[ccp4bb] iycr2014-symposium@innsbruck, 8-9 Sept 2014
Dear colleagues, A symposium/exhibition celebrating 100 years of crystallography in the International Year of Crystallography 2014 (www. iycr2014.org) will be held in Innsbruck (Austria) on8-9 September 2014. It aims to feature contributions from scientists having built the technical advances of (bio)molecular crystallography as well as from researchers having applied these advances to achieve milestone insights into hot topics in structural (bio)chemistry. Speakers include: Axel Brunger (Stanford University, USA), Geroge Sheldrick (Goettingen, DEU), Alex McPherson (Irvine, USA), Ken Holmes (Heidelberg, DEU), Patrick Cramer (Goettingen/Munich, DEU), Marat Yusupov (Strasbourg, FRA), Ilme Schlichting (Heidelberg, DEU), Wener Kühlbrandt (Frankfurt, DEU), Raimund Dutzler (Zurich, SUI), Giovanna Scapin (Merck, New Jersey, USA), and others Registration is free of charge, conference dinner 50EUR (regular) 25EUR (undergraduate and PhD students) see http://biocenter.i-med.ac.at/iycr2014 for more information incl. preliminary program. The spatio-temporal proximity with this year's http://www.murnauconference.de http://www.murnauconference.de/2014/schedule.html may inspire you to attend both meetings back-to-back. Organizers: K. Scheffzek, B. Rupp, T. Dunzendorfer-Matt, K. Liedl | Innsbruck; U. Wagner |Graz; K. Djinovic-Carugo , T. Clausen | Vienna; J. Brandstetter | Salzburg; M.S. Weiss | Berlin Hope to see you in Innsbruck, best wishes, Klaus -- Univ.-Prof. Klaus Scheffzek (Chair) Division of Biological Chemistry (Biocenter c/o Center of Chemistry and Biomedicine) Innsbruck Medical University Innrain 80-82 6020 Innsbruck, Austria E-mail: klaus.scheff...@i-med.ac.at mailto:klaus.scheff...@i-med.ac.at Phone: +43 512 9003 70330 (office), +43 512 9003 70300 (Secr)
[ccp4bb] cannot reproduce crystals
Dear all, I am trying to reproduce some protein crystals. The protein I am getting after cutting the his tag is very pure. I am using the reported protein concentration. The cofactor and EDTA needs to be added externally. The condition has calcium acetate, peg 4k and sodium acetate buffer. Unfortunately I am getting oil separation and light ppt. I have no clue what is wrong. Please help!
Re: [ccp4bb] cannot reproduce crystals
Hi, you changed something, and although it seems very small a change, this can make a difference between crystals and non-crystals. And removing the His-Tag is not a small change, since it influences the net charge. The first thing to try is micro and macro-seeding. If they don't work, you may need to screen again. Best, Tim On 07/14/2014 01:44 PM, dusky dew wrote: Dear all, I am trying to reproduce some protein crystals. The protein I am getting after cutting the his tag is very pure. I am using the reported protein concentration. The cofactor and EDTA needs to be added externally. The condition has calcium acetate, peg 4k and sodium acetate buffer. Unfortunately I am getting oil separation and light ppt. I have no clue what is wrong. Please help! -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] 2nd call, CCP4 structure solution workshop at the Photon Factory
Dear Colleagues, we are pleased to announce the CCP4 structure solution school at the Photon Factory, Tsukuba. All details can be found at http://www.ccp4.ac.uk/schools/Japan-2014 Title: CCP4 school: From data processing to structure refinement and beyond Dates: November 4 to 8, 2014 Site: The Photon Factory. Tsukuba, Japan The school content: Software workshop: The rest of the time after data collection will feature many modern crystallographic software packages taught by authors and other experts. It will be organized in three Sections - lectures, tutorials and hands-on trouble-shooting. There will be model data sets available for tutorials but data, provided by participants, will have higher priority for the hands-on sessions. Applicants: Graduate students, postdoctoral researchers and young scientists at the assistant professor level are encouraged to apply. Only 20 applicants will be selected for participation. Participants of the workshop are strongly encouraged to bring their own problem data sets so the problems can be addressed during data collection workshop and/or hands-on sessions. Application: Application deadline is 13 September. Application form, the program, contact info and other details can be found at http://www.ccp4.ac.uk/schools/Japan-2014/index.php Fees: There is no fee for the workshop, but the students will be responsible for their transportation and accommodation costs. A limited number of places will be available in the KEK dormitory on a first come basis. Naohiro Matsugaki, Charles Ballard -- Scanned by iCritical.
