Re: [ccp4bb] protein crystals?
Definitely magnesium salt. I've seen it many times. But try to repeat it anyway just to be safe. Nian On Sat, Dec 1, 2012 at 8:24 PM, Yibin Lin yyb...@gmail.com wrote: Dear Folks, I got some crystals at the conditions of 0.1M Lithium sulfate or 0.1 M MgCl2, 0.1M Sodium citrate 3.5 or Sodium acetic ph5.5, or 0.1M HEPES pH7.5, or 0.1M Tris pH8.5, 30% PEG400 or 12% PEG4000. Protein contains 0.017%DDM. Could anyone have the experience to say if it is protein crystal? And how to optimize these crystals. Thanks. Lin
Re: [ccp4bb] hkl2000 install
Not sure it is what you need. I put an external screen to my laptop when I use HKL2000 for remote collection. Best, Nian Huang, Ph.D. On Sun, Nov 18, 2012 at 8:48 PM, 王瑞 wangrui...@gmail.com wrote: OK,thank you all of you. I have installed one copy of HKL2000 on our desktop computer. But for my notebook's low 1366*768 resolution, the HKL2000 can't work ! So what could I do to resolve it ? 2012/11/13 王瑞 wangrui...@gmail.com: Dear everyone: I have got the returned cr_info.dat to /usr/local/lib and /usr/local/hklint,when I typing HKL2000,it still display: dell@ubuntu:~$ HKL2000 ERROR: Not a valid HKL-2000 license: Licence info file (cr_info) not found Error code: -1 So could anyone can help me ? 2012/11/9 王瑞 wangrui...@gmail.com: Dear everyone: I'm sorry for a little off-topic! I want to install HKL2000 on ubuntu11.10 32bits, but it produces a file named info not cr_info after run the access_prod program.And when I put info to /usr/local/lib directory and typingHKL2000 in terminal, it display: root@ubuntu:/usr/local/bin# HKL2000 ERROR: Not a valid HKL-2000 license: Licence info file (cr_info) not found Error code: -1 So could anyone tell me how to do it ? Rui Wang
Re: [ccp4bb] Glass Capillaries
Hi Michael, I would recommend an alternative http://www.mitegen.com/products/micrort/micrort.shtml Traditional capillary is a pain to handle, unless you have a rock sized crystal. Good luck, Nian Huang On Mon, Nov 12, 2012 at 11:13 AM, Michael Roberts mrobert...@talktalk.netwrote: Dear All, I would be interested to learn of other crystallographers' experience in their use of glass capillaries for protein crystal growth and X-ray diffraction clarity. There are many types of glass available - quartz, soda glass, borosilicate, etc. Are there specific types which people prefer for best results overall? Best wishes, Michael Roberts
Re: [ccp4bb] off topic: a Python online course and others
Hi, For those starters on python programming, I strongly recommend Hanns Petter Langtangen's book A primer on scientific programming with python. It specifically targets the scientific programming, which we care about the most. The only thing I am not sure is that this book was written using python 2.6. A transition to 3.2 maybe a pain in future. Nian Huang On Fri, Oct 19, 2012 at 3:12 PM, Aksyuk, Anastasia (NIH/NIAMS) [F] anastasia.aks...@nih.gov wrote: Hello all, Considering that many biologists come to the field with no background in programming (like me) and want to learn a scripting language, I thought that many young members of the community might find this useful. There's an ongoing FREE online course An Introduction to Interactive Programming in Python (no computer background needed), presented by the department of computer science from Rice University. It just started and as far as the first week goes, it is very well presented and entertaining. https://www.coursera.org/course/interactivepython There's plenty of other useful courses there as well. https://www.coursera.org Anastasia Anastasia A Aksyuk IRTA fellow Laboratory of Structural Biology, NIAMS Bldg 50, Room 1511, 50 South Drive MSC 8025 N.I.H., Bethesda, MD 20892-8025 email: anastasia.aks...@nih.gov phone: 301-451-8247
Re: [ccp4bb] Off Topic: Sucrose / Glycerol Gradient Maker Fractionator
Or make it yourself if you got time. Anal Biochem.http://www.ncbi.nlm.nih.gov/pubmed?term=a%20syringe%20based%20gradient%20former#1994 Sep;221(2):397-400. A syringe-based gradient former for linear and exponential gradients. Shearer G Jrhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Shearer%20G%20Jr%22%5BAuthor%5D. Nian On Thu, May 3, 2012 at 10:40 AM, Antony Oliver antony.oli...@sussex.ac.ukwrote: Dear all, I find myself working on a number of large multi-protein complexes, and am likely to need to use Sucrose / Glycerol gradients in preparing them. IWhilst I can do this manually in the short term, I was wondering if someone could recommend any manufacturer's (preferably Europe/UK based) that make suitable systems for: 1. Making the gradients in the first place 2. Fractionating the gradients after they have been in the centrifuge. With a great many thanks, Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512
Re: [ccp4bb] Anaerobic glovebox crystal cryo-cooling
Hi, Sometimes the forming of ice is due to the high humidity in the air. I recommend to put a dehumidifier in the room or silicone gel in your camber. Nian On Tue, Apr 24, 2012 at 9:52 AM, Kelly Daughtry kellydaugh...@gmail.comwrote: Is there room in the LN2 to plunge directly in, then maneuver into puck? That's what I did, with a different setup, but still anaerobic and moving into pucks. I did not run into ice problems. If there is not room within the LN2 bowl, can you use a larger one? I've found any hesitation when heading into LN2 (i.e.increased time spent hovering over LN2 in the gas/semi cool area) can cause ice. Thus I always try to plunge in quickly, then maneuver to the desired storage location (puck, cap). Kelly *** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** On Tue, Apr 24, 2012 at 10:20 AM, David Gallagher dt...@mrc-mbu.cam.ac.uk wrote: Hi all, I've been using a Belle technologies anaerobic glovebox with in built microscope and liquid nitrogen dewar port in an attempt to harvest and cryo-cool crystals in an oxygen free environment. I've been having some problems with icing and think this is most likely due to slow speed that it is possible to plunge loops into the liquid nitrogen. I've been plunging loops straight into vials preloaded into an ESRF puck (with pusher). This requires more care than what I would ordinarily do (i.e. plunging into bulk liquid nitrogen then and manouvering into a vial afterwards and then clipping said vial into a cane) and so takes longer - perhaps leading to icing. The latter technique is not available as the glovebox set up means that the dewar can only be reached by my right hand - the microscope is in the way! Does anyone have any experience of a similar set up and if so have some advice as to how I can overcome this problem? Many thanks, Dave Gallagher -- *David Gallagher * PhD Student MRC Mitochondrial Biology Unit Wellcome Trust / MRC Building Hills Road Cambridge CB2 0XY Email: dt...@mrc-mbu.cam.ac.uk Phone: 01223 252913
Re: [ccp4bb] very informative - Trends in Data Fabrication
I don't model zero occupancy in my model. But can't the refinement programs just treat those atoms with zero occupancy as missing atoms? Nian Huang On Sat, Mar 31, 2012 at 10:26 AM, Bosch, Juergen jubo...@jhsph.edu wrote: really fascinating, bringing back the discussion for a repository for your collected frames. Jürgen *Acta Cryst.* (2012). F*68*, 366-376 doi:10.1107/S1744309112008421http://dx.doi.org/10.1107/S1744309112008421 * * Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergenB. Rupphttp://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Rupp,%20B. *Abstract:* Physically improbable features in the model of the birch pollen structure Bet v 1d (PDB entry 3k78http://pdb.pdb.bnl.gov/pdb-bin/opdbshort?3k78) are faithfully reproduced in electron density generated with the deposited structure factors, but these structure factors themselves exhibit properties that are characteristic of data calculated from a simple model and are inconsistent with the data and error model obtained through experimental measurements. The refinement of the 3k78http://pdb.pdb.bnl.gov/pdb-bin/opdbshort?3k78model against these structure factors leads to an isomorphous structure different from the deposited model with an implausibly small *R* value (0.019). The abnormal refinement is compared with normal refinement of an isomorphous variant structure of Bet v 1l (PDB entry 1fm4http://pdb.pdb.bnl.gov/pdb-bin/opdbshort?1fm4). A variety of analytical tools, including the application of Diederichs plots, *R*[image: [sigma]] plots and bulk-solvent analysis are discussed as promising aids in validation. The examination of the Bet v 1d structure also cautions against the practice of indicating poorly defined protein chain residues through zero occupancies. The recommendation to preserve diffraction images is amplified. .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ sigma_rmgif.gif
Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays
I have seen people only use robot to optimize their crystal and get good diffraction (~2 A). If you keep having trouble, you can try this method instead, even though generally the case is the bigger the drop the bigger crystal. I remember the archive suggest us to use higher concentration of protein to reproduce the result when using bigger volume. Best, Nian On Mon, Mar 26, 2012 at 12:57 PM, Matthew Lalonde mattc...@gmail.com wrote: Dear All, I have searched the archives and would like more information about reproducing robot tray hits using 24-well hand trays. I reproducibly get crystals when I use small volumes (0.5 ul) in 3-well intelliplates but only precipitate in 1-2 ul sitting drops in 24-well hand plates. What parameters should I vary to reproduce crystals in hand plates? References that discuss this procedure exhaustively would also be appreciated. Thanks, Matt
[ccp4bb] OFFTOPIC: Plasmid for fast Baculovirus preparation.
