[ccp4bb]
Dear Prashant, I have been working with a protein-protein complex expressed in mammalian cells, and that complex in very poorly soluble. Even with 500mM NaCl in the buffer, I cannot concentrate the complex to above 3 mg/mL. I tried an old school technique and precipitated my protein complex with ammonium sulphate (~80% saturation) on ice. When I recovered my complex, I was able to get almost 9mg/mL without any precipitation at all. The sample still crystallizes, but I believe now that the sulphate ion is shielding/masking part of the protein better than the NaCl did. Perhaps this would be worth a try for you as well? Another thing you can try with a weakly soluble protein is to set up various ratios between the protein and the reservoir solution in your crystallization drop. One point I have not yet seen mentioned is that some proteins are hyper sensitive to changes in temperature. In my opinion, any protein sample should be kept cold, (on ice at the bench) UNLESS you have good reason and biophysical data to show otherwise. If you are concentrating at room temperature, try concentrating at 4C if you can, you might be pleasantly surprised. Good Luck! Bryan Prince From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Roger Rowlett rrowl...@colgate.edu Sent: Tuesday, August 19, 2014 5:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Some things to try to increase solubility: 1. Move the buffer pH away from the expected pI. Proteins have minimum solubility near their pI values. 2. Add solubilizing agents to the solution, e.g., 20-50% glycerol. (This may alter you crystallization screening strategy) 3. Include some inert salt in the solution to minimize electrostatic interactions, e.g. 100-500 mM NaCl. Ultimately, your protein just may not be very soluble. That is potentially OK...it will ppt and maybe xtallize well at low concentration. Roger Rowlett On Aug 19, 2014 1:52 PM, Prashant Deshmukh prashantbiophys...@gmail.commailto:prashantbiophys...@gmail.com wrote: Hi, i am concentrating my protein using centricon filter, but it is precipitated soon. Please help me solving this problem. Thanks. Prashant Deshmukh Dept. of Biophysics, NIMHANS, Bangalore 560 029, E-mail:prashantbiophys...@gmail.commailto:e-mail%3aprashantbiophys...@gmail.com Mob.No.: +919620986525tel:%2B919620986525 -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
[ccp4bb] FW: [ccp4bb] dynapro DLS cuvettes
Some memories from the dark recesses of my mind… Hope it helps! Bryan From: Prince, D Bryan Sent: Thursday, August 14, 2014 6:07 PM To: 'Gloria Borgstahl' Subject: RE: [ccp4bb] dynapro DLS cuvettes Dear Gloria, I seem to remember that we had a DynaPro DLS system and used disposable plastic cuvettes (low volume). I think these are the ones we used, and we were very happy with them. If memory serves, we could do about 25uL samples, and they were individually packed so cleanliness was not a concern. http://www.sigmaaldrich.com/catalog/product/sigma/z605050?lang=enregion=US Sigma has a supply of quartz cuvettes here that you might find helpful: http://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=104902943 GOOD LUCK Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gloria Borgstahl Sent: Thursday, August 14, 2014 5:35 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] dynapro DLS cuvettes Does any one know of a source of these cuvettes? Protein Solution doesn't exist anymore and Wyatt no longer has these. -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] emergency substitute for RT loop cover?
One thing you can try is to place modeling clay at the base of the pin. Add some mother liquor to a quartz capillary (1-2mm should work) and under a microscope carefully cover the loop with the capillary, pressing it into the clay to seal the crystal in a closed system. As long as you move or divert the cold stream, you should be ok. I actually collected a full dataset off one precious crystal this way at room temperature, and it diffracted to 2.2 Å. Good luck. Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Monday, July 07, 2014 12:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] emergency substitute for RT loop cover? Try to put your crystal into oil drop? Sent on a Sprint Samsung Galaxy S® III -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. Original message From: Frank von Delft Date:07/07/2014 12:32 PM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] emergency substitute for RT loop cover? Hi all Pretend you were stuck having to do RT data collection but without access to either Mitegen MicroRT Capillaries or the more old-fashioned quartz capillaries, to pop over the loop. Anybody have suggestions of alternative ways of doing this? I do want to use loops (I never learnt how to suck up crystals in capillaries). I have access to a passably stocked biochemistry teaching lab, and could at a pinch go rifle some more advanced research labs. (No, I'm not at home ;) Thanks! phx
Re: [ccp4bb] emergency substitute for RT loop cover?
Sorry, forgot you didn’t have any quartz capillaries. If you are in a biochem lab, do you have access to hematocrit capillaries? You might be able to use them to cover the loop in the fashion I posted just a minute ago. Again, good luck! Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: Monday, July 07, 2014 12:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] emergency substitute for RT loop cover? Hi all Pretend you were stuck having to do RT data collection but without access to either Mitegen MicroRT Capillaries or the more old-fashioned quartz capillaries, to pop over the loop. Anybody have suggestions of alternative ways of doing this? I do want to use loops (I never learnt how to suck up crystals in capillaries). I have access to a passably stocked biochemistry teaching lab, and could at a pinch go rifle some more advanced research labs. (No, I'm not at home ;) Thanks! phx
Re: [ccp4bb] L-Dopa Stabilization?