Re: [ccp4bb] cannot reproduce crystals
Hmmm…not enough information but I am wondering about the EDTA plus Ca business….the Ca++ ions are forming a strong chelate with the EDTA, so depending on your molar ratios either the free Ca ions would be consumed by EDTA or the EDTA by Ca …(?) – any variance in ratio here might prove troublesome. BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dusky dew Sent: Montag, 14. Juli 2014 13:44 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cannot reproduce crystals Dear all, I am trying to reproduce some protein crystals. The protein I am getting after cutting the his tag is very pure. I am using the reported protein concentration. The cofactor and EDTA needs to be added externally. The condition has calcium acetate, peg 4k and sodium acetate buffer. Unfortunately I am getting oil separation and light ppt. I have no clue what is wrong. Please help!
Re: [ccp4bb] cannot reproduce crystals
You may unleash a deluge of anecdotes and horror stories, but this is quite common. I have experienced this many times, and you just need to step back and ask yourself what is being done differently: 1. Are all materials used in the preparation of the protein the same (suppliers, sources, expression hosts, purification media, etc.)? I have had this happen several times when I have moved. If you have not rescreened with a broader condition matrix, do so. Slight changes in protein purity and quality (and they are not the same) can radically shift the crystallization behavior. 2. Did you or someone else improve the purification? Ultrapure pure protein sometimes crystallizes less well than less pure protein, for a number of reasons. One is that an extra step adding in could remove something critical. In number of cases in my lab, we have solved structures we thought were in the apo-form, but saw that ligands were bound. Making the true apo-form led to getting no crystals. 3. Are you trying to reproduce your work or work of others? If it is the latter, talk to the original people and don't rely on an old notebook. Very subtle differences crop up when doing lab work that impact crystallization, from how you make your PEG or buffer stocks (compared to those acquired in kits) to how someone sets up crystals. One case I know of, the group lost their crystals after a technician left. After some investigating, the slight pH difference caused by making up the buffer for a hanging drop reservoir differently was the source of the problem. Hope this helps, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jul 14, 2014, at 7:44 AM, dusky dew duskyde...@gmail.com wrote: Dear all, I am trying to reproduce some protein crystals. The protein I am getting after cutting the his tag is very pure. I am using the reported protein concentration. The cofactor and EDTA needs to be added externally. The condition has calcium acetate, peg 4k and sodium acetate buffer. Unfortunately I am getting oil separation and light ppt. I have no clue what is wrong. Please help!
Re: [ccp4bb] COOT - copy ncs residue range
Hi Alisa, almost the right syntax. The last one is a list, i.e. it should read: copy_residue_range_from_ncs_master_to_chains(0, C, 377, 402,[B]) ... and make sure you have the mast chain set to C. ... and there was a similar discussion earlier: https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg36035.html HTH, Bernhard P.S. There is a special bb for Coot related question... Hello everyone, I am trying to copy an NCS residue range from one NCS chain to another, but not to all of them. I. e I would like to copy residues 377-402 from chain C to chain B, without affecting chains A and D. (I have already turned the chain C into the master chain through NCS ghost control) Since extensions in coot does that for all chains simultaneously I am trying to use the Python or scheme commands. First of all, I am not sure I am using the right syntax, so please correct me if I am wrong. But here is what I have been writing for the Python command: copy_residue_range_from_ncs_master_to_chains(0, C, 377, 402,B) But then coot quits with the message: /usr/local/bin/coot: line 10: 34717 Segmentation fault: 11 /Library/Coot/bin/coot-real $@ logout I would greatly appreciate your help! Regards, Alisa -- Alisa Glukhova graduate student Tesmer lab
Re: [ccp4bb] cannot reproduce crystals
'oil separation and light ppt' is not necesserily an indicator that something is wrong with your crystallization condition. Actually, there are quite a few proteins that only crystallize in conditions with oil/water-like phase separation. Some crystals even appear WITHIN the oily drops (e. g. Xylanase). good luck! Clemens Zitat von dusky dew duskyde...@gmail.com: Dear all, I am trying to reproduce some protein crystals. The protein I am getting after cutting the his tag is very pure. I am using the reported protein concentration. The cofactor and EDTA needs to be added externally. The condition has calcium acetate, peg 4k and sodium acetate buffer. Unfortunately I am getting oil separation and light ppt. I have no clue what is wrong. Please help!