Dear All, Sorry for the non-ccp4 topic. I am trying make my Baculovirus preparation less time consuming and I found this paper: http://www.ncbi.nlm.nih.gov/pubmed/15939308 Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells. But my request of plasmid (GenBank Accession No. DQ003705) to the correspondent author got rejected by the email server. Is there any other way to contact the author or request the plasmid? Thank you very much. Nian Huang, Ph.D. Instructor, MC 8816 UT Southwestern Medical Center Dallas, TX 75390
Re: [ccp4bb] Crystal Structures as Snapshots
Is it possible the solution structure of SAXS, NMR and EM neglect the existence of a very small percentage conformation of the molecule due to the overwhelming signals from the majority conformations? But this state of the molecule is trapped and enriched by the crystallization condition. Nian On Sat, Feb 11, 2012 at 1:10 PM, Poul Nissen p...@mb.au.dk wrote: Another good lesson here: 2. The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure.http://www.ncbi.nlm.nih.gov/pubmed/16364917 *Vestergaard B*, Sanyal S, Roessle M, Mora L, Buckingham RH, Kastrup JS, Gajhede M, Svergun DI, Ehrenberg M. Mol Cell. 2005 Dec 22;20(6):929-38. On 11/02/2012, at 18.18, Joel Sussman wrote: 2012_02_11 Dear All, Two really striking examples of Intrinsically Flexible Proteins are: (1) *Adenylate kinase*: Vonrhein, Schlauderer Schulz (1995) *Structure* *3*, 483 “Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases” *http://portal.uni-freiburg.de/structbio/structuregallery/ak_folder/mpeg* in particular look at: *video as MPEG white background, closing opening enzyme (707kb*) Each black dot [upper left, in the morph] indicates an observed crystal structure. (2) *Lac repressor*: see *Proteopedia* page on lac repressor, morphing from the structure bound to its cognate DNA, to that of the structure bound to its the non-cognate DNA, at:* http://proteopedia.org/w/Lac_repressor* best regards, Joel * * On 10 Feb 2012, at 22:51, Jacob Keller wrote: Interesting to juxtapose these two responses: James Stroud: How could they not be snapshots of conformations adopted in solution? David Schuller: How could that possibly be the case when any structure is an average of all the unit cells of the crystal over the timespan of the diffraction experiment? JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Extra positive density seen after TLS refinement?
MolProbability score doesn't mean too much in your case, since you are essentially using a 1.5 A model against a 3 A database. The differences in the blobs might caused by the different delta sigma settings when you were viewing these two models. I have successfully used TLS for a 3 A dataset before. The blobs mean the discrepancies between you model and your data, no matter you are using TLS or not. If TLS doesn't give you better statics and map density, I would leave it out or change the TLS setting. I wouldn't put any molecule in the densities you provided, as the model looks already congested enough from the angle I see. I might be wrong w/o the information of map setting. Best, Nian On Sat, Feb 18, 2012 at 7:36 PM, Naveed A Nadvi nnad2...@uni.sydney.edu.auwrote: Dear crystallographers, I am fairly new in crystallographic work and structure determination, but I thought this would be the best place to post my questions. We had collected structural data for a protein that diffracted to 3 A. We had used a previously deposited structure (1.5 A) for molecular replacement. Our final structure used NCS restraints refinement over 4 chains within the assymetric unit. We were able to assign some water moleules using COOT and subsequently removed 'bad waters' manually. We used automated settings when dealing with these water molecules. In all cases these water molecules were found in the same positions as the initial structure (1.5 A) that we had used as a search model. This gave us confidence in the placement of our water molecules. Finally we had run validation tools (MolProbity) and our structure was found to be with Molprobity score within the 100th percentile. We then performed a TLS refinement (from TLSMD) to further improve R values. We used the final MolProbity-validated structure using 8 TLS groups and using PureTLS with constant B factor (50). We are observing large positive densities from the subsequent REFMAC5 refinement that are otherwise not observed in the absence of TLS refinement. My questions are: 1) Is TLS suitable for our dataset (3 A)? 2) Is TLS refinement independent of NCS refinement or should I define my NCS based on the 8 TLS groups? 3) Is it normal to see extra positive density after TLS refinement and what does it mean? 4) We had PEG4000 and Tris in our crystallization buffer. Could these 'blobs' represent these molecules or short water chains? I have attached images of the largest blob. Any comments and suggestions would be highly appreciated. Kind regards, Naveed A Nadvi Faculty of Pharmacy, University of Sydney, Australia
Re: [ccp4bb] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]
Just a reminder. Quickchange is not PCR. It is linear amplification. It is very hard to see a band in the gel if you follow the standard protocol. Nian On Fri, Feb 3, 2012 at 12:14 PM, Fred ccp4bb.l...@gmail.com wrote: Hi CCP4 list, Thanks everyone who have answered my post concerning to mutagenesis. From quick reading most of the answers, the following seems to be a consensus: 1) Do not concentrate your PCR product; 2) Too much DNA and/or impurities like salts or whatever can inhibits transformation; 3) Purify your PCR product before transformation if possible or use 3 of 4 microL of it. This is more or less the amount of DNA showed in the uploaded image. Kind regards, Fred P.S.: I'll let you know the results.
Re: [ccp4bb] [off topic] Control of crystals' direction and position in the drop.
I tried the bookend drop method. It didn't work for me but hopefully it will benefit other people. There is only one thing I need to add to the detailed protocol from Martyn. I had trouble not cutting through the plastic slides. Even I could do it, it was impossible to peel a layer up. So in the end, it became much easier for me by just cutting a cross in the middle of the coverslip, raising up one corner of the cross, and sealing it with a clear tape. Hopefully it can help. Nian On Tue, Nov 15, 2011 at 3:32 PM, MARTYN SYMMONS martainn_oshioma...@btinternet.com wrote: Attached is a picture of making the bookend setup - step b. shows the razor levering up the flap from the plastic coverslip - be careful not to cut right through (I have done that and the drop dries extra quick in such a case) also be careful to apply the protein/well droplet as shown with to the shiny side of the lifted flap. (Apologies for the attachment). hope your crystals go well. Martyn -- *From:* Nian Huang huangn...@gmail.com *To:* CCP4BB@JISCMAIL.AC.UK *Sent:* Tuesday, 15 November 2011, 17:44 *Subject:* Re: [ccp4bb] [off topic] Control of crystals' direction and position in the drop. Thank you guys. I basically tried almost everything that I can find in the hampton catalogue and in this bulletin, seeding, hanging, sitting, sandwitch drops (which made things worse), temperature, gel, oil batch, and additive screens. Manipulating the crystal with fiber increases the chance to make it grow twin. The book end coverslip sounds a fantasic idea. It might be just going to work. Best, Nian
Re: [ccp4bb] [off topic] Control of crystals' direction and position in the drop.
