[ccp4bb]
Hi Sajid, the NADP /NAD binding sites are often composed of two sites: one site that is specific for the adenine/adenosine moiety and another site for the nicotinamide moiety. It can happen that you see the adenine in the density but not the nictoninamid tangling around. HTH Guenter I have used NADP in my crystallisation condition; and the crystal diffracted well; I built the model completely and the r-factor and r-free are 0.26 and 0.29 respectively. The resolution is 2.3A. I am looking for NADP, in the binding region; I could see some density for NADP; But it is not continuous; there is no density for the middle phosphate group in fo-fc as well as 2fo-fc. Is it possible that NADP could hydrolyse? if so what could be the hydrolysed product thank you sajid Looking for local information? Find it on Yahoo! Local http://in.local.yahoo.com/ -- *** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: guenter.fr...@uni-konstanz.de Phone Office: +49-(0)7531 88 3205 Phone Lab : +49-(0)7531 88 3733 Fax: +49-(0)7531 88 2966
[ccp4bb] PhD fellowship in structure determination of cys-loop receptors at University of Copenhagen
General announcement and how to apply : http://www.farma.ku.dk/index.php?id=6811 Project description: http://www.farma.ku.dk/index.php/Project-10/6840/0/ Deadline for applications: Tuesday 25 August 2009 at 12 o'clock noon. About the group: http://www.farma.ku.dk/BR/ Please contact me if you wish further information. Best regards Michael Gajhede -- Professor Michael Gajhede Institute of Medicinal Chemistry University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen Ø Denmark Phone: +45 35306407 Email: m...@farma.ku.dk.dk
Re: [ccp4bb] pseudo-translational symmetry
First - there doesnt seem much to worry about. The Rs will be higher than usual when you have a strong pseudo-translation vector. There are many weak observations for the h k l=2n+1 reflections. But in cases like this is is very helpful to force the same indexing on all your different data sets so you can compare results easily: If your original structure has cell (a2= 47.3, b2=58.9, c2=67.6) and angles (alpha2=90, beta2=99.2, gamma2=90) and the second cell is (a1=67.5, b1=58.8, c1=98.9) and angles (alpha1=90, beta1=101.5, gamma1=90) with a pseudo translation of 0 0 0.5, then they are pretty obviously related. I would force my second cell to be 98.9 58.8 67.5 (alpha1=90, beta1=101.5, gamma1=90) by reindexing l h -k then you can just use the coordinate solution from the first cell plus a translated molecule. Ditto when processing in P1 make sure the cell is 98.9 58.8 67.5 89.9 101.5 89.9 and generate the 8 molecules Twinning checks can be misled when you have assigned the lower symmetry spacegroup - ie sent it P1 data instead of P21. And similarly they are less reliable when you have a strong pseudo-translation vector. Eleanor Hi all, I am trying to refine a structure to about 2.0A. Indexing in HKL2000 indicates the protein crystallized in P2 with unit cell lengths (a1=67.5, b1=58.8, c1=98.9) and angles (alpha1=90, beta1=101.5, gamma1=90). Molecular replacement with Phaser yields a solution in P1 21 1 with four molecules in the AU. Successive rounds of refinement lead to good quality maps with chemically appropriate packing of symmetry-related molecules. However, TLS and restrained refinement lead to continuing drops of Rfactor (around 20%, depending on weighting) but the stalling of Rfree around 27%. When the dataset is processed in P1, the unit cell has the dimensions (a=58.7, b=67.4, c=98.9, alpha=78.5, beta=89.9, gamma=89.9) and Phaser finds 8 molecules in the AU. Structure refinement for this scenario is ongoing, and it has yet to be seen whether this leads to improvement of the statistical parameters. I have solved other structures of this protein in complex with different substrates. One such structure was successfully solved in P1 21 1 with two molecules in the AU with the unit cell lengths (a2= 