Re: [ccp4bb] New PDB validation reports
Dear Katherine, Thanks for pointing this out! As far as I can tell the community has not reached a consensus on dealing with disordered side chains. I’m afraid it simply didn’t occur to us, when we were writing the validation task force report, that one approach would be favoured over the other, and we certainly didn’t intend to influence community practice by stealth! This unintended consequence really should have occurred to us, since Gerard Kleywegt and I had noticed earlier that structural genomics structures (particularly those from the SGC) have a systematically lower completeness than other structures, apparently because the choice was made more often to completely omit poorly-ordered residues rather than include them in the model. This leaves out many of the residues that would have poor Ramachandran and rotamer scores, thus raising those scores above the average in a similar way. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631636/) Some indication of completeness probably would be a good thing to include in the validation reports. Best wishes, Randy Read On 10 Jul 2014, at 20:26, Katherine Sippel katherine.sip...@gmail.com wrote: Hi all, I've been playing with the new PDB validation service. It is very pretty and kudos to all the hard work that has clearly gone into it. I did notice however that the way the information is presented, there seems to be a bias towards truncating side chains versus modeling them with higher b-factors. The disordered side chains have higher RSRZs (rightfully so), but there doesn't seem to be any indicator for missing atoms. As a results I can make my validation report prettier by truncating versus modeling with high Bs. I don't want to kick an ant pile here, but given this rather significant difference in quality reporting, I was wondering if the community had reached a consensus on this issue that I had missed. Cheers, Katherine -- Nil illegitimo carborundum - Didactylos -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] AMPLE failure
Hi Robert, Thanks for your interest in AMPLE and reporting this bug. There is a small bug that has come about as a result of the latest MRBUMP update last week that causes this problem. We're preparing a fix for it which should be in the next ccp4 update towards the end of this week or early next week. In the meantime I can send you (off list) the latest version of the code that you can manually install. Best wishes, Ronan On 12/07/14 18:58, Robert Kirchdoerfer wrote: Hi, I'm trying to run AMPLE from ccp4-6.4.0 on Linux Ubuntu. It looks like it found all the program dependencies that it needs and Rosetta looks like it ran okay and I think MRBUMP looks alright, but then the program stopped with the following error message: MR and shelx DONE ALL DONE (in 1.64101706141 hours) Saved results as file: /home/rob/Crystal/108_B7U_3/ccp4/ROSETTA_MR_1/resultsd.pkl *** * Information from CCP4Interface script *** The program run with command: /home/rob/Xstal_Programs/ccp4v640/destination/ccp4-6.4.0/bin/ccp4-python -u /home/rob/Xstal_Programs/ccp4v640/destination/ccp4-6.4.0/bin/ample -mtz /home/rob/Crystal/108_B7U_3/ccp4/XDS_ASCII_scaled2.mtz -fasta /home/rob/Crystal/108_B7U_3/phenix/RNK149.fasta -nmodels 1000 -name MVD0 -run_dir /home/rob/Crystal/108_B7U_3/ccp4 -nproc 8 -make_models True -rosetta_dir /home/rob/Xstal_Programs/Rosetta/rosetta-3.5 -frags_3mers /home/rob/Crystal/108_B7U_3/ccp4/aat000_03_05.200_v1_3 -frags_9mers /home/rob/Crystal/108_B7U_3/ccp4/aat000_09_05.200_v1_3 -make_frags False -F F_XDSdataset -SIGF SIGF_XDSdataset -FREE FreeR_flag -early_terminate True -use_shelxe True -use_arpwarp False has failed with error message Traceback (most recent call last): File /home/rob/Xstal_Programs/ccp4v640/destination/ccp4-6.4.0/bin/ample, line 838, in summary = amopt.final_summary() File /home/rob/Xstal_Programs/ccp4v640/destination/ccp4-6.4.0/share/ample/python/ample_options.py, line 197, in final_summary result_summary.append( getattr( result, title2attr[ h ] ) ) KeyError: 'SHELXE_ACL' Any thoughts on how I might troubleshoot this? Thanks and Best wishes, Rob -- Scanned by iCritical.