Thank you guys. I basically tried almost everything that I can find in the hampton catalogue and in this bulletin, seeding, hanging, sitting, sandwitch drops (which made things worse), temperature, gel, oil batch, and additive screens. Manipulating the crystal with fiber increases the chance to make it grow twin. The book end coverslip sounds a fantasic idea. It might be just going to work. Best, Nian
Re: [ccp4bb] data processing problem with ice rings
Hi, I agree with other people. You must have a wrong index here. Can you tell us what is the unit cell for this crystal from your determination? I can see very close spots in the high resolution shell from your image, which are overlapped into one spot in the low resolution shell. Try to use other frames to do the indexing. If it is still not working, it might be easier to collect another dataset with better crystal alignment. Best, Nian On Fri, Oct 14, 2011 at 12:12 AM, ChenTiantian chentiantian2...@gmail.comwrote: Hi there, I am processing a dataset which has bad ice rings (as you can see in the attach png file). I tried both XDS and imosflm, and got similar results, it seems that adding EXCLUDE_RESOLUTION_RANGE cannot get rid of the effects of the ice rings. the following is part of the CORRECT.LP which is the second attached file, you can find more details there. SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas Rmrgd-F Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 4.24 371525537 5545 99.9% 46.9% 52.7% 371502.4850.8%19.4% -28% 0.5135136 3.01 553449002 9840 91.5% 62.7% 65.1% 551161.7668.3%48.1% -28% 0.5207760 2.46 84636 12699 12703 100.0% 67.4% 84.7% 846341.5573.0%54.2% -19% 0.513 12104 2.13 97910 14743 14987 98.4% 254.5%199.3% 979080.16 276.2% 4899.9% -23% 0.473 14037 1.90 110260 16846 16940 99.4% 299.2%303.3% 1102450.06 325.0% -99.9% -17% 0.422 15995 1.74 118354 18629 18744 99.4%1062.0% 1043.6% 118317 -0.20 1156.4% -99.9% -13% 0.380 17414 1.61 122958 20193 20331 99.3% 967.5% 1571.1% 1228680.10 1059.7% 987.3%-2% 0.402 18348 1.51 125075 21554 21794 98.9% 838.9% 1355.1% 1249330.08 922.6% 1116.9%-1% 0.402 18977 1.42 72057 17042 23233 73.4% 640.8% 775.3%703910.08 732.5% 826.7%-8% 0.425 10003 total 823746 136245144117 94.5% 166.4%166.7% 8215620.40 181.1% 296.7% -15% 0.435 119774 Note that I/SIGMA of each resolution shell is 2.5, so how should I do to process the dataset properly? Any suggestion about this super ice rings? Thanks! Tiantian -- Shanghai Institute of Materia Medica, Chinese Academy of Sciences Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park, Shanghai, 201203
Re: [ccp4bb] Windows 7 and Xtal Software
A dual boot laptop is all you need. I always reinstall the windows to get rid of bloatware anyway. If you are going to buy a mac, you can also try the triple boot, but I don't think anybody is doing it. Although it is very convenient, a virtual machine will affect the performance of the software. Nowadays booting machine is very fast using a good SSD (under 7 second). So it is really not a big trouble comparing before. I am using a sub $400 laptop, and everything runs really well under Ubuntu including model building software. Nian On Sun, Aug 28, 2011 at 9:23 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, are there any additional problems or known issues running ccp4 or other xtal software on windows 7 (beyond those of Vista, etc.?) Your input would be really appreciate before I sink my own personal $$$ into a new laptop Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Windows 7 and Xtal Software
There are some windows only software, for example 3ds Max. But you have a point, you probably don't need linux system in this case. Nian On Tue, Aug 30, 2011 at 1:55 AM, Antony Oliver antony.oli...@sussex.ac.ukwrote: Erm, somewhat confused — if you are going to buy a Mac — why would you need (or want!) a triple boot system? It all seems to work just fine on OS X. Tony. On 30 Aug 2011, at 07:38, Nian Huang wrote: A dual boot laptop is all you need. I always reinstall the windows to get rid of bloatware anyway. If you are going to buy a mac, you can also try the triple boot, but I don't think anybody is doing it. Although it is very convenient, a virtual machine will affect the performance of the software. Nowadays booting machine is very fast using a good SSD (under 7 second). So it is really not a big trouble comparing before. I am using a sub $400 laptop, and everything runs really well under Ubuntu including model building software. Nian On Sun, Aug 28, 2011 at 9:23 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, are there any additional problems or known issues running ccp4 or other xtal software on windows 7 (beyond those of Vista, etc.?) Your input would be really appreciate before I sink my own personal $$$ into a new laptop Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Protein elution in Size Exclusion
Thanks, David. I believe the leaked column was probably my case, although I didn't see any visible leakage I re-tightened connections anyway. But the weird thing was that after I equilibrized the column for a while, the dry patches and cracks disappeared. Everything returned to normal even a standard ran fine. That's why I suspected the mixing of reagents in the column has something to do with it in the beginning. Nian Huang, Ph.D. UT Southwestern Medical Center On Sun, Aug 28, 2011 at 5:14 PM, David Briggs drdavidcbri...@gmail.com wrote: Hi Nian, It can be a number of things - but typically, air getting into the column, or a leaky seal somewhere letting it dry out. Switching from 150mM NaCl to 20% EtOH directly shouldn't cause a problem. The reason I suggest it is that I have had the same situation as described in the original post (protein comes off across entire volume of SEC column elution) and it turned out the column had a leak and had dried out in places. Repacking the column solved the problem. Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 28 August 2011 22:35, Nian Huang huangn...@gmail.com wrote: Hi, David, What is the common cause of knackered SEC column? Will equilibrizing a buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause the problem. There was no problem just after packing the column. Nian On Sun, Aug 28, 2011 at 9:24 AM, David Briggs drdavidcbri...@gmail.com wrote: Following on from Roger's fine suggestions: 8. Your column is knackered. Can you see fine lines or cracks in the column? Good packing is v.important for SEC columns. HTH, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 28 August 2011 10:25, Allan Pang a.p...@qmul.ac.uk wrote: Hi there everyone, What does it mean when you have proteins eluting in almost the whole column volume of S200? I ran a gel with fractions from 8ml to 20ml and saw band for my protein all throughout. Judging peaks on chromatogram is not useful as it doesn't have any aromatic rings. Cheers, Allan -- Allan Pang PhD Student G35 Joseph Priestley Building Queen Mary University of London London E1 4NS Phone number: 02078828480
Re: [ccp4bb] Protein elution in Size Exclusion
Hi, David, What is the common cause of knackered SEC column? Will equilibrizing a buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause the problem. There was no problem just after packing the column. Nian On Sun, Aug 28, 2011 at 9:24 AM, David Briggs drdavidcbri...@gmail.comwrote: Following on from Roger's fine suggestions: 8. Your column is knackered. Can you see fine lines or cracks in the column? Good packing is v.important for SEC columns. HTH, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 28 August 2011 10:25, Allan Pang a.p...@qmul.ac.uk wrote: Hi there everyone, What does it mean when you have proteins eluting in almost the whole column volume of S200? I ran a gel with fractions from 8ml to 20ml and saw band for my protein all throughout. Judging peaks on chromatogram is not useful as it doesn't have any aromatic rings. Cheers, Allan -- Allan Pang PhD Student G35 Joseph Priestley Building Queen Mary University of London London E1 4NS Phone number: 02078828480