47.3, b2=58.9, c2=67.6) and angles (alpha2=90, beta2=99.2, gamma2=90). Interestingly, these dimensions are highly similar to those of current structure, although a1 is equivalent to c2, and c1 is nearly double a2. I have run phenix.xtriage on the P2 scaled data, and the Patterson Analysis indicates that translational-pseudosymmetry is a posibility in my case. Alternatively, phenix.xtriage on the same dataset processed in P1 indicates the same translational-pseudosymmetry, but also suggests the presence of a pseudomerohedral twin. I am not sure if this twin simply relates the dataset to the higher symmetry P2 group. The sections of both log files pertaining to these analyses are copied below- sorry for all the text! At this stage, I am wondering what the next steps. Specificity, is this data suspicious for having pseudotranslational symmetry or twinning? If so, what should be my next steps to troubleshoot this problem? Any suggestions would be appreciated. Thanks in advance, Keith __ P2 PROCESSED DATA Patterson analyses: -- Largest Patterson peak with length larger than 15 Angstrom Frac. coord.:0.0000.0000.500 Distance to origin : 49.477 Height (origin=100) : 42.327 p_value(height) :2.073e-04 The reported p_value has the following meaning: The probability that a peak of the specified height or larger is found in a Patterson function of a macro molecule that does not have any translational pseudo symmetry is equal to 2.073e-04. p_values smaller than 0.05 might indicate weak translational pseudo symmetry, or the self vector of a large anomalous scatterer such as Hg, whereas values smaller than 1e-3 are a very strong indication for the presence of translational pseudo symmetry. The full list of Patterson peaks is: x y zheight p-value(height) ( 0.000, 0.000, 0.500 ) : 42.327 (2.073e-04) ( 0.111, 0.000,-0.476 ) :8.908 (2.385e-01) If the observed pseudo translationals are crystallographic the following spacegroups and unit cells are possible: space groupoperator unit cell of reference setting P 1 21 1 (a,b,2*c) x, y, z+1/2 (67.48, 58.78, 49.48, 90.00, 101.49, 90.00) ___ P1 PROCESSED DATA #### ## Twinning Analyses## ####
Re: [ccp4bb] pseudo-translational symmetry
Just a small comment on Eleanor's instructive advices : ... If your original structure has cell (a2= 47.3, b2=58.9, c2=67.6) and angles (alpha2=90, beta2=99.2, gamma2=90) and the second cell is (a1=67.5, b1=58.8, c1=98.9) and angles (alpha1=90, beta1=101.5, gamma1=90) ... I would force my second cell to be 98.9 58.8 67.5 (alpha1=90, beta1=101.5, gamma1=90) by reindexing l h -k I guess the proper reindexing would be l k -h, wouldn't it? (l h -k would not really do what you want AND it would also change the reference system to a left-handed one...) Cheers, Alejandro -- Unit of Protein Crystallography Institut Pasteur de Montevideo Uruguay
Re: [ccp4bb] pseudo-translational symmetry
Oops - sorry to mislead you. Yes - you are absolutely right.. Eleanor ale...@pasteur.fr wrote: Just a small comment on Eleanor's instructive advices : ... If your original structure has cell (a2= 47.3, b2=58.9, c2=67.6) and angles (alpha2=90, beta2=99.2, gamma2=90) and the second cell is (a1=67.5, b1=58.8, c1=98.9) and angles (alpha1=90, beta1=101.5, gamma1=90) ... I would force my second cell to be 98.9 58.8 67.5 (alpha1=90, beta1=101.5, gamma1=90) by reindexing l h -k I guess the proper reindexing would be l k -h, wouldn't it? (l h -k would not really do what you want AND it would also change the reference system to a left-handed one...) Cheers, Alejandro -- Unit of Protein Crystallography Institut Pasteur de Montevideo Uruguay
[ccp4bb] alternative graphics programs for OS X--summary