Dear Jacob, Please check the following references: http://www.ncbi.nlm.nih.gov/pubmed/8771063 http://www.ncbi.nlm.nih.gov/pubmed/9251095 From some limited reading of another paper, it seems like the dark solution could be the conversion of L-Dopa into melanin, which is accelerated in alkaline conditions. The authors were extracting L-Dopa from beans and showed that 3pH5 was ideal for maximizing solubility of L-Dopa in solution. They suggested citric acid from fruit juice (Lemon, 10mL/L) for extraction/solubilization of L-Dopa. (www.redalyc.org/pdf/939/93911288017.pdf) Good luck! Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Thursday, July 03, 2014 1:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] L-Dopa Stabilization? Dear Crystallographers, Does anyone have experience with co-crystallization of a protein with L-Dopa? In my hands, the L-Dopa degrades in water to a dark solution within 10 minutes. I am thinking of trying some reductants and metal chelation, but a verified successful formula would be much appreciated, and preferably it would be without going into a glove box. Jacob Keller *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Of course, if you can plan ahead, you can grow your crystals in shipping friendly plates. See the In-Situ plate at Mitegen for details. http://www.mitegen.com/mic_catalog.php?c=insituplates you also might try to add agarose to the drop to fix the crystal in place, but that could be tough with a very large drop (20uL). Good luck! Bryan On Jul 2, 2014, at 5:33 PM, Sampson, Jared wrote: Hi Meisam - We do this frequently, using pieces of spongy foam to pad the inside of a sturdy styrofoam shipping box, which keeps the temperature from fluctuating too much. This is then carried in a reusable grocery bag by one person whose sole responsibility it is to ensure it isn’t subjected to any jarring movements during the trip. We haven’t used anything quite so large as your 10+ μl drops, but this setup works fine with 24-well plates of ~2-4 μl hanging drops for the 1.5-hour drive to our friendly neighborhood synchrotron. Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center http://kong.med.nyu.edu/ On Jul 2, 2014, at 4:17 PM, Meisam Nosrati meisam.nosr...@gmail.commailto:meisam.nosr...@gmail.com wrote: Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. = -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Recovering crystals from dry drops
I recently had a similar situation for a protein crystal that was sitting in very thick, sticky goop. I put three drops of Paratone N on a coverslip and harvested the crystal by using a Mitegen yoke tool to dig around the crystal, then a regular nylon loop to move the crystal into a drop of Paratone N. A bit of the semi solid goop came over with the crystal, but I was able to swish it away in the Paratone N. I repeated this in a total of three drops and then flash froze it in liquid nitrogen. The crystal diffracted to better than 2 Å at the synchrotron. Good luck on recovering your crystals! Regards, Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish Chattopadhyay Sent: Thursday, February 20, 2014 11:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Recovering crystals from dry drops Would you please share your experience and comments on recovering protein crystals from dry (or almost dry hanging drops) for data collection. I found some beautiful crystals in hanging drops that were set up three years ago; from the color of the crystals ( the protein binds a colored substrate) and the their shape I know these are crystals of my protein. I had collected data using some of these crystals when they were fresh; resolution was poor and the overall I/sigma was low. I am curious if the dehydration would improve the diffraction now. Thanks Debasish Ph: (205)934-0124; Fax: (205)934-0480 -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
[ccp4bb] Postdoctoral Position Available at AstraZeneca in Waltham, MA
At AstraZeneca, we're constantly striving to make new discoveries. Discoveries that will make a meaningful difference to health globally. We already have an exceptional product pipeline, and everyone here has a part to play in making sure that pipeline fulfills its potential. This is your opportunity to join them - and to fulfill your own. We have an exciting opportunity for a biochemist / biophysicist / crystallographer to join the Structure Biophysics group at AstraZeneca in Waltham, MA. This is a collaborative effort with the laboratory of Professor Jamie Cate at the University of California Berkeley to understand the structural basis of mechanism-based toxicity to antibiotic treatment. Success in generating chimeric ribosomes will result in novel means to assess antibacterial sensitivity using a suite of genetic, biochemical and biophysical approaches. This position provides an opportunity to explore the gene-to-structure process in an industrial setting. You'll be exposed to a variety of aspects within the drug discovery cascade, and will interact with scientists in a cross-disciplinary environment. You'll also be expected to deliver your novel research results within AstraZeneca, and externally via scientific conferences and peer-reviewed publications. * Build an understanding of antibiotic selectivity for the bacterial ribosome through the design and analysis of the functional activity of chimeric ribosomes * Assess the antibacterial sensitivity of recombinant bacterial strains containing chimeric ribosomes against a panel of known ribosomal inhibitors * Understand the structural basis of ribosomal inhibitor selectivity by performing crystallography on chimeric ribosomes in complex with antibiotics * Work collaboratively with scientists across various disciplines to achieve project objectives * Externally present results at scientific conferences, and publish scientific papers in high quality peer-reviewed journals A PhD in one or more of the following areas: * Protein/RNA biochemistry * Biophysics, crystallography, ribosome biology, molecular microbiology * Molecular biology, expression and purification skills to independently produce high-quality RNA-protein complexes * Experience with multiple biochemical and biophysical techniques to assess the quality, stability and function of RNA-protein complexes * Experience in performing antibacterial sensitivity assays * Experience in designing complex reagents for biophysical and crystallographic studies * Crystallographic experience is highly desirable * Strong written and oral communication skills As one of the world's leading pharmaceutical companies, our business is focused on providing innovative, effective medicines that make a real difference in important areas of healthcare. What's more, we support and encourage our people in discovering their own potential. Excellent learning and development opportunities will be available to you throughout your career here. Please follow this link to view the full advertisement and online application: http://jobs.astrazeneca.com/jobs/23176-postdoctoral-position-synthetic-b iology-to-probe-ribosomal-inhibitor-selectivity -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Izit dye stained crystal
Are you referring to the I3C magic phasing triangle by any chance? Beck, et al Acta Cryst D 61(?) (2008) is the reference I think. Good luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Richard Gillilan Sent: Friday, November 30, 2012 5:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Izit dye stained crystal We've found that high PEG concentration seems to compete with dye binding, so I'm not surprised your didn't see good uptake. I doubt the anomalous signal is from the dye ... if you calculate the molar dye concentration you would need to have significant occupancy in the lattice, you'll probably find that the crystals would look almost black ... unless there was a color change. I recall seeing a poster at the annual ACA meeting 3-4 years ago maybe, in which somebody was using polybrominated or polyiodinated aromatics to both dye the crystals and phase the structures at once. In fact, I think we gave the poster an award for that. I don't have my past notes handy to remember, but could look it up if you're interested. Richard Gillilan MacCHESS On Nov 30, 2012, at 4:19 PM, Sarathy Karunan Partha wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn't take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.INP -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Problems in crystallization