Re: [ccp4bb] Another paper structure retracted
To make my idea a little bit clearer, the reviewers first make the acceptance decision just based on the paper itself, on the condition the coordinate matches the description of the paper. Then the editor promises the publication date and the pdb can be subjected to final quick review, either by the reviewers or a special team from the journal. Nian 2011/8/10 Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com the editor should agree to publish the paper swiftly in advance before the data become accessible to reviewers. This seems to miss the point - how is the reviewer then supposed to judge the map? BR ** ** Nian On Wed, Aug 10, 2011 at 5:25 PM, Filip Van Petegem filip.vanpete...@gmail.com wrote: Just another example of where it would have been good for the reviewers to get access to the data during the review process... and where at least one of the reviewers *should* be a protein crystallographer... ** ** Filip Van Petegem On Wed, Aug 10, 2011 at 2:01 PM, David Schuller dj...@cornell.edu wrote: Time to fuel up the gossip engines for the approaching weekend: http://www.sciencedirect.com/science/article/pii/S096921260800186X RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer Gβ1γ2 Structure, Volume 16, Issue 7http://www.sciencedirect.com/science?_ob=PublicationURL_tockey=%23TOC%236269%232008%23999839992%23693753%23FLA%23_cdi=6269_pubType=Jview=c_auth=y_acct=C22719_version=1_urlVersion=0_userid=492137md5=9dc4b8953d3fa243dc98e395b6ac590d, 9 July 2008, Pages 1086-1094 Structure 2QNS withdrawn. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ ** **
Re: [ccp4bb] Another paper structure retracted
I Agree with the idea of adding crystallographer reviewers. But accessing to data is not feasible unless there is a good way to protect authors. For example, the editor should agree to publish the paper swiftly in advance before the data become accessible to reviewers. In any case, the flaw of this structure is very clear in the table. Nian On Wed, Aug 10, 2011 at 5:25 PM, Filip Van Petegem filip.vanpete...@gmail.com wrote: Just another example of where it would have been good for the reviewers to get access to the data during the review process... and where at least one of the reviewers *should* be a protein crystallographer... Filip Van Petegem On Wed, Aug 10, 2011 at 2:01 PM, David Schuller dj...@cornell.edu wrote: Time to fuel up the gossip engines for the approaching weekend: http://www.sciencedirect.com/science/article/pii/S096921260800186X RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer Gβ1γ2 Structure, Volume 16, Issue 7http://www.sciencedirect.com/science?_ob=PublicationURL_tockey=%23TOC%236269%232008%23999839992%23693753%23FLA%23_cdi=6269_pubType=Jview=c_auth=y_acct=C22719_version=1_urlVersion=0_userid=492137md5=9dc4b8953d3fa243dc98e395b6ac590d, 9 July 2008, Pages 1086-1094 Structure 2QNS withdrawn. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Protein truncation problem in crystal.
The R and Rfree are low enough so your space group is correct. I don't see a resolution in the log file. If it is 3 A dataset, 28% is not bad for a start at all. The N terminal region is probably completely disordered if it is not degraded. Nian On Sat, Jul 16, 2011 at 3:47 AM, Appu kumar appu.kum...@gmail.com wrote: Dear CCP4 User, I am new to crystallography and i need your valuable suggestion to help me out. We have crystallized full length protein and I am solving protein structure by molecular replacement method and molecular weight of protein is 37kDa. Template pdb i am using have only C-terminal 24kDa protein structure. HKL2000 gave a space group C2221 and Mathews Coefficient indicate one molecule of 37 kDa with solvent content 17% only. When i am running phaser, it gave me a most probable molecualr weight of protein in ASU to be 23kDa. and also i am not getting any density for N-terminal of protein. Right now my Rwrk and Rfree are 28% and 37% but it not decreasing further. When i am indexing in lower symetry sapce group P1211, most probable molecular weight ofpreotin com comes to be 46kDa in ASU. Would please anyone can help, and suggest me what is the real situation?. Thanks in advance Mathews table Data line--- CELL 57.8080 70.0520 105.7080 90. 90. 90. Data line--- SYMM 'C 2 2 21' Cell volume:428071.531 For estimated molecular weight 36000. Nmol/asym Matthews Coeff %solvent P(2.30) P(tot) _ 1 1.4917.30 1.00 1.00 _ Space-Group Name: C 2 2 21 Space-Group Number: 20 Unit Cell: 57.81 70.05 105.71 90.00 90.00 90.00 MW of average protein to which Matthews applies: 36000 Resolution for Matthews calculation: 2.30 Z MW VM% solvent rel. freq. 1 36000 1.49 17.25 0.002 == most probable Z is the number of multiples of the total composition In most cases the most probable Z value should be 1 If it is not 1, you may need to consider other compositions Histogram of relative frequencies of VM values -- Frequency of most common VM value normalized to 1 VM values plotted in increments of 1/VM (0.02) --- relative frequency --- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ||||||||||| 10.00 - 8.33 - 7.14 - 6.25 - 5.56 - 5.00 - 4.55 - 4.17 -- 3.85 --- 3.57 - 3.33 3.12 - 2.94 --- 2.78 --- 2.63 2.50 2.38 - 2.27 -- 2.17 - 2.08 --- 2.00 --- 1.92 - 1.85 -- 1.79 -- 1.72 - 1.67 - 1.61 - 1.56 - 1.52 - 1.47 * (COMPOSITION*1) 1.43 - 1.39 - 1.35 - 1.32 - 1.28 - 1.25 - Most probable VM for resolution = 2.30957 Most probable MW of protein in asu for resolution = 23168.4 Thanks in advance Appu Kumar singh.
Re: [ccp4bb] crystallization of a weird protein
It might be SDS precipitation. Although 0.1% SDS is generally considered not high enough to precipitate at 4 degree, it might interact with other components in your solution to form even less soluble material. Nian On Fri, May 20, 2011 at 9:36 AM, WEI MIN butiany...@gmail.com wrote: Dear All I have a difficulty to crystallize my membrane protein(his tagged). I got the salt crystals from many different screening conditions. The protein is in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree although it forms the milk solution. The solution is getting clear in the room temperature within 5 mins when I set up the trays. I would like to take any advice. I would appreciate your input. Best Min
Re: [ccp4bb] Stereo solution with Nvidia '3D vision' or '3D vision pro'
Check pymolwiki for the full description of setup. It should work for coot too. http://www.pymolwiki.org/index.php/Stereo_3D_Display_Options Nian On Fri, May 6, 2011 at 10:27 AM, zhang yu ccp4f...@gmail.com wrote: Dear colleagues, Sorry to present the stereo issue to the board again. Since my old SGI CRT monitor only has 75 HZ refresh rate, the flickering in stereo mode bothered me a lot. Recently, I want to update my old CRT to 120 HZ LCD. I have a Nvidia Quadro FX3800 in my workstation. I would like to make sure some issues before I make the upgrade. 1. Can I apply the previous stereo emitter (Purchased from Real D, Model #E-2) to 120HZ LCD? Although the company told me this emitter is not compatible with LCD, could some one tell me why? Is it true that the Nvidia 3D vision is the only solution for the stereo in LCD? 2. Nvidia supply two kinds of 3D emitters. One of them is 3D vision, while the other one is 3D vision pro. Which one is sufficient for crystallographier user? (3D vision pro is much more expensive than 3D vision) It seems that 3D vision is for home user and powered by the Nvidia GeForce series graphic cards. While 3D vision pro is for professional user and powered by Nvidia Quardro series graphic card . 3. It looks that the Nvidia 3D glasses are very compact. Is it comfortable for someone like me already with eyeglasses? Thanks Yu -- Yu Zhang HHMI associate Waksman Institute, Rutgers University 190 Frelinghuysen Rd. Piscataway, NJ, 08904
Re: [ccp4bb] OT: Covalent modification of Cys by reducing agents?