[ccp4bb] definitions of unique reflections
There is some disagreement on terms used to deposit data. We need a definition and an algorithm for each definition. Unique Reflections My definition is all the possible reflections out to the high resolution reported not related by symmetry. Where can I find this? The .mtz contains a list of all HKL calculated to the highest resolution. Usually, we are not able to measure all these diffraction spots due to limits of the detector, mechanical limits, crystal orientation, etc. 'Total reflections' The depositions server asks for total reflections. I assume it wants only those unique reflections we were able to collect, regardless of the sigma cut off. These are called 'observed'. The total we use in refinement will be a subset of the 'unique observed' that are cut on sigma. However, some crystallographers believe that we should not cut on sigma since some of the intensities may in fact be zero. Is this a question for both the Refmac and Phenix people? Please give us some guidance and maybe a reference or two that we can use. -- Kenneth A. Satyshur, M.S.,Ph.D. Senior Scientist University of Wisconsin Madison, Wisconsin 53706 608-215-5207 attachment: satyshur.vcf
[ccp4bb] How to store PEG screens
Dear all, I have 3 short questions about PEG solutions: Does anyone know the best way to store crystallization screening blocks that contain PEG 3350? Is it a good idea to freeze the PEG solutions at -80°C and thaw them before use? Would the freeze-thaw process considerably alter the PEG chain lengths? Thank you, -Jerome Jerome Nwachukwu
Re: [ccp4bb] definitions of unique reflections
My preference is to use the term 'observed' for reflections whose intensities have been integrated, and the term 'informative' for those that satisfy some statistical criteria of being useful for structure determination. Programs like Truncate have hidden criteria of rejecting some observed reflections from the informative group, so this issue has been around for a long time. For a typical, properly done data collection, resolution limit is a widely used criterion of informativity. For anisotropic diffraction, a single number is definitely not a proper way to define the resolution limit. So we need something like signal-to-noise ratio cut-off to define a better equivalent of the resolution limit. The question is what we mean by signal-to-noise: it can be individual (unique/merged) reflection values (a wide-spread practice in small molecule crystallography, and for a good reason) or either signal, noise, or both of them, which are group averages rather than individual estimates, Personally, I prefer a ratio of an average signal to an individual uncertainty as a criterion that defines the informativity limit equivalent to a resolution cut-off. The second aspect of the issue is what value of the signal-to-noise ratio (however defined) should be the limiting criterion. The value around 2 represents a limit of what is 'fully' informative, and, as has been discussed, lower values of signal-to-noise provide some extra information. Around the ratio of 1, the value of the information becomes minimal. So for me, there are 2 types of data completeness: one, in terms of Bragg's condition, which defines if we missed part of reciprocal space during the experiment; and second, in terms of what is informative for structure solution. The second type will typically be low for resolution range close to the limit in the case of anisotropic diffraction. There is, therefore, nothing wrong in terms of how the experiment was done, if such completeness is low; on the other hand, the first type can tell us whether the experiment could be done better. So there are good reasons to report both types of completeness in the publication and in the deposit, even if there is no such custom yet. Zbyszek Otwinowski There is some disagreement on terms used to deposit data. We need a definition and an algorithm for each definition. Unique Reflections My definition is all the possible reflections out to the high resolution reported not related by symmetry. Where can I find this? The .mtz contains a list of all HKL calculated to the highest resolution. Usually, we are not able to measure all these diffraction spots due to limits of the detector, mechanical limits, crystal orientation, etc. 'Total reflections' The depositions server asks for total reflections. I assume it wants only those unique reflections we were able to collect, regardless of the sigma cut off. These are called 'observed'. The total we use in refinement will be a subset of the 'unique observed' that are cut on sigma. However, some crystallographers believe that we should not cut on sigma since some of the intensities may in fact be zero. Is this a question for both the Refmac and Phenix people? Please give us some guidance and maybe a reference or two that we can use. -- Kenneth A. Satyshur, M.S.,Ph.D. Senior Scientist University of Wisconsin Madison, Wisconsin 53706 608-215-5207 Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] How to store PEG screens