Those are the EasyXtal 15 well tools with the EasyXtal DG-Crystal Supports. Look at this link to find them: http://www.qiagen.com/products/protein/crystallization/default.aspx I agree that they keep the drops from spreading out, but I have experienced trouble harvesting smaller crystals from these xtal tools. Good luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rob Gillespie Sent: Wednesday, November 07, 2012 9:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems in crystallization Qiagen used to sell hanging-drop trays with screw-cap tops, and the tops had six rings on them (3 large, 3 small). The rings had a lip on them that kept the drop from spreading beyond the edge. I found these quite useful when setting up drops that had detergent in them. I unfortunately don't know what they are called, as I have several at my bench but not the packaging they arrived in. On Wed, Nov 7, 2012 at 9:04 AM, Eva Bligt-Lindén eva.bl...@abo.fi wrote: Thank you for your replies. To my knowledge the protein is not a surface-tension-reducing-protein and there is no detergent in the sample. I have tried different plates and still the same result. I will try a different crystallization technique such as batch under oil or counter-diffusion if I do not solve this problem. But now I am curious to know what is causing the disturbed surface tension and if someone else has experienced and dealt with something similar. Kind regards, Eva Quoting anna anna marmottalb...@gmail.com: Why don't you try batch under oil? 2012/11/7 Eva Bligt-Lindén eva.bl...@abo.fi Dear ccp4 users, I have a problem in the crystallization of my target protein. Whenever I set up a vapour diffusion experiment, either hanging or sitting drops, the drops spread out. The surface tension is completely lost in 80-90% of the droplets. Have any one experienced something similar? What could be the reason for this strange behaviour? I have tried three different commercial screens with 96 condition each and there is no difference between the screens. There is no difference between manual or robotic setups either. The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer controls are all ok. Kind regards, Eva __**__ Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Crystal Optimization
Dear Muhammed, In my experience, crystals like that are likely made up of contaminated or non-homogeneous protein. Have you run NATIVE PAGE and IEF gels to determine the purity of your sample? Is it the correct MW by mass spec without contaminating peaks? Good Luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Greg Costakes Sent: Tuesday, July 10, 2012 2:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Optimization Have you tried multiple rounds of micro-seeding? --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** From: Muhammed bashir Khan muhammad.bashir.k...@univie.ac.at To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, July 10, 2012 1:22:28 PM Subject: [ccp4bb] Crystal Optimization Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] crystallization analysis software
Ed, I looked into this a number of years ago, and remember the following papers. I did not actually use any of the databases described due to IT issues at the time. I hope this helps. Kind regards, Bryan LISA: an intranet-based flexible database for protein crystallography project management http://scripts.iucr.org/cgi-bin/paper?S0907444901009295 HalX: an open-source LIMS (Laboratory Information Management System) for small- to large-scale laboratories http://scripts.iucr.org/cgi-bin/paper?S0907444905001290 Xtrack - a web-based crystallographic notebook http://scripts.iucr.org/cgi-bin/paper?S0907444902012696 CLIMS: Crystallography Laboratory Information Management System http://scripts.iucr.org/cgi-bin/paper?za5055 These I found while looking up the others. MOLE: A data management application based on a protein production data model http://onlinelibrary.wiley.com/doi/10.1002/prot.20291/full Design of a data model for developing laboratory information management and analysis systems for protein production http://onlinelibrary.wiley.com/doi/10.1002/prot.20303/full The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory http://scripts.iucr.org/cgi-bin/paper?S0907444911007943 -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Wednesday, May 23, 2012 12:24 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization analysis software Does anyone know of a (non-commercial) software that can analyze results of a crystallization screen? What I am looking for is some way to tell what components/factors favor protein solubility/precipitation based on binary input (clear drop/precipitate). I did some googling, but please feel free to use lmgtfy on me :) Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
[ccp4bb] Propane still?
Good afternoon fellow ccp4bb'rs, I was wondering if anyone knows if a still to condense gaseous propane to liquid propane using dry ice is commercially available. I want to make sure that it is not something I can purchase before I build one fit to purpose. I appreciate any advice and knowledge you can share. Regards, Bryan Prince -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] very informative - Trends in Data Fabrication
I thought we had evidence for hackers doing this already. J http://www.nature.com/nature/journal/v477/n7365/full/477373e.html (no flames, please-'tis intended to be funny, not factual) Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Monday, April 02, 2012 1:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] very informative - Trends in Data Fabrication I like your point--somehow we should enlist the evil inclination to power our science, a la Faust. How is it that those hackers are so innovative for so little reward? I remember a Smithsonian article years ago which quoted the calculated mean $/hr rate of money counterfeiters as being ~pennies/hr, and I assume hackers would fit right in there... JPK On Sun, Apr 1, 2012 at 11:45 PM, Artem Evdokimov artem.evdoki...@gmail.com wrote: I can't resist asking: If we assume that the data fabrication techniques and the techniques for discovery of such activities should have the same sort of arms race as the development of viruses and anti-malvare software (but of course on a much more modest scale since structural biology is a relatively niche discipline) - can we then speculate further that eventually the most sophisticated fabrication techniques would be equivalent to de novo structure prediction :) It's really too bad that there's no real money in this (again, relatively speaking - not as much money as there is in software development), because if there was then the structural biology equivalent of 'virus hackers' would in reality approximate the same development trajectory as the most successful (and legitimate) protein modelers. Given the ingenuity of hackers and like-minded people in general, I sometimes wonder if this isn't a better way to develop structure prediction tools... Artem On Sun, Apr 1, 2012 at 10:09 AM, Paul Emsley paul.ems...@bioch.ox.ac.uk wrote: On 31/03/12 23:08, Kevin Jin wrote: I really wish PDB could have some people to review those important structures, like paper reviewer. So do the wwPDB, I would imagine. But they can't just magic funding and positions into existence... If the coordinate is downloaded for modeling and docking, people may not check the density and model by themself. However this is not the worst case, since the original data was fabricated. 1. All of data was correct and real, Hmmm... It will be very difficult for people to check the density and coordinated if he/she is not a well-trained crystallographer. I hope and believe that this is not the case. Even basically-trained crystallographers should be able to calculate and interpret difference maps of the kind described by Bernhard. And with the EDS and PDB_REDO server, one does not even need to know how to make generate a difference map... Paul. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Desalting columns
Actually, I would refer the ccp4-bbs to Journal of Structural Biology 175 (2011) pp216-223 for the use of fluorescence in relation to protein crystallization. Regards, Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Christian Roth Sent: Tuesday, February 28, 2012 8:09 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Desalting columns If you want test a lot of different conditions a Thermofluorecence assay might work for your protein and you may find a condition which stabilise your protein. However there is no warranty that it crystallise better afterwards. Christian Am Montag 27 Februar 2012 17:01:33 schrieb Sangeetha Vedula: Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] Freezing crystal
Dear Theresa, Gary Gilliland's paper on cryosalts would seem to be useful for your problem. http://scripts.iucr.org/cgi-bin/paper?en0028 Also, I have used 15% glycerol in a synthetic mother liquor to effectively freeze crystals grown in 2M Ammonium Sulfate. Another method that Jim Pflugrath teaches is to use ordinary table sugar (sucrose) dissolved in the reservoir solution to cryoprotect your crystal. A webinar is located on the rigaku website here: http://www.msc.com/protein/webinar-001.html Also, Hampton Research has a good list of tips in the Tech Support portion of their webpage. www.hamptonresearch.com Whichever you choose, good luck with your crystals! Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa H. Hsu Sent: Sunday, February 05, 2012 5:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Freezing crystal 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
Re: [ccp4bb] sealing slides on VDX plates?