Thank you for such great explanation. Hopefully people won't consider me hijacking this thread. Nian On Sun, Apr 17, 2011 at 11:08 AM, Artem Evdokimov artem.evdoki...@gmail.com wrote: TCEP relies on a completely different chemistry to achieve the same goal as BME or DTT. DTT/BME use S(-)SS with redox potentials of -0.26 to -0.33 V (at pH 7) whereasTCEP uses P(3+)-P(5+) oxidation with redox potential that's a lot higher (I don't know of a reference with a stated redox potential for this system) because TCEP readily reduces oxidized forms of both DTT and BME in solution. TCEP also does not readly break S-Hg bonds unlike DTT or BME. TCEP does not readily react with oxygen in solution (both DTT and BME react rapidly) and has long shelf life as buffered 1M solution at pH 5.6-6.3. TCEP has two non-trivial disadvantages that are for the most part not relevant for the purposes of crystallography: one is that it does not work very well in high Phosphate concentration, and the other is that it hinders the reaction of thiols with haloacetamides and suchlike (because it itself can react with haloacetamides). If you're labeling with haloacetamides you may want to use tri- tertbutul phosphine instead (it's much less soluble than TCEP, but 1 mM solution in water can be made). TCEP does not permeate biological membranes and therefore has been used to reduce thiols outside the cell while keeping intracellular ones intact. Due to its size and charge, it also is quite selective which protein disulphides are readily reduced - ones on or near protein surface are reduced quickly whereas buried or shielded ones are often not reduced at all w/o the use of a chaotrope. That's rather useful to us as it often allows us to reduce the unwanted inter-molecular disulphides (bad: aggregates) while at the same time preserve the valuable intra-molecular ones (good: structure) Artem On Sat, Apr 16, 2011 at 11:46 PM, Nian Huang huangn...@gmail.com wrote: Dear Horacio, How does TECEP compare to BME or DTT? People claim it is better, but I want some crystallographers' opinion? Nian On Sat, Apr 16, 2011 at 4:24 PM, Horacio Botti hbo...@pasteur.edu.uywrote: Dear Mike BME readily autooxidizes (need for metal traces and dissolved O2). Is yours a metalloprotein? Is your buffer contaminated with metals? Those situations would make the case a bit different. If not, unless your BME stock is already oxidized, blocking of the accesible thiols with BME should take some time. If you treat your protein for 40 min with fresh BME you should not observe thiol blocking. If you let the preparation to stay for several days, even at 4-6 °C you may observe the blocking that you may be observing. If you want to prevent Cys blocking you can also change to DTT (it is a dithiol, does not readily form mixed disulfides) and use it with caution (for thiol reduction it is advisable to use stoichiometric DTT (with respect to the number of Cys you need to reduce) and 10 fold excess of BME, look for their redox potentials). Take care of not over-reducing your protein if internal disulfide bonds are expected. Once reduced I suggest you to remove any reducing agent and store the protein at -80 °C. External Cys can be easily oxidized, they are highly expossed to metals and oxidants (H2O2, BME disulfides, etc). Diffusion is for sure much faster than SS bond formation, although some cys react at almost diffusion-controlled rates with oxidants (is yours a thiol'dependen t peroxidase?) You can take a look at the following reference (advertising): 2011. Factors Affecting Protein Thiol Reactivity and Specificity in Peroxide Reduction. Chem Res Toxicol. Metals can contaminate bad quality materials (water, salts, buffers, etc), take care of that too. If you need to control the redox state of your protein you should use DTNB (Ellman´s reagent), or DTDPy, to measure accesible reduced thiol groups. Good luck! Horacio Quoting Kendall Nettles knett...@scripps.edu: We see BME adducts in all of our estrogen receptor structures, though we don't always put them in the models. Sometimes we only see one or two atoms of the adduct, and in others it is completely ordered. We only see it on the solvent accessible cysteines. We do it on purpose. We used to treat the protein with iodoacetic acid to generate uniform modification of the cysteines, but then we realized we could get then same homogeneity with 20-50mM BME. Kendall Nettles On Apr 15, 2011, at 4:09 PM, Michael Thompson mi...@chem.ucla.edu wrote: Hi All, I was wondering if anyone knew whether or not it is possible for reducing agents with thiol groups, such as DTT or beta-mercaptoethanol (BME), to form covalent S-S bonds with Cys residues, particularly solvent-exposed Cys? I have some puzzling biochemical results, and in the absence of a structure (thus far), I was wondering if this might be something to try to control
Re: [ccp4bb] OT: Covalent modification of Cys by reducing agents?
Dear Horacio, How does TECEP compare to BME or DTT? People claim it is better, but I want some crystallographers' opinion? Nian On Sat, Apr 16, 2011 at 4:24 PM, Horacio Botti hbo...@pasteur.edu.uywrote: Dear Mike BME readily autooxidizes (need for metal traces and dissolved O2). Is yours a metalloprotein? Is your buffer contaminated with metals? Those situations would make the case a bit different. If not, unless your BME stock is already oxidized, blocking of the accesible thiols with BME should take some time. If you treat your protein for 40 min with fresh BME you should not observe thiol blocking. If you let the preparation to stay for several days, even at 4-6 °C you may observe the blocking that you may be observing. If you want to prevent Cys blocking you can also change to DTT (it is a dithiol, does not readily form mixed disulfides) and use it with caution (for thiol reduction it is advisable to use stoichiometric DTT (with respect to the number of Cys you need to reduce) and 10 fold excess of BME, look for their redox potentials). Take care of not over-reducing your protein if internal disulfide bonds are expected. Once reduced I suggest you to remove any reducing agent and store the protein at -80 °C. External Cys can be easily oxidized, they are highly expossed to metals and oxidants (H2O2, BME disulfides, etc). Diffusion is for sure much faster than SS bond formation, although some cys react at almost diffusion-controlled rates with oxidants (is yours a thiol'dependen t peroxidase?) You can take a look at the following reference (advertising): 2011. Factors Affecting Protein Thiol Reactivity and Specificity in Peroxide Reduction. Chem Res Toxicol. Metals can contaminate bad quality materials (water, salts, buffers, etc), take care of that too. If you need to control the redox state of your protein you should use DTNB (Ellman´s reagent), or DTDPy, to measure accesible reduced thiol groups. Good luck! Horacio Quoting Kendall Nettles knett...@scripps.edu: We see BME adducts in all of our estrogen receptor structures, though we don't always put them in the models. Sometimes we only see one or two atoms of the adduct, and in others it is completely ordered. We only see it on the solvent accessible cysteines. We do it on purpose. We used to treat the protein with iodoacetic acid to generate uniform modification of the cysteines, but then we realized we could get then same homogeneity with 20-50mM BME. Kendall Nettles On Apr 15, 2011, at 4:09 PM, Michael Thompson mi...@chem.ucla.edu wrote: Hi All, I was wondering if anyone knew whether or not it is possible for reducing agents with thiol groups, such as DTT or beta-mercaptoethanol (BME), to form covalent S-S bonds with Cys residues, particularly solvent-exposed Cys? I have some puzzling biochemical results, and in the absence of a structure (thus far), I was wondering if this might be something to try to control for. I have never heard of this happening (or seen a structure where there was density for this type of adduct), but I can't really think of a good reason for why this wouldn't happen. Especially for something like BME, where the molecule is very much like the Cys sidechain and seems to me like it should have similar reactivity. The only thing I can think of is if there is a kinetic effect taking place. Perhaps the rate of diffusion of these small molecules is much faster that the formation of the S-S bond? Does anyone know whether or not this is possible, and why it does or does not happen? Thanks, Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] immobilized DNA resin
Heparin simulates the structure of DNA and RNA, so it has nonspecific affinity towards DNA or RNA binding protein. It has also been used as DNAse or RNase inhibitor but it is not very good one. Nian Huang, Ph.D. UT Southwestern Medical Center On Sat, Apr 9, 2011 at 7:44 PM, Alexandra Deaconescu deac...@brandeis.eduwrote: Hello ccp4 enthusiasts: I am afraid this is a non-ccp4 related question. Can anyone recommend an immobilized dsDNA chromatographic resin for purification of DNA-binding proteins? GE seems to have something - I was wondering if people have other recommendations? In the age of GST and His tags etc., these are not very much used, but I do not have a tag in this case... Thanks a lot, Alex
Re: [ccp4bb] Question about GST cleavage
I once cleaved a GST tag on the resin using TEV by rocking overnight at 4 C. I would say it is 100% cutting judging from the gel. One thing to add is that the protein bound so tight to the beads that cutting tag is the only way to elute it except by SDS. I haven't had any trouble with TEV and thrombin with rocking or shaking. The important part is that you have to have good protease to start with. Nian Huang, Ph.D. UT Southwestern Medical Center On Thu, Mar 31, 2011 at 2:41 PM, gauri misra kamga...@gmail.com wrote: Just an offshoot of the same Question.. I would like to ask whether the same applies for GST-tag digestion using thrombin.. No agitation gives better results in the above case too... Any personal experiences On Thu, Mar 31, 2011 at 11:29 AM, Klaus Piontek klaus.pion...@ocbc.uni-freiburg.de wrote: And not at full moon! Klaus Am 31.03.2011 um 16:23 schrieb Xiaopeng Hu: Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] S-200 buffer-based peak shift?