Hi Jerome, -I have heard that PEG solutions can become unstable in light. We usually store our block in the fridge, where photons are scant anyway. For any stocks that I prepare, I wrap the tube/bottle in aluminum foil. I'm not sure about freezing them. -Some labs (not ours) evidently prepare buffered stocks of PEG solutions, as their pHs tend to shift with time. This is something I've been meaning to try. Of course, you may need to worry about buffer components that are incompatible with crystal hits. Hope this helps, Chris On Mon, Jul 14, 2014 at 9:33 AM, Jerome Nwachukwu jnwac...@scripps.edu wrote: Dear all, I have 3 short questions about PEG solutions: Does anyone know the best way to store crystallization screening blocks that contain PEG 3350? Is it a good idea to freeze the PEG solutions at -80°C and thaw them before use? Would the freeze-thaw process considerably alter the PEG chain lengths? Thank you, -Jerome Jerome Nwachukwu
Re: [ccp4bb] How to store PEG screens
I don't think storage matters. I doubt Hampton stores their PEG stock solutions at -80 before they ship out to customers. I've solved tons of structures leaving my PEGS and PEG screens at RT in the light. Nick On Mon, Jul 14, 2014 at 12:32 PM, Chris Fage cdf...@gmail.com wrote: Hi Jerome, -I have heard that PEG solutions can become unstable in light. We usually store our block in the fridge, where photons are scant anyway. For any stocks that I prepare, I wrap the tube/bottle in aluminum foil. I'm not sure about freezing them. -Some labs (not ours) evidently prepare buffered stocks of PEG solutions, as their pHs tend to shift with time. This is something I've been meaning to try. Of course, you may need to worry about buffer components that are incompatible with crystal hits. Hope this helps, Chris On Mon, Jul 14, 2014 at 9:33 AM, Jerome Nwachukwu jnwac...@scripps.edu wrote: Dear all, I have 3 short questions about PEG solutions: Does anyone know the best way to store crystallization screening blocks that contain PEG 3350? Is it a good idea to freeze the PEG solutions at -80°C and thaw them before use? Would the freeze-thaw process considerably alter the PEG chain lengths? Thank you, -Jerome Jerome Nwachukwu -- [This e-mail message may contain privileged, confidential and/or proprietary information of H3 Biomedicine. If you believe that it has been sent to you in error, please contact the sender immediately and delete the message including any attachments, without copying, using, or distributing any of the information contained therein. This e-mail message should not be interpreted to include a digital or electronic signature that can be used to authenticate an agreement, contract or other legal document, nor to reflect an intention to be bound to any legally-binding agreement or contract.]
Re: [ccp4bb] COOT - copy ncs residue range
Hi Alisa, This is how I would do this. 1. In COOT got to Extensions -- Modelling -- Copy Fragment. In the dialog box that opens change Atom selection to //C/350-402 then hit OK. 2. To move the newly created fragment into position in B, go to Calculate -- LSQ Superpose. Change Reference Molecule to chain B of your original model and Moving Molecule to the newly created fragment (should be at the bottom of the list). Change the Residue Ranges from 350 to 377 then fit. 3. Merge the fragment into your model and save the merged PDB. Delete the extra residues (350-369) and use a text editor to change the chain ID of the new fragment to B. There might be some other cleanup you have to do but that should get you most of the way. -Donald . Donald Damian Raymond, Ph.D. Postdoctoral Research Fellow Stephen C. Harrison Lab Harvard Medical School CLSB 3082 email: raym...@crystal.harvard.edu http://www.linkedin.com/in/donalddraymond/ On Jul 13, 2014, at 1:44 PM, Alisa Glukhova alis...@umich.edu wrote: Hello everyone, I am trying to copy an NCS residue range from one NCS chain to another, but not to all of them. I. e I would like to copy residues 377-402 from chain C to chain B, without affecting chains A and D. (I have already turned the chain C into the master chain through NCS ghost control) Since extensions in coot does that for all chains simultaneously I am trying to use the Python or scheme commands. First of all, I am not sure I am using the right syntax, so please correct me if I am wrong. But here is what I have been writing for the Python command: copy_residue_range_from_ncs_master_to_chains(0, C, 377, 402,B) But then coot quits with the message: /usr/local/bin/coot: line 10: 34717 Segmentation fault: 11 /Library/Coot/bin/coot-real $@ logout I would greatly appreciate your help! Regards, Alisa -- Alisa Glukhova graduate student Tesmer lab