I remember a technique that used warmed Vaseline (liquefied) in a shallow pan. People would invert the plate and dip it into the liquefied Vaseline, then flip it over to dry. I suppose one could use masking tape to cover the outer edge of the plate to keep it Vaseline free. I am not sure how it would work with cryschem (24-well sitting drop) plates. Good luck! Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of malho...@miami.edu Sent: Friday, November 18, 2011 1:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] sealing slides on VDX plates? Vaseline (pure, with no additives) from CVS/Walgreens in a 10 ml syringe and a pipette tip (wide bore) also works very well. On 11/18/11 12:58 PM, Bosch, Juergen wrote: Dow Corning Heavy Vacuum Grease, a 10 ml syringe and a shorter cut 200 µl tip attached to the syringe. Jürgen On Nov 18, 2011, at 12:55 PM, Dima Klenchin wrote: Hello, I wonder what everyone is using for sealing hanging drop slides on VDX plates? For the most part, we paraffin oil but I am unhappy with it because it is too think and too frequently there is break in the oil and the drop dries too much. I find vacuum grease to be not terribly practical because it takes too much time - particularly when the well needs to be opened (seeding, modifying well content, etc). In ideal world I would like to find a much thicker oil that 1) contains as little volatiles as paraffin oil, and 2) allows no more water vapor diffusion through it than paraffin oil. Hampton suggests Cargille immersion oils for this purpose but the MSDS for these oils states that they have slight odor, so I am a bit concerned with unknown volatiles getting into crystallization drop. So, what do you like to use? Thanks much, - Dima -- Arun Malhotra Phone: (305) 243-2826 Associate ProfessorLab: (305) 243-2890 Dept. of Biochemistry Molecular Biology Fax: (305) 243-3955 University of Miami School of Medicine PO Box 016129 E-Mail: malho...@miami.edu Miami, FL 33101 Web: http://structure.med.miami.edu
Re: [ccp4bb] not so good news (Steve Jobs RIP)
I thought I would have nothing to say about this topic, but I am sitting here at my desk listing to my iPod, remembering fondly my apple IIc computer I started with in elementary school. I would like to share a story with you, if you would be kind enough to listen. I have a son who is severely developmentally delayed, he is nine, but functions as an infant due to cerebral palsy and other complications. We were at Children's Hospital during a recent specialist visit, and sat near another family who have a child much like mine. This child was playing with an iPad doing simple spelling games. He, too has been developmentally delayed, but his parents purchased an iPad for him because of the interactive touch screen. Then they started with very simple games like popping soap bubbles. In a year he has gained better fine motor control in his hands, has learned to communicate better with his family and has started to truly blossom. I pray that my son will experience a similar epiphany with his new iPad, but only time will tell. So, I say thank you, Steve- you will be truly missed. Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Richard Gillilan Sent: Thursday, October 06, 2011 5:31 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] not so good news (Steve Jobs RIP) Jobs and Apple not only deeply shaped my career, but also my son's path as well. I bought an Apple II+ with my first Summer's earnings as an undergraduate lab assistant in organic chemistry in the very early 80's. Spent winter break writing a printer driver in Apple 6502 code so I could plot molecular orbitals. Skipped the first Macs when they came out, but returned to Apple when the Performa was popular. I may have been the first to announce on this list that OS X would be based on unix ... and I enthusiastically got a copy as soon as I could get my hands on it. It's true there will never be another Jobs, but I fully expect more great surprises ... the universe is only just getting started. Richard
Re: [ccp4bb] Do manufacturers change their crystallogenesis screens?