Superdex 200 instruction manual suggests a minimal 150mM NaCl is required to prevent binding of protein to the resin. But it seems more to the side of preventing loss of protein instead of misjudging protein size. Nian Huang, Ph.D. UT Southwestern Medical Center On Tue, Mar 22, 2011 at 7:23 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] linux flavors
I vouch for Ubuntu too. FC always has the stability problem for me and I am too tired to find right drivers and compile the programs. I switched after FC8. Ubuntu's repositories seems to be much more reliable and it worked with every program that I am using, including 3D imaging (I am using Nvidia shuttle glass though). You can easily retrieve the Nvidia driver from the repository if Ubuntu hasn't already done it for you. But the bad side is that you are going lose your computation skill since you have less problem to handle. Nian Huang, Ph.D. UT Southwestern Medical Center On Tue, Feb 22, 2011 at 9:16 AM, David Roberts drobe...@depauw.edu wrote: Hello all, Quick question on linux varieties. For years (and years) I have used fedora (after Ultrix of course). In fact, most of my computers are running FC7 (that long ago), it's very stable and works fine. However, since it is no longer supported, I'm toying with upgrading. I upgraded one machine to FC13. However, this nouveau driver thing is killing me, and getting my nvidia drivers installed is hopeless (I have followed every thread on this and I simply give up - it's not worth it). With a Zalman monitor it doesn't matter - nouveau works fine and my stereo is good - so I don't really care (or do I). The question is this - what flavors of linux out there are simplest to install - work instantly with various hardwares, and run stereo seamlessly (either Zalman stereo or hardware stereo with an emitter). For zalman anything works - which is why I'm going that way - but I still need hardware stereo on a few machines. So, for hardware, I need my nvidia drivers to install easily. I'm downloading ubuntu - is that a good choice? Can I run different flavors of linux with nfs and share drives in a local network (so one has fc7, one has fc13, and another has ubuntu)? Thanks Dave
Re: [ccp4bb] linux flavors
I'm downloading ubuntu - is that a good choice? Can I run different flavors of linux with nfs and share drives in a local network (so one has fc7, one has fc13, and another has ubuntu)? Replied too fast and didn't finish your message. I have Ubuntu and most of other machines in NFS are running Redhat. So it shouldn't be a problem. Nian On Tue, Feb 22, 2011 at 9:16 AM, David Roberts drobe...@depauw.edu wrote: Hello all, Quick question on linux varieties. For years (and years) I have used fedora (after Ultrix of course). In fact, most of my computers are running FC7 (that long ago), it's very stable and works fine. However, since it is no longer supported, I'm toying with upgrading. I upgraded one machine to FC13. However, this nouveau driver thing is killing me, and getting my nvidia drivers installed is hopeless (I have followed every thread on this and I simply give up - it's not worth it). With a Zalman monitor it doesn't matter - nouveau works fine and my stereo is good - so I don't really care (or do I). The question is this - what flavors of linux out there are simplest to install - work instantly with various hardwares, and run stereo seamlessly (either Zalman stereo or hardware stereo with an emitter). For zalman anything works - which is why I'm going that way - but I still need hardware stereo on a few machines. So, for hardware, I need my nvidia drivers to install easily. I'm downloading ubuntu - is that a good choice? Can I run different flavors of linux with nfs and share drives in a local network (so one has fc7, one has fc13, and another has ubuntu)? Thanks Dave
Re: [ccp4bb] Freezing crystals in a contained system
Mitegen plastic capillaries can actually last about a week from a Mitegen seminar I attended. I also got ice when I tried to freeze it once and I didn't further pursue it. Maybe coat the capillary with glycerol will help? Nian Huang, Ph.D. UT Southwestern Medical Center On Thu, Feb 17, 2011 at 11:03 AM, R Conners, Biochemistry r.conn...@bristol.ac.uk wrote: Dear all, We are working on a Category 3 protein which must be contained so we have our crystals mounted in a loop and then covered with a plastic Mitegen cover which is glued in place. We're currently collecting at room temperature, but wondered if anyone has any experience of using a contained system at low temperatures? Any attempts I've had so far at freezing through either the plastic or a glass capillary have resulted in formation of ice on the surface so it is not even possible to see the crystal to centre it. Best wishes, Becky - Dr Becky Conners School of Biochemistry University of Bristol, UK http://www.bris.ac.uk/biochemistry/brady r.conn...@bristol.ac.uk 0117 3312149
Re: [ccp4bb] Anyway to change the toolbar position in coot?
That's great. The 3D screen we got is only 23 inch. A full screen GUI like in the game will certainly be helpful. Nian Huang, Ph.D. Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 On Fri, Dec 31, 2010 at 5:00 PM, Nian Huang huangn...@gmail.com wrote: Dear all, The python version coot has two nicely made toolbars. I can change the position or get rid of one of them by going to the preferences. In the current wide screen dominant world, the other toolbar takes too much precious horizontal space. Is there a way that I can make it vertical? Thanks. Nian Huang, Ph.D. Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390
[ccp4bb] Anyway to change the toolbar position in coot?
Dear all, The python version coot has two nicely made toolbars. I can change the position or get rid of one of them by going to the preferences. In the current wide screen dominant world, the other toolbar takes too much precious horizontal space. Is there a way that I can make it vertical? Thanks. Nian Huang, Ph.D. Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390
Re: [ccp4bb] salt or protein crystals?