Hi, Thanks to the many replies to my queries about cheap/easy alternatives to Photoshop and CorelDraw on OS X. In a rare display of unanimity, the bulletin board spoke with essentially one voice: ALTERNATIVE TO PHOTOSHOP: Gimp (not gimpshop) http://www.gimp.org/ As one sage respondent said, Gimp is no less intuitive than Photoshop. I could certainly easily figure out how to do the types of trivial manipulations for which I previously used Photoshop; power users may have a different experience. ALTERNATIVE TO CORELDRAW/ILLUSTRATOR: Inkscape http://www.inkscape.org/ Again, straightforward to use, and I sense that I will find it a completely satisfactory replacement for CorelDraw (if only it could read .cdr files, sigh). Notes: 1. At least one of these programs requires updating the version of X11 that comes with 10.5 (e.g., XQuartz 2.3 something, http://xquartz.macosforge.org/trac/wiki) . If you are using crystallographic software you've probably already done this. 2. One kind soul pointed out that I already have GraphicConvertor installed as part of the OS, and this program can do simple manipulations (brightness, contrast, cropping, etc.). Hidden in plain sight. 3. There was one vote (out of dozens total) for ImageMagick, but as far as I know this is command line only. It also occurs to me that ImageJ would do at least some of the simple things that I need (e.g., getting an image of a gel ready for publication). 4. There was one vote for OmniGraffle (but this is commercial, and costs a few dollars). Thanks again. The ccp4bb rules. Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] Electron Density Maps
Dear Pascal, 2009/8/10 Pascal Egea pas...@msg.ucsf.edu: Dear All, I am currently carrying the refinement of a structure and comparing the results obtained in Refmac, Phenix and CNS. While Phenix and Refmac write maps and their corresponding coefficients in mtz format allowing display of 2Fo-Fc and Fo-Fc maps in COOT, the corresponding Fo-Fc map as written by CNS does not show positive and negatives peaks. There is density but it does not look like why I would expect for a Fo-Fc map. Why is that? Is your map read as an Fo-Fc map? I guess not... I believe there is a way to tell COOT that you want to load you CNS map as a difference map. This section http://www.ysbl.york.ac.uk/~emsley/coot/doc/chapters/user-manual_6.html#SEC160 of the user manual might explain better. Hope this helps, Folmer Fredslund
Re: [ccp4bb] Electron Density Maps
If you want to display CNS maps in Coot and have them on the right scale, then you need to do one of two things. Either: 1. Don't use the map file, use the reflection file containing the map coefficients, or 2. Change the setting in CNS which controls the extent of the output map. Instead of outputting a map covering a single molecule, you need to output a map covering a whole asymmetric unit (or if you prefer a whole unit cell). The Xplor maps output by CNS are output in O mode, not Coot mode, by default. This is due to a fundamental difference in understanding what a map is between programs which work crystal space (e.g. Coot, Quanta, XtalView) and those which don't. Kevin Pascal Egea wrote: Dear All, I am currently carrying the refinement of a structure and comparing the results obtained in Refmac, Phenix and CNS. While Phenix and Refmac write maps and their corresponding coefficients in mtz format allowing display of 2Fo-Fc and Fo-Fc maps in COOT, the corresponding Fo-Fc map as written by CNS does not show positive and negatives peaks. There is density but it does not look like why I would expect for a Fo-Fc map. Why is that? I am also surprised by the differences between maps and their contouring levels between Refmac/Phenix and CNS. Is the way to scale ED maps different between those programs? Can differences in the way to perform bulk solvent correction account for those differences? Thanks in advance for your suggestions. Pascal Egea
[ccp4bb] calculation of radii
Dear all i want to know how can i calculate the alpha helical radii of 3-D structure of a protein. Is there any programme to calculate the radii of alpha helix.
Re: [ccp4bb] How to model apo protein structure from solved ligand bound high resolution structure?