Re: [ccp4bb] How to store PEG screens
Jerome, Does anyone know the best way to store crystallization screening blocks that contain PEG 3350? I would recommend storing them in a fridge or a clean coldroom (mold-free). Lower temperature and low light does help. Is it a good idea to freeze the PEG solutions at -80°C and thaw them before use? Good idea, if they were pre-aliquoted into useful volumes. We do that occasionally. However, -20°C is just as good. Would the freeze-thaw process considerably alter the PEG chain lengths? No, the real issue is the generation of oxygen reactive species that cause aldehyde and peroxide formation, which in turn can modify your protein. It also causes cross-linked polymer formation. Also avoid metal ion contamination. Like lipids, plain PEG solutions in water and most detergents with PEG head groups (C12E8, octyl-POE, Brij, Triton, Tween, etc.) should be stored under argon and at -20°C. So, if you are tempted to use the 5-year old bottle of Triton X-100 or old 50% stock of PEG on the bench top, caveat emptor. Take a look at an old paper by Fran Jurnak (J. Cryst, Growth, 76, 577-582, 1986) for the trials and tribulations of working with PEG from different manufacturers of PEG (as well as how the purify it if you really get worried). Regards, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jul 14, 2014, at 11:33 AM, Jerome Nwachukwu jnwac...@scripps.edu wrote: Dear all, I have 3 short questions about PEG solutions: Does anyone know the best way to store crystallization screening blocks that contain PEG 3350? Is it a good idea to freeze the PEG solutions at -80°C and thaw them before use? Would the freeze-thaw process considerably alter the PEG chain lengths? Thank you, -Jerome Jerome Nwachukwu
Re: [ccp4bb] How to store PEG screens
I also used to store my PEG solutions in light, and my stocks do sit out on the bench. I can't say for sure whether light or temperature make a difference, but I like to heed what seem like superstitions in crystallography to eliminate variables. We purchase our screens from Qiagen, who suggests that blocks be stored in the fridge and warmed to RT before use. Best, Chris On 7/14/14, Nicholas Larsen nicholas_lar...@h3biomedicine.com wrote: I don't think storage matters. I doubt Hampton stores their PEG stock solutions at -80 before they ship out to customers. I've solved tons of structures leaving my PEGS and PEG screens at RT in the light. Nick On Mon, Jul 14, 2014 at 12:32 PM, Chris Fage cdf...@gmail.com wrote: Hi Jerome, -I have heard that PEG solutions can become unstable in light. We usually store our block in the fridge, where photons are scant anyway. For any stocks that I prepare, I wrap the tube/bottle in aluminum foil. I'm not sure about freezing them. -Some labs (not ours) evidently prepare buffered stocks of PEG solutions, as their pHs tend to shift with time. This is something I've been meaning to try. Of course, you may need to worry about buffer components that are incompatible with crystal hits. Hope this helps, Chris On Mon, Jul 14, 2014 at 9:33 AM, Jerome Nwachukwu jnwac...@scripps.edu wrote: Dear all, I have 3 short questions about PEG solutions: Does anyone know the best way to store crystallization screening blocks that contain PEG 3350? Is it a good idea to freeze the PEG solutions at -80°C and thaw them before use? Would the freeze-thaw process considerably alter the PEG chain lengths? Thank you, -Jerome Jerome Nwachukwu -- [This e-mail message may contain privileged, confidential and/or proprietary information of H3 Biomedicine. If you believe that it has been sent to you in error, please contact the sender immediately and delete the message including any attachments, without copying, using, or distributing any of the information contained therein. This e-mail message should not be interpreted to include a digital or electronic signature that can be used to authenticate an agreement, contract or other legal document, nor to reflect an intention to be bound to any legally-binding agreement or contract.]