Dear Flip, I think with respect to the Formulatrix database, it would be useful to have the date of entry into the database for each screen input. I agree that there are discrepancies in the database, but they can generally be traced to a change from one catalog to the next. If you have the date of entry, then you can find the correct catalog to look at for your formulation information. It would be useful if the various crystallography suppliers would place their plate information in some format easy to incorporate into the Rock Maker database. Maybe this is something that can be discussed at the RAMC in France this fall. Kind regards, Bryan Prince -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Flip Hoedemaeker Sent: Wednesday, August 31, 2011 5:01 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Do manufacturers change their crystallogenesis screens? Op 8/24/2011 17:21, Chris Morris schreef: Hi Chris, Yes, I have seen quite a few inconsistencies in screen formulations. Errors in listed conditions include recipe changes, but also typos both in the vendor description and in the database entries. At the moment I'm building a list of all discrepancies of the screens in the Formulatrix database together with the date found. This is a tedious work in progress but I'm happy to share the list sofar with people interested (offline). It will be published on the web site when complete. In the meantime, it is wise to double-check each hit condition found on the vendor web site, and in the cases where the exact composition is not published ask the vendor directly. Flip HI, I've recently seen two examples where the description of a screen in a local database was different to the current one on the manufacturer's web site. This happened in two different labs, using different software, and with different screen manufacturers. This could potentially lead to an optimisation screen that finds no hits, because the wrong condition is being optimised. Does anyone have experience of this? Am I just looking at a few one-off errors, or is there a general problem here? The ideal solution is for screen manufacturers to give version numbers to their screens. Failing that, a good fix at the laboratory is to download the screen description every time a deep-well plate is received, and second best would be to download it every time a trial plate is set up. If there is a real concern here, we will implement one of these in xtalPiMS. Regards, Chris Chris Morris chris.mor...@stfc.ac.uk Tel: +44 1925 603689 Fax: +44 1925 603825 Mobile: 07921-717915 http://pims.instruct-fp7.eu/ STFC, Daresbury Lab, Daresbury, Warrington, UK, WA4 4AD
Re: [ccp4bb] Protein preps become a jelly
I had a similar problem with crystals that were obtained from PEG-based precipitants over long periods of time. If I harvested the crystals in about 5 days, they would diffract to ~3 Angstrom. If I let them grow any time past about 7 days, they became PEG-alated and didn't diffract at all. I personally have not had that happen in salt-based precipitant conditions. I would try adding up to 10mM DTT or 5mM TCEP to minimize proteinaceous skin formation, which I believed was crosslinked by the PEG. Attempts to change PEG's did not work and I couldn't find a salt based precipitant condition that would give us crystals where the active site was not occluded by the salt. BTW, have you purified the prep over SEC followed by IEX or RP columns? Have you dissolved the gel and run mass spec to see if there is any proteolysis going on? If you have an active proteolytic fragment, there may be one or more hydrophobic patches being exposed and this could cause the protein to gum up in order to bury those patches. Anyway, good luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Laurie Betts Sent: Tuesday, August 30, 2011 11:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein preps become a jelly I have had protein crystals (so very high protein concentration) that turn into gummy bear-like objects, where instead of crumbling they are like, well, a gummy bear or a piece of rubber. I attributed it to oxidation or other chemical ageing processes. I am sure others will have suggestions for preventing this. On Tue, Aug 30, 2011 at 11:31 AM, aidong a...@xmu.edu.cn wrote: Dear Buddies, Sorry for bothering you with an off-ccp4 question. We recently are experiencing a very strange phenomena. A couple of protein preps with reasonably high concentration (10-20mg/ml) become a jelly after storages for overnight or a couple of days at 4C. All of them have been purified by gel filtration. Some of these proteins behave like this from very first preps but some of them had been very kind to us previously. We have googled extensively in CCP4BB and www but it appears this only happens to us. It would be highly appreciated that you could exchange their experiences or provide your suggestions. Aidong Han, Ph.D Department of Biomedical Sciences School of Life Sciences Xiamen University Xiamen, Fujian 361005 China Phone: 0592-218-8172 (O) 0592-218-8173 (L) Web: http://life.xmu.edu.cn/adhanlab/ -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] co-crystallization
Dear TY, Typically between 5-10x molar concentration over the protein is enough to ensure binding when the IC50 is uM to low mM. For tighter binding compounds (nM to low uM), 2-5x is sufficient. Whatever you do, when the precipitate occurs DO NOT REMOVE it. I learned to my chagrin that you change all the dynamics of the drop when you do. I ended up with empty crystals until I left the precipitate in place. Think of it this way- Free protein + compound ↔ protein:compound complex + precipitate (mix of protein + compound) If you change the equilibrium by removing the precipitate, you remove the “pressure” on the P:C complex, and it will dissociate to P + C. The precipitate acts as a reserve of protein and compound, thus favoring (or stabilizing) the P:C complex. I set drops up as a slurry frequently, and if I get crystals, they always have the compound bound. Pay attention to the drops if you are screening, because it will be important to note what makes the precipitated solution better (clear drops=solubilizing) or worse (aggregated drops=decreased solubility of your complex). You can also try suspending your compound in LMW PEG’s (200-400 FW) instead of DMSO. Either way, try using DMSO (~20%) or LMW PEG (~30%) depending on your crystallization conditions as a cryoprotectant. Any crystals that grow have some small amount of those agents in them already, so they should be more tolerant of them in higher concentrations. Best of luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yvonne TAN Yih Wan Sent: Thursday, August 25, 2011 2:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] co-crystallization Hi , I am co-crystallizing a protein with compound and would like to know how much of compound to add to protein solution to start with. I know that the protein binds compound in a 1 to 1 ratio but also noticed that the compound precipitates out of solution when DMSO is diluted off. Where should I start of? A 1 protein :2 compound ratio or more? And what is the best method to determine if the binding is homogeneous (that all protein has got a compound in it)? Any suggestions would help. Thanks TY -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] spherulites and PEG3350