Hi Donghui, It’s going to be tricky. Perhaps, you can predict some local rotamer variations around the ligand interacting regions. Predicting the global changes is difficult, particularly if you are anticipating a big conformational change. For example, in one of our structure, the active site of the unliganded protein is in an open conformation and adopted closed conformation upon ligand binding (JBC, Vol. 282, 27334-27342). You can find many such examples in the literature, including the text book example of cAMP receptor protein. I would try to crystallize the apo form. If this is not possible, you can try studying the dynamics by solution studies or through molecular dynamics simulation (http://ambermd.org/). All the best Satheesh Palaninathan Satheesh , PhD Research Scientist Department of Biochemistry and Biophysics Texas AM University, 2128 TAMU College Station, TX - 77843-2128 USA. Ph. 1-979-862-7639 Date: Mon, 10 Aug 2009 11:10:42 +0800 From: wdh0...@gmail.com Subject: Re: [ccp4bb] How to model apo protein structure from solved ligand bound high resolution structure? To: CCP4BB@JISCMAIL.AC.UK More information about the two ligand bound structures, each protein is composed of two globular domains, between the two globular domains, there lies a deep cleft and ligand sits or buried there comfortably. These two domains are connected by a hinge region, here we want to model how this hinge region moves upon ligand binding. Any recommendation for any program which can do this task well. Thanks ahead. Donghui On Sun, Aug 9, 2009 at 8:37 PM, wu donghui wdh0...@gmail.com wrote: Dear CCP4ers, Recently we have solved two structures from E.coli in high resolution, which have bound two different ligands tightly already. Here I want to know if there is any program which can let us model the structure of our apo proteins confidently. Thanks ahead for any comment and input. Regards, Donghui _ We all see it as it is. But on MSN India, the difference lies in perspective. http://in.msn.com
[ccp4bb] Heavy atom searching with disulfides
Hi all Has there been any work/reports of using disulfide restraints (number of heavy atoms as well as distance) for heavy atom searching/scoring for anomalous sulfur phasing? What resolution range would this be most effective? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Heavy atom searching with disulfides
The paper Acta Cryst. D59 (2003) 2125-2132 discusses searching for disulfides. In general this works for data truncated for phasing to between 2.1 and 2.9 A. If the resoltuions is higher than 2.1, you can search for individual sulfurs, if it is worse than 2.9 even fitting them as disulfides is unreliable and they have to be treated as 'super-sulfurs'. The main advantage of incorporating disulfides in this resolution range is that the resulting experimental phases extend better to higher resolution. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Mon, 10 Aug 2009, Francis E Reyes wrote: Hi all Has there been any work/reports of using disulfide restraints (number of heavy atoms as well as distance) for heavy atom searching/scoring for anomalous sulfur phasing? What resolution range would this be most effective? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Pictures of DDM crystals?
Hi all, I am doing initial crystallization screening for a membrane protein purified in DDM. The actual concentration of DDM in the protein solution is 2 CMC after the protein concentration step (Amicon MWCO 50 kD used). I just found over 50 conditions out of 1000 give me crystals. Having worked on soluble proteins, frankly they all look ugly to me to different degrees. I have selected a few conditions to go after, hoping that they will grow big enough for me to verify the crystal identity by gels. I wonder if there is a quick way to tell if they are detergent crystals. I know a microscope with UV capacity would do the job, but we probably will not get one very soon. I have read through all threads on CCP4BBS regarding detergent crystals. I have an impression that it is actually not easy to crystallize detergents--please correct me if I am wrong. Your thoughts and comments are helpful. Thank you for your attention. -- Best regards, Joe
[ccp4bb] Disulfide bond survival in the presence of DTT?