Re: [ccp4bb] How to store PEG screens
Michael, Chris and Nick, Thank you so much for your help. -Jerome Jerome Nwachukwu jnwac...@scripps.edumailto:jnwac...@scripps.edu On Jul 14, 2014, at 1:48 PM, R. M. Garavito rmgarav...@gmail.commailto:rmgarav...@gmail.com wrote: Jerome, Does anyone know the best way to store crystallization screening blocks that contain PEG 3350? I would recommend storing them in a fridge or a clean coldroom (mold-free). Lower temperature and low light does help. Is it a good idea to freeze the PEG solutions at -80°C and thaw them before use? Good idea, if they were pre-aliquoted into useful volumes. We do that occasionally. However, -20°C is just as good. Would the freeze-thaw process considerably alter the PEG chain lengths? No, the real issue is the generation of oxygen reactive species that cause aldehyde and peroxide formation, which in turn can modify your protein. It also causes cross-linked polymer formation. Also avoid metal ion contamination. Like lipids, plain PEG solutions in water and most detergents with PEG head groups (C12E8, octyl-POE, Brij, Triton, Tween, etc.) should be stored under argon and at -20°C. So, if you are tempted to use the 5-year old bottle of Triton X-100 or old 50% stock of PEG on the bench top, caveat emptor. Take a look at an old paper by Fran Jurnak (J. Cryst, Growth, 76, 577-582, 1986) for the trials and tribulations of working with PEG from different manufacturers of PEG (as well as how the purify it if you really get worried). Regards, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.commailto:garav...@gmail.com I also used to store my PEG solutions in light, and my stocks do sit out on the bench. I can't say for sure whether light or temperature make a difference, but I like to heed what seem like superstitions in crystallography to eliminate variables. We purchase our screens from Qiagen, who suggests that blocks be stored in the fridge and warmed to RT before use. Best, Chris On 7/14/14, Nicholas Larsen nicholas_lar...@h3biomedicine.commailto:nicholas_lar...@h3biomedicine.com wrote: I don't think storage matters. I doubt Hampton stores their PEG stock solutions at -80 before they ship out to customers. I've solved tons of structures leaving my PEGS and PEG screens at RT in the light. Nick On Jul 14, 2014, at 11:33 AM, Jerome Nwachukwu jnwac...@scripps.edumailto:jnwac...@scripps.edu wrote: Dear all, I have 3 short questions about PEG solutions: Does anyone know the best way to store crystallization screening blocks that contain PEG 3350? Is it a good idea to freeze the PEG solutions at -80°C and thaw them before use? Would the freeze-thaw process considerably alter the PEG chain lengths? Thank you, -Jerome Jerome Nwachukwu
[ccp4bb] how to apply non-protein crystallographic symmetry
I have a pdb file for a non-protein having C 1 2/c symmetry. PyMol can't recognize the spacegroup. Can someone recommend software that can apply the crystallographic symmetry to give the full structure? Richard Gillilan MacCHESS Cornell University
Re: [ccp4bb] how to apply non-protein crystallographic symmetry
Dear Richard, Coot shouldn't have an issue with non-Sohnke space groups. I used it to build a structure in P-1. If you want to edit the molecule, you could also try ShelXle, available at http://ewald.ac.chemie.uni-goettingen.de/shelx/eingabe.php. Best, Tim On 07/14/2014 10:26 PM, Richard Gillilan wrote: I have a pdb file for a non-protein having C 1 2/c symmetry. PyMol can't recognize the spacegroup. Can someone recommend software that can apply the crystallographic symmetry to give the full structure? Richard Gillilan MacCHESS Cornell University -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] how to apply non-protein crystallographic symmetry
Thanks to all who responded so quickly! Coot worked. Richard On Jul 14, 2014, at 4:33 PM, Tim Gruene wrote: Dear Richard, Coot shouldn't have an issue with non-Sohnke space groups. I used it to build a structure in P-1. If you want to edit the molecule, you could also try ShelXle, available at http://ewald.ac.chemie.uni-goettingen.de/shelx/eingabe.php. Best, Tim On 07/14/2014 10:26 PM, Richard Gillilan wrote: I have a pdb file for a non-protein having C 1 2/c symmetry. PyMol can't recognize the spacegroup. Can someone recommend software that can apply the crystallographic symmetry to give the full structure? Richard Gillilan MacCHESS Cornell University -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] Crystallization Scientist position at Syngenta