Something else to try would be the Protic Ionic Liquid kit from Hampton. I recently had crystals of a protein that would only grow as laminated stacks of plates. Optimizing the conditions and using an additive screen didn't improve crystal morphology. I tried the PIL kit from Hampton and was able to get single, thick plates in several conditions. At the ACA meeting in Hawaii a few years back, there was a poster about ageing your PEG solutions by microwaving them and letting them cool on the bench. This was the only way that the poster's author could get reproducible crystals of her target protein. Good Luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Regina Kettering Sent: Wednesday, August 24, 2011 2:47 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] spherulites and PEG3350 Something to consider is the quality of the PEG 3350. We have found that different qualities of PEG 3350 can give different results, depending on the type and amount of contaminants. What used to be the Fluka PEG 3350 is now the pharm grade of PEG 3350 (aka Miralax). We use high quality PEG 3350 for normal screening, but switch to the highest quality grade we can get for optimizing. Regina From: Jan van Agthoven janc...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, August 24, 2011 2:05 PM Subject: [ccp4bb] spherulites and PEG3350 Dear all, I recently obtained some spherulites while trying to crystallize my protein. The spherulites are manually reproducible, but changing pH, protein concentration, and salt concentration does not result in crystal formation. Microseeding with crushed spherulites isn't a solution either as it only yields new spherulites. Next stepp is the use of an optimization kit but I have a limited amount of material, and I start doubting that these are protein spherulites, as the spherulites are not particularly soft. The condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms easily spherulites around that concentration? Thanks, -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Aging PEGs
For those of us truly controlling types :), I used to make the PEG solutions and filter them over a Bio-Rad resin that filtered out all the junk added to stabilize the PEG solution. Then, of course I had to freeze all my PEG solutions in aliquots, or wrap them in foil and store at 4C in the dark. This would take several days, depending on the FW of the PEG. If you are really sensitive about what is in your PEG solutions, try GC-grade PEG's. The FW profile is much more restricted around the reported value (i.e. PEG 3350 molecular biology grade has a broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter peak profile.) Back before you could buy Crystal Screen I, II or HT, you had to make the stock solutions, then make the screen. But at least when you did that, you had all the stocks. Now, I just buy pre-made solutions, and keep them in a drawer with a date opened written on the bottle. Isn't progress grand? :) Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Wednesday, August 24, 2011 3:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Aging PEGs A while ago I measured the pH's of old and new PEGs and found them very different, and internally attributed all old vs new PEG issues to pH. Upon reflection, this seems too simplistic. Are there other known mechanisms of crystallization capacities of PEGs of various ages? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
Dear Arnon, I have a Nanodrop2000, which reads from the post or a user supplied cuvette. I have had NO complaints about using the Nanodrop for reading protein concentration immediately prior to crystallization setup. When I have observed differences in OD280 vs Bradford, it is usually due to one of the following: 1) The protein is deficient in the amino acid residues that provide the 280nm signal. This can be corrected with the extinction coefficient, and can be programmed into the Nanodrop so that the readout is correct. 2) The protein is behaving badly in the Bradford assay (interference with some component of the protein buffer) 3) The dilution used in the Bradford is contributing to large concentration errors (and can be combined with 2, above) The value of the Nanodrop, is that you get a OD280 that you can reproduce prior to every experiment at a low cost of protein sample. The N2000 can also do the Bradford or other assay on the post, or with a cuvette if you really want to do it that way. When there have been no mitigating factors, my Nanodrop OD280 readings have been within 5% of the values I get from SEC-MALS, MassSpec or Bradford. I have no experience with the Pearl, but I am very happy with my Nanodrop 2000. Good luck with your choice! Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Arnon Lavie Sent: Thursday, June 16, 2011 3:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford. 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 correlation between Nanodrop and Bradford? While researching the Nanodrop machine, I heard about the Implen NanoPhotmeter Pearl. So Question 2: Is the Pearl better/worse/same as the Nanodrop for our purpose? Thank you for helping us to advance to the next millennium, even if it is nearly a dozen years late. Arnon -- *** Arnon Lavie, Professor Dept. of Biochemistry and Molecular Genetics University of Illinois at Chicago 900 S. Ashland Ave. Molecular Biology Research Building, Room 1108 (M/C 669) Chicago, IL 60607 U.S.A. Tel:(312) 355-5029 Fax:(312) 355-4535 E-mail: la...@uic.edu http://www.uic.edu/labs/lavie/ ***
[ccp4bb] Question about TEV cleavage
Hello all: I am curious about what level of recovery is reasonable when performing a TEV cleavage to remove 6HIS tags (N-terminal) from a protein. Currently we are experiencing 50% loss of soluble protein after TEV cleavage, and feel that this is too much. Are there better systems for his tag cleavage that have better soluble protein recoveries than TEV? Many thanks (in advance) Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] optimization of dumbbell like crystal
Dear Harvey, Microseeding and adding 1-5% glycerol to the drops helped me when I obtained crystals like these. Good Luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Harvey Rodriguez Sent: Sunday, February 20, 2011 9:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] optimization of dumbbell like crystal Dear CCP4BBers, Recently I got a crystal which appeared in the condition 2.1M DL-Malic Acid, pH 7.0. The crystal looks like a dumbbell but was composed of a cluster of very thin needles as shown in the picture. The crystal can be repeated but the diffraction is poor. I have tried the grid optimization by pH and salt concentration gradients, however there was no improvement in either the shape or the quality of the crystal. Does anyone have some experiences in optimizing this kind of crystals? Any suggestion is appreciated! Harvey -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] AMP-PNP Hydrolysis
Hello Steve, You can also check out this paper: Bystrom, Pettigrew, Remington and Branchaud (1997) Bioorganic Medicinal Chemistry Letters, Vol 7 No 20 pp2613-2616. It describes the creation of AMPPCF2P, which I had opportunity to use a few years back and it worked great! Good luck, Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Derek Logan Sent: Monday, February 14, 2011 4:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AMP-PNP Hydrolysis Hi Steve, Funnily enough I just read the following paper today, which describes exactly this phenomenon: http://www.ncbi.nlm.nih.gov/pubmed/21093442 Is AMPPCP as sensitive to acid conditions? I would suspect not. Best wishes Derek ___ Derek Logantel: +46 46 222 1443 Associate Professorfax: +46 46 222 4692 Dept. of Biochemistry and Structural Biology mob: +46 76 8585 707 Centre for Molecular Protein Science www.cmps.lu.se Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com On Feb 14, 2011, at 15:05, Young-Jin Cho wrote: Hi Steve, With my experience, it is (very) common to see AMPPNP is hydrolyzed to AMPPN (supposedly) with my protein. Although the literature often reported AMPPNP as a stable ATP mimic, such a luck wasn't true with my case, maybe same as you. If you go to Sigma website where I purchased, it may say it is not stable in an acidic condition. My mother liquor was in an acidic condition. So you'd better consider if you used it in an acidic condition, otherwise, your protein inherently has a strong power to hydrolyze it. In addition to the pH, I often see it can go hydrolysis easily. However, you can try more as you mentioned it may contain impurity. I just want to inform you that it is not surprising to see this hydrolysis. Good luck~ Young-Jin On Mon, Feb 14, 2011 at 8:30 AM, Soisson, Stephen M stephen_sois...@merck.com wrote: Hi there, Was recently looking at a structure of an enzyme with AMP-PNP added to the crystallization mix, and all I see is density for ADP. I was wondering if hydrolysis of AMP-PNP to ADP is relatively common - either as a result of extended time in crystallization or exposure of the resultant crystals to synchrotron radiation? I know that there can be up to 10% contamination of ADP in the purchased material, so it could just be that we have selected that form in the crystal, or that there was endogenous ADP bound that failed to substitute. Just curious if hydrolysis is a common observation. Thanks in advance- Steve Stephen M. Soisson, Ph.D. Structural Chemistry Site Lead, WP Merck Research Laboratories 770 Sumneytown Pike, WP14-1101 West Point, PA 19486 Phone: (215) 652-6185 Fax:(215) 652-9051 stephen_sois...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] N-terminal sequencing