Hi all- Got a perplexing thiol chemistry/phasing issue. To prevent non-native disulfide bonds from forming between free Cys but to preserve native disulfides, I have heard of using a very low (say 0.5 mM) concentration of DTT. I've recently come across a paper where the assignment of 3 disulfides in a protein structure was carried out by calculating a 4A resolution anomalous difference map (detecting the sulfur anomalous signal with 1.7A x-rays). However, at every step in the protein prep and in the crystallization solution, there exists mM concentrations of DTT. Every protein is different, so the microenvironment and the protection of a disulfide will be different for every one you come across. But I am surprised that a disulfide can withstand that much DTT. I guess the proof is in the pudding, which would be the anomalous peaks observed in the maps. What is the upper limit on DTT (or B-ME or TCEP) concentration that people have used and still maintained native disulfides? Thanks- Brad
[ccp4bb] DNA binding protein
Hi all, I had a simple question about DNA binding protein. Is there an easy way to detect if your heterologously expressed protein is bound to DNA post purification. Also is there an easy way to strip the protein of DNA without any damage done to the protein in doing so. I would appreciate if someone can provide any valuable tips for the same. thanks, Neeraj -- Neeraj Kapoor TPCB Graduate Fellow Sakmar Lab/ Molecular Biology Biochemistry The Rockefeller University 1230 York Avenue, RRB 510 New York, NY 10021 lab.1.212.327.8284:fax.7904 mobile: 917.535.2030 http://www.rockefeller.edu/labheads/sakmar/sakmar-lab.html
[ccp4bb] hardware stereo in Coot on Mac
Two questions RE hardware (dual images on CRT monitor, 3-D with stereo glasses) stereo on a Macintosh. 1 We have a configuration: Mac dual Intel processors OS 10.4.11 NVIDIA Quadro FX 4500 graphics card X11 version 1.1.3 It runs hardware stereo in O and Pymol , which don't require an X- window terminal, and will do side-by-side stereo in Coot run from an X11 window, but when we try to do hardware stereo, we get message, This computer appears not to be able to do hardware stereo. This must have a simple fix, but I haven't found it. 2. Is there a combination of Mac OS 10.5.x and Xquartz 2.x.x that does hardware stereo reliably? Dave McKay Department of Chemistry Biochemistry University of Colorado, Boulder
Re: [ccp4bb] DNA binding protein
Hi Neeraj, An absorption spectra between 220 and 400 nm (for example) should show you if there is DNA coming along with your protein. In theory A280 is about 1.7 times A260 for a pure protein sample. This is a rough estimate. If your peak is shifted towards 260 instead of 280 then you can suspect the presence of contaminating DNA or RNA. As to get rid of the DNA, I can suggest several ways to do it. 1-An heparin affinity column could out-compete your contaminating DNA while binding your protein. They work really well. 2-Ion exchange is worth trying. It has worked for me with an extremely basic archeal RNA binding protein purified in E.coli and bringing along some endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the contaminating nucleic acid, however with some loss of protein. 3-DNAse treatment may be worth trying but the presence of traces of DNase may be a problem with subsequent crystallization trials in presence of the bona-fide target DNA sequence. 4-Precipitation of the DNA with streptomycine sulfate. You could also loss some protein. 1 and 2 are in my opinion the less invasive soutions. Hope this helps. Best regards Pascal Egea
Re: [ccp4bb] Pictures of DDM crystals?