Definitely small molecule crystals. You might want to push the detector closer and use better cryo solution for further confirmation. Nian On Tue, Dec 7, 2010 at 8:14 AM, xiuwen zhang congru...@gmail.com wrote: Dear Colleagues, Currently we got several very tiny crystals. After exposuring a cluster of crystals one hour in home source, we could find some weak diffraction spots. As the spots are too few for indexing, I am not quite sure whether these tiny crystals are salt crystals or protein crystals. I appreciate your experience on similar case. Attached files are two diffraction images at 0 and 90 degree. Thank you very much for your kind help. Cheers, Xiuwen
Re: [ccp4bb] brute force MR
Try this, assuming you have good data. Use one molecule you got to do refinement (rigid body or restraint refinement with tight restraint) and phase the map. Then do a phased map molecular replacement. You might want only use the core of your protein to do the molecular replacement search to avoid the clashing problem. It worked once for me even the map was very bad. Nian On Tue, Dec 7, 2010 at 3:38 PM, Arnon Lavie la...@uic.edu wrote: Hi there: The situation: We are facing difficult molecular replacement: we believe we have two molecules in the ASU, but phaser/molrep find only one. Using the electron density calculated using this single molecule, we have manually placed the 2nd molecule, albeit not good enough for rigid body refinement. Our strategy: We are looking for a program to do a 3 dimensional search around the current position of the 2nd molecule. Maybe one that calculates R-factor at the different positions, to allow to identify the correct one. ... Does anyone know of such a program, or an alternative approach? Thanks. 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/ ***
Re: [ccp4bb] Removing a tight binding ligand
Kd is crucial. If this is something like streptavidin and biotin, there is no way to separate them without denature the protein. You can try varying pH, binding it to ion exchange column and then washing it with large volume of buffer, as mentioned in MBP manual from NEB, or running it through a hydrophobic column after treating the protein with really high salt. The last one worked for one of my colleagues. Although I still doubt you can completely get rid of the ligands. Nian Huang, Ph,D. Dept. of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 On Thu, Oct 7, 2010 at 8:05 PM, SIPPEL,KATHERINE H ksip...@ufl.edu wrote: Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
Re: [ccp4bb] phenix target weight refinement
Hi, Salameh, As Pavel said, you might have a ligand with incorrect cif file. A quick fix is to run phenix.elbow on that cif file, which worked for me. I will try the new version of phenix and to see if it can fix the problem. Hopefully you already solved the problem. Nian Huang, Ph.D. UT Southwestern Medical Center On Mon, Mar 15, 2010 at 9:14 AM, Salameh, Mohd A., Ph.D. salameh.m...@mayo.edu wrote: In fact I'm using an older version of phenix 1.26b and never faced a problem, and worked beautifully on twinned data. do you suggest to upgrade it to the latest version? Thanks Huang, M ** Mohd A. Salameh, Ph.D. Mayo Clinic Cancer Center Griffin Cancer Research building,Rm 331 4500 San Pablo Rd Jacksonville, FL 32224 Tel: (904) 953-0046 Fax: (904) 953-0277 salameh.m...@mayo.edu ** -Original Message- From: Nian Huang [mailto:huangn...@gmail.com] Sent: Friday, March 12, 2010 5:49 PM To: Salameh, Mohd A., Ph.D. Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] phenix target weight refinement Which version did you use? I remember the old version of phenix (1.3) will automatically change your setting (wxc and wxu) each round. And v1.5 will automatically choose wxu if you use tls. I haven't tried 1.6 yet. Maybe there are some bugs associated with it? The default wxc is 0.5 and wxu is 1. So the default has tighter weight than your specification. Just use default setting instead. Nian Huang, Ph.D. UT Southwestern Medical Center On Fri, Mar 12, 2010 at 11:59 AM, Salameh, Mohd A., Ph.D. salameh.m...@mayo.edu wrote: Yes Im using wxc_scale and wxu_scale but I also use tls refinement,so in this case if the weights need to be tightly restrained, I should not use tls refinement! ** Mohd A. Salameh, Ph.D. Mayo Clinic Cancer Center Griffin Cancer Research building,Rm 331 4500 San Pablo Rd Jacksonville, FL 32224 Tel: (904) 953-0046 Fax: (904) 953-0277 salameh.m...@mayo.edu ** -Original Message- From: Phil Jeffrey [mailto:pjeff...@princeton.edu] Sent: Friday, March 12, 2010 12:37 PM To: Salameh, Mohd A., Ph.D. Subject: Re: [ccp4bb] phenix target weight refinement Use wxc_scale and wxu_scale not wxc and wxu. If you're doing TLS it will ignore wxu_scale either way. Salameh, Mohd A., Ph.D. wrote: *Dear All,* *In every** refinement** round** the** wxc and wcu** are out of control, although I modify the def file and** change the values** ** of** wxc=1 and wxu=1** so they are tightly restrained**, but it fail**ed** to** fix the problem,** wxc and wxu** values keep fluctuating, in my latest** refinement** round wxc=256!!** wxu=3. The** Stereochemistry looks** very** loosely restrained** and the** gap between R-free and R-work** is** too big**. I**'**ve been using phenix for almost 4 years and** never had this problem,** or once** I** set the values of wxc and wxu that will be it, never change.** I** also tried to fix wxc from the command line and** didn't** work either (phenix.**refine refine.def fix_wxc=1.0). I** will certainly** appreciate** any help**. Thanks, Mohd ** * ** *Mohd A. Salameh, Ph.D.* Mayo Clinic Cancer Center Griffin Cancer Research building,Rm 331 4500 San Pablo Rd Jacksonville, FL 32224 Tel: (904) 953-0046 Fax: (904) 953-0277 salameh.m...@mayo.edu
Re: [ccp4bb] phenix target weight refinement
Which version did you use? I remember the old version of phenix (1.3) will automatically change your setting (wxc and wxu) each round. And v1.5 will automatically choose wxu if you use tls. I haven't tried 1.6 yet. Maybe there are some bugs associated with it? The default wxc is 0.5 and wxu is 1. So the default has tighter weight than your specification. Just use default setting instead. Nian Huang, Ph.D. UT Southwestern Medical Center On Fri, Mar 12, 2010 at 11:59 AM, Salameh, Mohd A., Ph.D. salameh.m...@mayo.edu wrote: Yes Im using wxc_scale and wxu_scale but I also use tls refinement,so in this case if the weights need to be tightly restrained, I should not use tls refinement! ** Mohd A. Salameh, Ph.D. Mayo Clinic Cancer Center Griffin Cancer Research building,Rm 331 4500 San Pablo Rd Jacksonville, FL 32224 Tel: (904) 953-0046 Fax: (904) 953-0277 salameh.m...@mayo.edu ** -Original Message- From: Phil Jeffrey [mailto:pjeff...@princeton.edu] Sent: Friday, March 12, 2010 12:37 PM To: Salameh, Mohd A., Ph.D. Subject: Re: [ccp4bb] phenix target weight refinement Use wxc_scale and wxu_scale not wxc and wxu. If you're doing TLS it will ignore wxu_scale either way. Salameh, Mohd A., Ph.D. wrote: *Dear All,* *In every** refinement** round** the** wxc and wcu** are out of control, although I modify the def file and** change the values** ** of** wxc=1 and wxu=1** so they are tightly restrained**, but it fail**ed** to** fix the problem,** wxc and wxu** values keep fluctuating, in my latest** refinement** round wxc=256!!** wxu=3. The** Stereochemistry looks** very** loosely restrained** and the** gap between R-free and R-work** is** too big**. I**'**ve been using phenix for almost 4 years and** never had this problem,** or once** I** set the values of wxc and wxu that will be it, never change.** I** also tried to fix wxc from the command line and** didn't** work either (phenix.**refine refine.def fix_wxc=1.0). I** will certainly** appreciate** any help**. Thanks, Mohd ** * ** *Mohd A. Salameh, Ph.D.* Mayo Clinic Cancer Center Griffin Cancer Research building,Rm 331 4500 San Pablo Rd Jacksonville, FL 32224 Tel: (904) 953-0046 Fax: (904) 953-0277 salameh.m...@mayo.edu
Re: [ccp4bb] SDS-PAGE of peptides
You can include urea in your gel to resolve ~5 kDa without using gradient gel. I am sorry that I forgot what was the percentage. Please take a look at Gel Electrophoresis of Proteins: A Practical Approach. Or you can try the premade gradient gel from Invitrogen. http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.99253.File.dat/O-063575-NuPage_fin.pdf. The Bis-Tris/Mes gel can resolve 2.5KDa peptide. Regards, Nian Huang, Ph.D. Univ. of Texas Southwestern Medical Center On Thu, Feb 11, 2010 at 1:13 PM, Jacob Keller j-kell...@md.northwestern.edu wrote: Dear Crystallographers, does anybody have any good tricks for getting nice, sharp SDS-PAGE bands of peptides 10kD? Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] multi domain protein
Deleting the loop /unessential regions is a more robust way to go. You will be surprise to see that how much those regions can contribute the clashes. Increasing clash tolerance in Phaser makes the program runs really slow and gives ambiguous results. Best, Nian On Wed, Oct 21, 2009 at 11:41 PM, Vandana Kukshal v_kuks...@cdri.res.in wrote: hello i have 3.25 A data of multidomain protein with 4 individual domain .one domains structure is already known . and for others domain 40 % simmilar structure is known . when i am running phaser with one known domain i am getting the solution but after getting solution i am serching other domains but clashes are coming . how should i proceed . thanx
