = .8399 Cruickshanks DPI for coordinate error= 3.4923 Overall figure of merit = .6952 ML based su of positional parameters = .6421 ML based su of thermal parameters=29.9144 - Trying gamma equal 0.00E+00 Not converging with gamma equal 0.00E+00 Trying gamma equal 0.550343E-01 Not converging with gamma equal 0.550343E-01 Trying gamma equal 0.115531 Not converging with gamma equal 0.115531 Trying gamma equal 0.1820500046 Not converging with gamma equal 0.1820500046 Trying gamma equal 0.2552550137 Not converging with gamma equal 0.2552550137 Trying gamma equal 0.3357805014 Not converging with gamma equal 0.3357805014 Trying gamma equal 0.4243585467 Not converging with gamma equal 0.4243585467 Trying gamma equal 0.5217944384 Not converging with gamma equal 0.5217944384 Trying gamma equal 0.6289739013 Gamma decreased to 0.6075379848 I am runing this on a mac --two different mac platroms, powerpc does this and goes on intel mac pro (or whatever it was) gamma shoots up and the program gets stuck in a loop... so, a) WHAT IS GAMMA?? b) why the difference between the machines??, is this a bug, or diagnostic of some problem...? (with my refiment.. which isnt really working...) related to that, any opinions whether it is a good idea to include HL restraints in arp/warp buidling/refinment at 2.6Å and poor starting phases.. (starting from exp. phases, i recall Tassos suggested this but for which case??? i am running vs 7) (i still have the partial model but little success to go beyond it by any meanstweaking parameters in arpwarp now suggestions??) many thanks..., tommi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] auto-tracing programs
...sorry but the quickfold2 didnt work that well for me... ;( resolve does deliver some main chain to get an idea that i have one domain clearly there but different vs of resolve e.g 2.06 and newest vs give very different results... i dont care about side chains right now.. what is a not so good map, right good question... well i meant 3-2.8 Å and well dont have any quantitavive measure right now.. of course you can see indeed by eye whats in the map... just takes some time, doing it once is ok of course... i guess indeed i'll just try different things... (still interested to hear opinions though...) cheers, tommi Quoting Anastassis Perrakis [EMAIL PROTECTED]: On Jul 31, 2007, at 15:24, Tommi Kajander wrote: Hi, i would be interested in hearing about people's preferences on programs for doign auto-tracing of protein chains (with not so great maps), I do like ARP/wARP (objective opinion). so far my feeling has been nothing is at least much better than resolve in doing this. but i was wondering if people would care to share examples on cases where there was some difference to what you started with... ..of course one can always complete and correct by hand but when you are doing this with phasing iteratively it would be interesting to hear opinions.. A bit more seriously now, my feeling is as follows: 1. If the automated program does not deliver more than 80-90% of the structure, all of it reliably in sequence and without out of register errors, I would personally go back and do it in O. Being a big fun of Coot, I still like O better for building form scratch - I guess it is most likely just habit though. 2. Even if I would do things by hand, having a resolve, arp/warp, bucaneer, textal model in parallel can be helpful as guidance. I would run all these, it takes less time to run them than think if they can be useful or not. 3. arp/warp (.. yes,yes) can deal with extremely bad maps if high resolution data is available. I have seen it doing things I could not do by hand. I have also seen it (more often ...) to fail to trace 2.5 A maps that it would only take me a day or two to trace completely. so, what is a 'not so great map' is not clear to me. an awful looking 1.1 A map with 70 deg. phase error, is not the same as an awful looking 3.2 A map with similar errors, and a MAD 3.2 A map can be easy to trace either by hand or automatically. And sorry for the shameless plug as usual, but arp/warp does work at low resolution. not as well as in high resolution, but we do get successes (75%) with some real 3.0 a datasets .. although not often ... but its worth a try. And of course the Quick Fold (albe) module of ARP/wARP can also be useful and it takes for 500 residues less time to run than me writing this email ;-) Tassos thanks, tommi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940 -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] heavyatom soaking problem
Quoting Priyank Maindola [EMAIL PROTECTED]: Hello all, I am struggling with getting the phases out for my protein. Heavyatom database shows that only mercury and samarium have the binding motifs in it. Sodium iodide soak is killing the crystal even as less as 0.2 Molar concentration for 30sec. With Potassium iodide crystal is okay upto 0.75M for 1 a half min but the incorporation is not at relevent sites.(Iodide signal is not distinct and is weak) how much data and what is anomalous redundancy? what space group? could you do Sulphur-SAD? Hg Au salts precipitate it. i dont understand this, precipitate the crystals? what concentrations and soak times do you use and what is the pH? ...ie more details would be useful. With Pt signal is weak. again how about a longer soak??? higher concentration??? ... or try Pd, should be more reactive. Somewhere I read that high salt causes problems for heavy atom to bind to protein.My protein needs high salt for stability (750mM NaCl) I think that would mainyl affect electrostatic binding, but imidazole of cys attachement of Hg, Pr etc probably not. (??) and again, why no se-met?? Please help with some suggestion!! tommi
Re: [ccp4bb] High overall b-factor
probably that of course and unfortunatelly wilson plot will not work at that resolution, you need the linear bit. also one can always take a look at other less than 3 Å structures in the PDB... another question is how did you refine the b-factor, hopefully just an overall b-factor...? this is probably best you can get at that resolution.. whether you can trust the ligand density depends on (size, shape and chemical properties of) the ligand. tommi Quoting ezra peisach [EMAIL PROTECTED]: I have never worked at 3.2A - but I suspect that the overal temperature factor is being determined from the slope of a Wilson plot. However, Wilson plots only really work at higher resolution (2.8 or better). Look at the output from truncate and see what it is predicting - and look at the plot - it should be pretty obvious why you have such a high B. Ezra On Fri, 9 Mar 2007, George Lountos wrote: Hello all: I just recently collected data on initial crystals I grew of an enzyme with inhibitor. The crystals diffract to only 3.2 A but I was able to get phases by molecular replacement to see if there is any inhibitor bound. Although the data processed well in HKL2000 with good statistics and the current structure refinement is at R-factor of 22% and R-free 30% at 3.2 A, the overall B-factor of the protein is very, high (100 A^2). I can see difference density for the ligand in the active site and after refinement it fits well in the density but the B-factor for the ligand is 110. I have not come across a refinement with such high B-factors where the protein density and ligand density can be distinguished at such high B-factors. Does anyone have any suggestions if there is something going wrong here? Thanks, George Get a FREE Web site, company branded e-mail and more from Microsoft Office Live! -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