I forgot the mention this: does anyone happen to have some pictures of DDM crystals? It would be very very helpful for me to take a look and get a sense how they look like. I appreciate your inputs and help. Thanks, Joe On Mon, Aug 10, 2009 at 4:47 PM, Joe gch...@gmail.com wrote: Hi all, I am doing initial crystallization screening for a membrane protein purified in DDM. The actual concentration of DDM in the protein solution is 2 CMC after the protein concentration step (Amicon MWCO 50 kD used). I just found over 50 conditions out of 1000 give me crystals. Having worked on soluble proteins, frankly they all look ugly to me to different degrees. I have selected a few conditions to go after, hoping that they will grow big enough for me to verify the crystal identity by gels. I wonder if there is a quick way to tell if they are detergent crystals. I know a microscope with UV capacity would do the job, but we probably will not get one very soon. I have read through all threads on CCP4BBS regarding detergent crystals. I have an impression that it is actually not easy to crystallize detergents--please correct me if I am wrong. Your thoughts and comments are helpful. Thank you for your attention. -- Best regards, Joe -- Best regards, Joe
Re: [ccp4bb] DNA binding protein
I had a simple question about DNA binding protein. Is there an easy way to detect if your heterologously expressed protein is bound to DNA post purification. Yes. UV absorbance. DNA absorbs UV strongly, proteins do not. DNA absorbs 260 more thn 280, the opposite is true for proteins. In fact, it's always a good idea to check 260/280 ratio because quite frequently even non-DNA binding proteins purified by a single tag affinity chromatography contain enough DNA to skew estimation of protein concentration from 280 nm absorbance by several fold (9X in one case of my personal experience). Also is there an easy way to strip the protein of DNA without any damage done to the protein in doing so. No generic solution. Greatly depends on the protein and its properties. Dima
Re: [ccp4bb] DNA binding protein
TurboNuclease digests DNA and RNA to 1-4 base long fragments. It's very useful to remove nucleic acids during protein purification and virus purification. Another benefit of using TurboNuclease at cell lysis is to significantly reduce lysate viscosity. So it reduces the lysate volume for column loading. We can make a lysate of only 1L for 300 grams of insect cells and load a Ni column without back pressure problem. We can use lower g-force for lysate clearing. We got much lower 260 reading in the Ni pool as well. The specific activity of TurboNuclease (~1.3 units per ng) is thousands of fold higher than DNaseI. So the minute quantity of TurboNuclease used has no interference with down stream applications. Crystals of many proteins have been obtained when the cells were lyzed with TurboNuclease, a few without using affinity purification. TurboNuclease is protease-free and endotoxin-free. --Chun Competing Interests Statement Accelagen is the maker of TurboNuclease. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Pascal Egea Sent: Monday, August 10, 2009 4:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] DNA binding protein Hi Neeraj, An absorption spectra between 220 and 400 nm (for example) should show you if there is DNA coming along with your protein. In theory A280 is about 1.7 times A260 for a pure protein sample. This is a rough estimate. If your peak is shifted towards 260 instead of 280 then you can suspect the presence of contaminating DNA or RNA. As to get rid of the DNA, I can suggest several ways to do it. 1-An heparin affinity column could out-compete your contaminating DNA while binding your protein. They work really well. 2-Ion exchange is worth trying. It has worked for me with an extremely basic archeal RNA binding protein purified in E.coli and bringing along some endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the contaminating nucleic acid, however with some loss of protein. 3-DNAse treatment may be worth trying but the presence of traces of DNase may be a problem with subsequent crystallization trials in presence of the bona-fide target DNA sequence. 4-Precipitation of the DNA with streptomycine sulfate. You could also loss some protein. 1 and 2 are in my opinion the less invasive soutions. Hope this helps. Best regards Pascal Egea
Re: [ccp4bb] DNA binding protein