I think that this CRO can help you: Proteos 4717 Campus Drive Kalamazoo, Michigan 49008 269.372.3423 http://www.proteos.net Good luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Junyu Xiao Sent: Tuesday, February 08, 2011 2:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] N-terminal sequencing Hi, Sorry for non-crystallography related questions. I am seeking protein N-terminal sequencing service. However, the facilities I have worked with previously (Michigan state and UCSD) were both closed. Does anyone know any companies or core facilities that can do this? Thanks, Junyu --- Junyu Xiao, Ph.D. University of California, San Diego Leichtag Room 283 9500 Gilman Drive, 0721 La Jolla, CA 92093-0721 Lab phone: 858-822-0684 -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] buffers for crystallization
Dear Chandan, 388 buffers are quite a bit. I presume you are talking about intervals of pH and not 388 buffers all at one pH. Can you explain a bit more about how you determined that the buffer is the problem, and what results led you to that conclusion? From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of chandan kishore Sent: Thursday, January 27, 2011 1:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] buffers for crystallization Hi Everyone, I have to crystallize one protein which contains a zinc fingure motifs, I am facing a problem in crystallization. I already used 388 buffers . Can anyone suggest me some books or papers freely available online on Buffers required for crystallization. Thanks -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate
Quickly passing the crystal through Paratone N has worked well for me when I crystallize in ammonium sulfate or sodium citrate conditions. Another trick is to dissolve sucrose (table sugar) in 10uL of the reservoir solution until it is saturated. Then separate the sucrose-reservoir mix into two 5ul drops. Add 5ul of the reservoir solution to one of the drops to make a 50% solution. Pass the crystal through the 50% saturated sucrose solution, then the 100% saturated sucrose solution and freeze. Good luck with the crystal! Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Robert Kirchdoerfer Sent: Wednesday, December 15, 2010 4:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate I've had good luck cryoprotecting high salt crystal conditions with sodium malonate (2.0-2.5M). Start with a 5M sodium malonate solution and dilute to 40-50% with mother liquor. good luck, Rob From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jerry McCully [for-crystallizai...@hotmail.com] Sent: Wednesday, December 15, 2010 1:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate Dear All; Recently we got some crystals from the condition #51 in the SaltRx crystallization kit from Hampton research. It contains 1.5M Na2HPO4 and 0.1M Tris(pH8.5). We am going to do a test diffraction ASAP. What cryoprotectant did you use for this condition? Thanks a lot and have a nice holiday season! Jinghua
Re: [ccp4bb] Crystal gel band
I recently ran mass spec analysis on some crystals that I had obtained from an optimization screen. I was looking for modifications in the protein. In order to get enough signal, I had to harvest and dissolve about 8 crystals roughly 0.3 x 0.15 x 0.15mm into the MS loading buffer in order to get a strong enough signal for the experiment. Alas, I did not find the modification I had been looking for, but I am not surprised that one single crystal, or even a small cluster of them did not show a band on a coomassie-stained gel. Do you A) have a MS handy, and B) can you sacrifice ~8 crystals to get enough S/N for a good experiment? Good luck! From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Tuesday, November 02, 2010 10:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal gel band It reads like you need to run a lane or two with a positive control of some kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals of a protein around the same expected molecular weight and try run on the gel lanes with about the same amount of crystalline volume as your putative protein crystals? From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of xaravich ivan Sent: Monday, November 01, 2010 9:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal gel band Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] I-TASSER predicts NADPH binding, need to confirm with experiment
Dear Xuan, I am not certain, but I think that Jacob was referring to a spectrophotometer called a Nanodrop. It is available from ThermoFisher Scientific and can provide absorbance data on as little as 2uL of sample. I think that if you have access to a Nanodrop, and use Ultrafree 0.5mL concentrators, you can achieve the results that Jacob described. Hope that helps, Bryan From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Xuan Yang Sent: Wednesday, August 04, 2010 9:46 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I-TASSER predicts NADPH binding, need to confirm with experiment Dear Jacob, Nanodrop ultrafiltration sounds really fancy to me. I am afraid that I have no access to such equipment yet. Thanks for letting me know about this new technology. Sincerely, Xuan Yang 2010/8/4 Jacob Keller j-kell...@fsm.northwestern.edu I like nanodrop ultrafiltration: concentrate your protein to the highest stable concentration possible figure out what is the lowest possible robustly-detectable nadph signal on your nanodrop combine the two in such a way in the top of a microcon of appropriate MWCO to acheive the highest possible protein concentration with the lowest possible nadph concentration. Take a baseline spec reading before spinning. spin long enough to get enough flowthrough to measure on the nano (~10uL is plenty.) Flowthrough should be the free nadph concentration L. Total L should be known, as well as total P, so you can figure out bound concentrations PL easily. you should probable do this in triplicate or so, with appropriate controls. I found 50uL/microcon to be a good balance of pipette-ability and economy of protein. If you want to get fancier, you can do more samples varying concentrations (or do some other more sophisticated method.) Jacob On Wed, Aug 4, 2010 at 3:10 AM, Xuan Yang pattisy...@gmail.com wrote: Dear All, 3D structure modeling server I-TASSER predicts a binding site for NADPH and I want to test this prediction. What would be the nice quick way to tell whether this protein bind NADPH or not, when I have a lot of recombinant protein? Sincerely, Xuan Yang -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Beginning crystallography text