Re: [ccp4bb] Detection of DNA in protein complex crystals
Hi, Kevin, Wash your crystal very very well to get rid of unbound DNA. Then run your crystal with SDS PAGE gel, with protein and DNA as markers. After staining the gel with silver stain, the DNA will show with different color and migrate about where the dye locates. So don't run too long time. Regards, Nian On Tue, Jun 23, 2009 at 3:38 PM, Kevin Judekj...@berkeley.edu wrote: Note that, despite the claim otherwise in Kettenberger and Cramer, SYBR Gold does stain at least some proteins, so be sure to run the appropriate controls. kmj On Tue, Jun 23, 2009 at 11:52 AM, Allyn Schoeffler asch...@berkeley.edu wrote: Dear Nick, If you have access to a fluorescent microscope, you can try staining crystals with PicoGreen and seeing if they fluoresce. (see ref: Kettenberger H Cramer P, 2006, Acta Cryst D v62 pp146-150: Kettenberger H, Cramer P.Fluorescence detection of nucleic acids and proteins in multi-component crystals.) I've had good luck harvesting dissolving crystals and then running a gel (stain with SYBR-Gold to increase sensitivity). If you can't get enough material to detect a band, you can radioactively kinase the dissolved crystals and then run a gel. Best Regards, Allyn Dear all, I am trying to solve the structure of a transcription factor in complex with its DNA. I got crystals of the complex under different conditions than the protein alone and they also look different. Unfortunately, they only diffract to 6Å so far. Before I continue to optimize the crystals I would like to confirm that the crystals really contain the bound DNA. Thus I tried to crystallize the protein alone under the same conditions as the complex which did not give crystals. I also tried to stain the crystals with methylene blue, but that did not work (but staining with IZIT did not work either). Additionally I dissolved a crystal and measured the absorption. The ratio between A260 and A280 was 1.3. So there seems to be DNA, but less than there should be. Does any of you know a good way to quickly but reliably confirm the presence of DNA in my crystals? Thanks a lot in advance. Nick -- Allyn J. Schoeffler Berger Lab Dept. of Molecular and Cell Biology UC Berkeley phone: (510) 643-9491
Re: [ccp4bb] high number of lysines
Hi, Amit If you mostly got clear drops, you might first increase your protein's concentration before you try anything else. Of course, I assume you have a lot of protein, since you are think about doing lysine methylation. Best, Nian On Wed, Jun 10, 2009 at 6:24 AM, amit sharma3112a...@gmail.com wrote: Dear All, I have a trimeric molecule (~39 kDa, pI=9.1) carrying 15 lysines in each monomer. I have tried several crystallization screens, however, I mostly get clear drops. The only conditions that I tend to get precipitates in, are the ones carrying citrate. I am beginning to methylate the lysines or remove a few of those by mutagenesis. I would be grateful if somebody could share their experience in crystallization of proteins with a high number of lysines. Also, any inputs on strategies to crystallize such molecules would be greatly appreciated. Many thanks in advance. -- Amit Sharma
Re: [ccp4bb] Halide soaking
You always get the entry of Bromide into crystal by quick soaking, because it does not require the incorporation of Bromide into the protein. But whether the signal is good enough for phasing is another story. You have to collect the full data set to know the answer. Nian 2009/3/31 tat cheung cheng theif...@yahoo.com.hk: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供網上安全攻略，教你如何防範黑客! 請前往 http://hk.promo.yahoo.com/security/ 了解更多!
Re: [ccp4bb] Software for Drawing Protein Secondary Structure
You can try TOPS. http://www.tops.leeds.ac.uk/ Best, Nian On Fri, Aug 22, 2008 at 8:57 AM, Buz Barstow [EMAIL PROTECTED] wrote: Dear All, Does anyone know of a program that is capable of drawing the secondary structure of a protein molecule? I'd really like to be able to take a protein molecule, and draw the secondary structure out along a line, indicating the positions of secondary structural elements like sheets and helices. Thanks! and all the best, --Buz
Re: [ccp4bb] Refinement in half occupancy
It should be straight forward. A good example you can use is to make an alternate conformation of a residue in coot. Then you can follow the generated pdb to modify your ligand. Are you sure refmac won't run? What is the error message from refmac? Nian Huang, Ph.D. Dept. of Biochemistry UT Southwestern Medical Center On Mon, Jun 9, 2008 at 5:21 PM, Matthew Chu [EMAIL PROTECTED] wrote: Dear All, Can anyone teach me how to refine a ligand in a protein structure with half occupancy in refmac? I have tried to combine the coordinates of the two different conformations of that particular ligand in one pdb, after modeling in Coot individually, and then changed the occupancy to 0.5 for each of the conformation. However, I couldn't manage to refine this pdb in Refmac. Thanks in advance! Best regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] Crosslinking reagents
I will try that. But I don't think people can tell the final concentration of glutaraldehyde in the drop by using its vapor. Maybe using direct soaking is what I should do. By the way, should I quench it by ammonium or not? Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 On Sun, Mar 2, 2008 at 12:28 AM, Nian Huang [EMAIL PROTECTED] wrote: Dear all, I know most people use glutaraldehyde as the crosslinking reagent for their crystals. But there are a lot of other crosslinking chemicals available both in protein chemistry and histology, such as simple formaldehyde. Did anybody have experience with other chemicals and how did they compared with glutaradehyde? I have a crystal very sensitive to glutaradehyde but I really wants to try the crosslinking method. Thanks, Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390
Re: [ccp4bb] Crosslinking reagents
I used glutaraldehyde vapor by placing crystal drop above a bridge with 15% glutaradehyde for various amount of time. It worked for me for many crystals, but not for the one that I am trying now. The crystal didn't diffract any more. I intended to make the crystal tougher for heavy metal soaking. I am just wondering what will happen if I use some other crosslinking chemicals. Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 On Sun, Mar 2, 2008 at 12:28 AM, Nian Huang [EMAIL PROTECTED] wrote: Dear all, I know most people use glutaraldehyde as the crosslinking reagent for their crystals. But there are a lot of other crosslinking chemicals available both in protein chemistry and histology, such as simple formaldehyde. Did anybody have experience with other chemicals and how did they compared with glutaradehyde? I have a crystal very sensitive to glutaradehyde but I really wants to try the crosslinking method. Thanks, Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390
[ccp4bb] Crosslinking reagents
Dear all, I know most people use glutaraldehyde as the crosslinking reagent for their crystals. But there are a lot of other crosslinking chemicals available both in protein chemistry and histology, such as simple formaldehyde. Did anybody have experience with other chemicals and how did they compared with glutaradehyde? I have a crystal very sensitive to glutaradehyde but I really wants to try the crosslinking method. Thanks, Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390
[ccp4bb] Tough ligand to bind?
Dear All, I have been trying to get a protein-ligand complex crystal for a long time. Here, ligand is either substrate or product for this kinase. I have tried many methods: soaking with apo-crystal, co-crystal with different concentration of ligand and screen for new conditions. I got a couple of co-crystals with the new conditions, but still no ligand was found in the active site. Anybody has some new ideas that I can try? I have been using glycerol as my cryoprotectant. Could cryoprotectant have some negative effect on co-crystal? I will try to collect some data at room temperature in the future. Thank you in advance for your suggestion. Nian Dept of Biochemistry UT Southwestern Medical Center
[ccp4bb] Disordered active sites
Hi, all, I met some crystal structures with disordered active sites. Soaking common ligands can not make it become ordered. I am wondering what people generally do in such situation. Thanks, Nian Huang
Re: [ccp4bb] Program to find the center of mass
WOW. There are so many ways to do it. Thank you all for replying. Nian Huang Department of Biochemistry Univ of Texas Southwestern Medical Center On 5/10/07, Charlie Bond [EMAIL PROTECTED] wrote: Just to complete the set, in pdb-mode for emacs, if you do pdb-increment-centroid 0 0 0 (e.g. to move a molecule to the origin), it reports the original centroid. Cheers, Charlie -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310 35 Stirling Highway Crawley WA 6009 Australia [EMAIL PROTECTED] +61 8 6488 4406
[ccp4bb] Program to find the center of mass
Dear all, I was trying to do a NCS averaging of the map using Resolve. I had a partial model, which generated a rotation and translation matrix by program lsqkab. Resolve also requires a center of mass input. Do you know any program can estimate it either by map or pdb file? Thanks. Nian Huang Department of Biochemistry Univ of Texas Southwestern Medical Center