I'd agree with Pascal as well, and can add that if you have a protein for which you use an affinity tag, often high salt (500mM) will strip the DNA off your protein, and might even keep it stable. Cheers - Miles On Aug 10, 2009, at 3:59 PM, Pascal Egea wrote: Hi Neeraj, An absorption spectra between 220 and 400 nm (for example) should show you if there is DNA coming along with your protein. In theory A280 is about 1.7 times A260 for a pure protein sample. This is a rough estimate. If your peak is shifted towards 260 instead of 280 then you can suspect the presence of contaminating DNA or RNA. As to get rid of the DNA, I can suggest several ways to do it. 1-An heparin affinity column could out-compete your contaminating DNA while binding your protein. They work really well. 2-Ion exchange is worth trying. It has worked for me with an extremely basic archeal RNA binding protein purified in E.coli and bringing along some endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the contaminating nucleic acid, however with some loss of protein. 3-DNAse treatment may be worth trying but the presence of traces of DNase may be a problem with subsequent crystallization trials in presence of the bona-fide target DNA sequence. 4-Precipitation of the DNA with streptomycine sulfate. You could also loss some protein. 1 and 2 are in my opinion the less invasive soutions. Hope this helps. Best regards Pascal Egea Miles Pufall Postdoctoral Scholar Yamamoto Lab UC San Francisco Cellular and Molecular Pharmacology Mail Stop 2280 600 16th Street, Genentech Hall S-574 San Francisco, California 94158-2517 (415)476-4480
[ccp4bb] DS Visualizer ActiveX Control in PowerPoint
Hi, This is non-CCP4 related question, I apologize to those who are not interested. But I hope there might be someone over there with a good answer. Program: Accelry DS Visualizer 2.0 (Free ware). It has some interesting features. My intention: Create an image from pdb files and save as .msv file. Then in PowerPoint this file can be inserted via an ActiveX Control window. In 2003 version it works this way: (1) Open ppt, then bring in “Visual Basic” and “Control Toolbox” panel. (2) Click “More Controls” in “Control Toolbox” penal, then select “Accelrys DSVisualizer ControlR1” (3) The cursor becomes cross then makes a window in ppt slide. (4) Switch ppt to “Full Screen” then right-click on black window to import molecule file. (5) Now you can manipulate the molecule as in DS Visualizer when ppt is in “Full Screen” However, in 2007 version the layout is different and the Visual Basic and Control ToolBox exist in the Developer menu. I could not locate the Accelrys DSVisualizer ControlR1. Instead there are entries such as DSDisplayPanel Class and DSStatusBar Class. Choosing either one and trying to draw a window will give the same error: Could not access system registry. I also posted a similar question to the Accelrys forum but no reply yet. Thanks in advance. Yong-Fu Li IAVI, Brooklyn, NY
Re: [ccp4bb] DS Visualizer ActiveX Control in PowerPoint
Yong-Fu, Microsoft in its infinite wisdom decided to bury ActiveX functionality in Office 2007. To access Controls, you need to click on the big round button in the upper-left, select PowerPoint Options, choose then Popular tab and then check the Show Developer tab in the Ribbon option. The Developer tab will give you access to the Control Toolbox. Cheers, Warren (PS. For a similar free and unsupported control for PyMOL Sessions, see http://delsci.com/axpymol/ ) From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Yong-Fu Li Sent: Monday, August 10, 2009 6:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DS Visualizer ActiveX Control in PowerPoint Hi, This is non-CCP4 related question, I apologize to those who are not interested. But I hope there might be someone over there with a good answer. Program: Accelry DS Visualizer 2.0 (Free ware). It has some interesting features. My intention: Create an image from pdb files and save as .msv file. Then in PowerPoint this file can be inserted via an ActiveX Control window. In 2003 version it works this way: (1) Open ppt, then bring in Visual Basic and Control Toolbox panel. (2) Click More Controls in Control Toolbox penal, then select Accelrys DSVisualizer ControlR1 (3) The cursor becomes cross then makes a window in ppt slide. (4) Switch ppt to Full Screen then right-click on black window to import molecule file. (5) Now you can manipulate the molecule as in DS Visualizer when ppt is in Full Screen However, in 2007 version the layout is different and the Visual Basic and Control ToolBox exist in the Developer menu. I could not locate the Accelrys DSVisualizer ControlR1. Instead there are entries such as DSDisplayPanel Class and DSStatusBar Class. Choosing either one and trying to draw a window will give the same error: Could not access system registry. I also posted a similar question to the Accelrys forum but no reply yet. Thanks in advance. Yong-Fu Li IAVI, Brooklyn, NY
Re: [ccp4bb] Disulfide bond survival in the presence of DTT?