*Senior Crystallization Scientist* Jealott's Hill, Bracknell, Berkshire, UK We are seeking a highly talented and motivated crystallization scientist who is enthusiastic about working on a wide variety of biological systems. The successful candidate will have a strong background in structural biology and will be skilled at protein crystallization, crystal optimization and protein engineering for crystallography. Experience in X-ray data collection and knowledge of x-ray structure analysis is a plus. The successful candidate will work as part of a team providing x-ray crystallography outputs to support chemistry design throughout RD. Excellent oral and written communication skills and the ability to work in multidisciplinary project teams are highly desirable qualities for this position. *Responsibilities* • Carry out crystallization trials, crystal optimization and initial characterization by x-ray diffraction for proteins and protein-ligand complexes • Perform biophysical characterization of proteins intended for crystallization • Provide guidance for the design of optimal constructs for protein crystallography • Effectively communicate results through presentations, e-mails and reports. • Develop and further own skills in theoretical and practical crystallography and structural biology through appropriate reading and attendance at courses and external meetings. Actively seek to broaden understanding of other disciplines. *Experience required* • PhD or MSc degree in structural biology or a related field • 3-5 years experience in practical macromolecular crystallography, ideally across a wide range of protein families • Working experience in design, production and purification of proteins and protein-ligand complexes specifically for protein crystallography • Knowledge of protein crystallization and optimization techniques • Knowledge of x-ray diffraction and basic data collection strategies • Knowledge of software for molecular biology and protein structure analysis *About Syngenta* Syngenta is one of the world’s leading companies with more than 27.000 employees in over 90 countries dedicated to our purpose: Bringing plant potential to life. Through world-class science, global reach and commitment to our customers we help to increase crop productivity, protect the environment and improve health and quality of life. For more information about us please go to http://www.syngenta.com/. For a full job profile and to apply to this position, please visit http://www.syngentajobs.com/Taleo_apply.html?CountryCode=2gblang=enlocation=23236270036668portal=101430233 - job number 14038910 I am happy to answer informal questions about the position, but if you wish to apply please do so via the Syngenta Careers website linked above. The position will be open until August 4. Thanks, Daniel Kloer Head of Protein Analysis and Computational Chemistry Syngenta
[ccp4bb] Beamline scientist position at SLS and SwissFEL
http://www.psi.ch/pa/offenestellen/0829 As an emerging method, serial crystallography with bright micro-focused X-ray beams from both synchrotron and free electron laser sources has shown great potentials for advancing the frontiers in structural biology. Delivery of hundreds and thousands of crystals in a controlled and nevertheless high-throughput and cost effective manner also presents unprecedented challenges. In a collaboration of SLS and SwissFEL, we are looking for a Beamline Scientist Sample Delivery for Serial Crystallography Your tasks Develop and implement solid-support sample delivery systems for micro-/nano-crystallography at synchrotron-radiation (SR) and X-ray free-electron laser (XFEL) sources Actively participate in serial crystallography experiments at XFEL sources Further develop the SLS protein crystallography beamlines Your profile You hold a PhD degree in a natural science or related field and have several years of experience with the use of SR and related instrumentation. Working knowledge for protein crystallography would be a significant advantage. If you are a good team player with fine communication skills and sense of responsibility, this position will offer a great opportunity for you to develop your research career in an exciting and highly multidisciplinary environment. Exceptional candidates in the general area of MX with any additional skills relevant to SwissFEL may apply as well. We offer Our institution is based on an interdisciplinary, innovative and dynamic collaboration. You will profit from a systematic training on the job, in addition to personal development possibilities and our pronounced vocational training culture. If you wish to optimally combine work and family life or other personal interests, we are able to support you with our modern employment conditions and the on-site infrastructure. For further information please contact Dr Meitian Wang, phone +41 56 310 41 75 or Dr Rafael Abela, phone +41 56 310 32 71. Please submit your application online (including list of publications and addresses of referees) for the position as a Beamline Scientist (index no. 6112-01). Paul Scherrer Institute, Human Resources Management, Elke Baumann, 5232 Villigen PSI, Switzerland __ Meitian Wang Swiss Light Source at Paul Scherrer Institut CH-5232 Villigen PSI - http://www.psi.ch/sls/ Phone: +41 56 310 4175