Having recently completed the CSHL Macromolecular crystallography course, I can recommend Introduction to Macromolecular Crystallography by Alexander McPherson (ISBN 987-0-470-18590-2). I am posting the link below: http://www.amazon.com/Introduction-Macromolecular-Crystallography-Alexander-McPherson/dp/0470185902/ref=sr_1_1?ie=UTF8s=booksqid=1278619717sr=1-1 Kind regards and good luck! Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Peter Hsu Sent: Thursday, July 08, 2010 3:36 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Beginning crystallography text Hi all, I haven't gotten past the phase of growing the crystal, but I'd certainly still like to learn the actual theories of crystallography. Can anyone recommend a good beginner to mid-level text on macromolecular crystallography? Thanks, Peter
Re: [ccp4bb] Elspeth Garman's husband
My most sincere and heartfelt condolences to Elspeth and her family. May they find peace of mind and strength of heart during this most difficult time. Kind regards, Bryan Prince On Jul 3, 2010, at 6:28 AM, Frank von Delft wrote: Dear all Last night, John Barnett, physicist and husband of Elspeth Garman, passed away after a long battle with cancer. Please join me in passing condolences to Elspeth and her family. She mentioned that she may not be particularly responsive in the next while. Frank -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] UV microscope
If you check http://formulatrix.com/product_crystallizationimaging_muvis.html, you will find a stand-alone UV microscope. I have enjoyed using the RockImager UV microscope, which is an integrated hotel/imaging station. The MUVIS is a bench-top standalone imaging system that should supply what you need. Cheers, Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ho Leung Ng Sent: Wednesday, June 30, 2010 8:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] UV microscope I used a Korima a few times and didn't like it. Poor image quality and you have to worry about nuking your crystals with UV. However, I haven't tried any other UV microscopes to compare. For most purposes outside of high throughput imaging, I'd rather just shoot the mystery crystals with xrays. You may want to read Gill, Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals, Acta Cryst F 66:364, which compares several microscopes. http://www.ncbi.nlm.nih.gov/pubmed/20208182 ho Hi there. I don't know how much it costs, but I've used a Korima PRS-1000http://www.korimainc.com/prs1000.html serval times, and it appears to be fairly good. Although the image quality isn't great and there is still a bit of a learning curve for identifying small crystals and/or crystals buried in precipitate, I've found this microscope to be a very valuable tool. -Joel = Joel M. Guenther PhD Candidate, Department of Chemistry Kuriyan Laboratory http://jkweb.berkeley.edu/ University of California, Berkeley 176 Stanley Hall, QB3 Berkeley, CA 94720-3220 =
Re: [ccp4bb] PEG 1000
When the ACA meeting was in Hawaii (2006?), there was information about microwaving PEG solutions to artificially age new samples so that they would crystallize like the older PEG's. So I would infer that heat does have a significant effect on solid PEG's. All PEG's with MW =600 are solids at room temperature. What I used to do was to make a large hot water bath (37-42C) and put the container of PEG in the bath so that it was 2/3 covered by water and let it melt. It would take 3-4 hours to fully melt the sample, but then you can make a 50-70% (v/v) solution that will remain liquid at room temperature for your crystallization. Good Luck! Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Flip Hoedemaeker Sent: Wednesday, June 23, 2010 6:26 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] PEG 1000 The meting point of PEG 1000 is around 38C. Obviously, if Sigma has heated the batch to fill the bottles in the first place this is a futile exercise... I'd ask them first. Flip Vellieux Frederic wrote: R.Srinivasan wrote: Dear All, I have got initial crystals in a condition with PEG 1000. The PEG 1000 stock we had in our lab was rock solid and when i heated it to about 50 degrees for 15 to 20 minutes it became a solution. We thought the compound has got out dated or something like that and bought a brand new bottle from Sigma and this is rock solid too. Is this something characteristic of PEG1000? The hit condition says its 12.5% w/v PEG 1000 but it apparently seems i could never get a powder of it. So my question is, Can i go ahead using this melted solution form of PEG1000 for setting up optimizations? Thank you all in anticiaption, Vasan All higher molecular weight PEG's are solid. Some are stored as flakes, but as you mention some are rock solid. And it's very difficult to get them out of the container (breaking the bottle is sometimes necessary). What I would go for personally would be mechanical grinding because I do not know what happens to the PEG when it's heated to 50 deg or higher. But perhaps you could take your bottle, cut the content in half, make a powder out of the one half and use the other one with this heating method and see if there are any differences. Or else if you happen to have an analytical chemistry service at hand, provide them with a small sample of each of the 2 and ask them to check if there is any difference. Fred.
Re: [ccp4bb] crystallization of highly-glycosylated protein complex-avoid phase separation
Dear Jerry, First of all, it will be hard to reproduce the conditions with the glycosylated protein because by its nature, it is heterogeneous. One thing I would try with the glycosylated protein is a detergent screen, or if you don't have one, use a few NDSB's. Second, I would try setting up a lower PEG condition concentration and seed into it with the phase separated material, or with polystyrene nanobeads. Third, I would try FID (Free Interface Diffusion- like Microlytic's Crystal Former- www.microlytic.com) experiments, or use Enrico Sturza's technique with the Granada (?) Crystallization Boxes. Have you tried shifts in temperature? Start by warming up the protein to between 37-42C, then set up the drops. Allow the plate to equilibrate at some temperature between 37-20C then transfer to 4C after 24H. Something I suggest is that you go back to construct design and mutate the amino acids being glycosylated to something like GLY or ALA. If you can, express the protein in a different system (if you used Ecoli, try Insect cells or vice-versa). Alternately, you can use SGC's method of dilute protease mixed with your protein prior to setting up the drops. I have tried 1:1 dilution, but I think that is too weak. I would start with 1:100 or so. Good Luck, and let us know how it turns out. Regards, Bryan Prince. From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jerry McCully Sent: Wednesday, March 03, 2010 10:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization of highly-glycosylated protein complex-avoid phase separation Dear ALL: Recently we've been trying hard to crystallize a highly glycosylated protein complex ( 30% percent of carbohydrate in the total 120KD molecular weight). IT is a high affinity protein complex. One component can be crystallized in high salt condition and the other can be crystallized in PEG. Through screening, we happened to get some very ugly crystals in PEG condition using gel-fil purified complex sample. The problem is that it is very hard to repeat the screening condition. In contrast, it is very easy to get phase separation in the drop although we tried to optimize the protein and precipitant concentration. We tried to de-glycosylated using PNGase and EndoH but failed in non-denaturing condition. Hampton additive screen did not help either. We tried seeding several times but so far we have not got any better luck. Can anyone give some guidance here? Thanks a lot, Jerry McCully Hotmail: Powerful Free email with security by Microsoft. Get it now. http://clk.atdmt.com/GBL/go/201469230/direct/01/ -- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