Hi, I am sorry that I cannot recall the highest concentration of DTT that I ever used (inadvertently) when working with disulphide-containing proteins. However I have the following mildly useful suggestion: If you have properly formed -S-S- as well as a bunch of free -SH perhaps a safer strategy is to modify the available -SH with something - it could be a reversible protective group or a permanent one such as iodiacetamide. This way you avoid playing with -S-S- shuffling entirely. Notably, this will reduce your chances of obaining a derivative since protected -SR are much less likely to react with heavy atom agents. As a useful bonus, if you modify the SH with e.g. iodoacetamide, then run MS on the intact protein - you will find out how many -SH remain unmodified. Among those, some are in -S-S- bonds and some are buried and therefore inaccessible to the reagent. By repeating iodoacetamide treatment post-reduction or on an unfolded sample you can further figure out the pattern. Good luck, Artem Nothing is built on stone; all is built on sand, but we must build as if the sand were stone Jorge Luis Borges _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Brad Bennett Sent: Monday, August 10, 2009 5:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Disulfide bond survival in the presence of DTT? Hi all- Got a perplexing thiol chemistry/phasing issue. To prevent non-native disulfide bonds from forming between free Cys but to preserve native disulfides, I have heard of using a very low (say 0.5 mM) concentration of DTT. I've recently come across a paper where the assignment of 3 disulfides in a protein structure was carried out by calculating a 4A resolution anomalous difference map (detecting the sulfur anomalous signal with 1.7A x-rays). However, at every step in the protein prep and in the crystallization solution, there exists mM concentrations of DTT. Every protein is different, so the microenvironment and the protection of a disulfide will be different for every one you come across. But I am surprised that a disulfide can withstand that much DTT. I guess the proof is in the pudding, which would be the anomalous peaks observed in the maps. What is the upper limit on DTT (or B-ME or TCEP) concentration that people have used and still maintained native disulfides? Thanks- Brad
[ccp4bb] {Spam?} quick purification?
Hi All A little off topic, but I am thinking about expressing and purifying 40+ mutants, for an assay, at maybe 1mg total protein each. I'd like the purification to be quick and easy (ie. one step) but cleaner than just a 6his-tag purification. Currently, my options are either GST, a longer his tag or a strep-tag. Any thoughts on the comparison between GST and strep and Ni purifications would be much appreciated. OR are there any other (better) high affinity purifications, I've missed, excluding expense Ab resins. Thanks, Mark Collins
Re: [ccp4bb] {Spam?} quick purification?
Hi,
Since you're considering a serious undertaking, it would be good to know
whether your protein is decently expressed and reasonably characterized in
its native state - before advising you on the process :)
If your protein is normally expressed well, is soluble, and not aggregated -
there's going to be little to no difference between the results of major
methods used for primary affinity capture. If your protein is not very
abundant in the lysate, then higher fidelity methods may give you better
purity - in this case StrepII is somewhat better than GST. You can always
try the good old Biotin tag - this will however require that your E. coli
carry excess of BirA (typically supplied from a separate plasmid) as normal
biotinylation levels in most E. coli strains are fairly low.
With 40+ mutants you can expect a certain amount of attrition - some of the
mutants will not express well and some will not purify well.
In general there's nothing wrong with single-step IMAC provided that you can
work out reasonable purification conditions in advance and can assume that
most of your 40+ mutants behave reasonably well (and close to the test
case). For outlier mutants there will undoubtedly be issues with
purification however primary capture method is not likely to fix them since
it did not cause them in the first place.
For abundant proteins - if you use a 'high fidelity' IMAC resin such as
His-SELECT or Talon and if your protein of interest is present (in the
lysate) in reasonable quantity then you can expect the purity of your
single-step product to be compatible between GST and IMAC purification.
StrepII tag is of course a good choice as well.
Regards,
Artem
Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone
Jorge Luis Borges
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mark
Collins
Sent: Monday, August 10, 2009 10:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] {Spam?} quick purification?
Hi All
A little off topic, but I am thinking about expressing and purifying 40+
mutants, for an assay, at maybe 1mg total protein each. I'd like the
purification to be quick and easy (ie. one step) but cleaner than just a
6his-tag purification.
Currently, my options are either GST, a longer his tag or a strep-tag.
Any thoughts on the comparison between GST and strep and Ni purifications
would be much appreciated. OR are there any other (better) high affinity
purifications, I've missed, excluding expense Ab resins.
Thanks,
Mark Collins
