Re: [ccp4bb] How to determine water number?
AntonioLeung wrote: I am a novice working on protein structure. When I pick water using COOT, too many waters picked, filling in the whole cell. Too many - hmm. Did you, by any chance, peak-search a difference map without first changing the default sigma cut-off to (say) 3.5? Paul.
Re: [ccp4bb] imosflm with multiple data sets
Thanks Frank, Luke forgot to tell me that he had actually implemented this ! For those wishing to use this option, the Matrix line has to be dragged and dropped on the Sector line of the segment of data that you want to assign the matrix to, NOT to the Matrix line of that sector. I agree Frank, it is very tedious to have to have to drag past all the images, we will change things so that the collapsing the sector still leaves the Matrix visible. While on this topic, Clemens Vonrhein kindly pointed out to me that I had not read the original Email carefully enough, in that the second sector of data had an oscillation range of 0-180, the same as the first sector, but the phi value had been changed by 180 degrees between the two runs. Thus, in order to get the matrix from the first sector to correctly predict images from the second sector, 180 degrees needs to be added to the phi values of each image. This is easily done by manually entering the phi values for the first image of the second sector, then this will be automatically propagated for all following images. Andrew On 18 Aug 2009, at 06:55, Frank von Delft wrote: Actually, drag-and-drop DOES work, and is *dead* handy! (But a considerable annoyance: you HAVE to open the sector to be able to click on the matrix line -- and then you have to drag that matrix past all the 300 (or whatever) images to get to the next sector. For many images, this really slow. Better to put matrix and images on separate sub-nodes.) Andrew Leslie wrote: Dear Tom, There is a straightforward way to do what you want. It is probably simplest to start by reading in only the images from the first segment (0-180). Then do the indexing, cell refinement and integration in the usual way. Then read in the second segment of data. You will notice that in this second segment, underneath the Sector name, there is a line starting Matrix and this will be Unknown. If you go to the Matrix line of the first segment, the matrix will have a name (based on the image template). Double click on the name of the matrix. A popup window (Matrix properties) will appear. Click on the save matrix file icon (a blue disc) and save the matrix with an appropriate filename. Now go to the Matrix line of the second segment, double click (on Unknown) as before and this time click on the Open matrix file icon (a folder) and read in the matrix that you saved from the first sector. You can now process the second segment using this matrix. It would be even nicer if you could drag and drop the matrix, this is on our to do list. Best wishes, Andrew On 17 Aug 2009, at 13:33, Brett, Thomas wrote: I am an imosflm novice and have a relatively simple question. I have a 360 deg data set collected in two swathes of 180 deg (one with phi=0 and omega going 0-180 and the second with phi = 180 and omega going 0-180). What is the easiest way to process the two datasets using a matching orientation matrix (or one rotated by 180 deg as it were) so that all the data can be merged together. Is there an easy way to do it in imosflm or must one process the two sets separately and then manipulate later with pointless before scalling and merging everything together? Thanks in advance. -Tom Tom J. Brett, PhD Assistant Professor of Medicine Division of Pulmonary and Critical Care Washington University School of Medicine Campus Box 8052, 660 S. Euclid Saint Louis, MO 63110
[ccp4bb] Vacancy for crystallographer at Heptares
This is posted on behalf of Dr. Fiona Marshall at Heptares. The Heptares web site for applications is: http://home.btconnect.com/heptares/vacancies.html Please respond using the web site or the Email address below, not to me. Andrew Leslie Structural Biologist/Crystallographer Heptares is a new drug discovery company located in Hertfordshire UK, working on G protein coupled receptors across a range of therapeutic areas. We are using a unique structure based approach, which originated from work at the MRC Laboratory of Molecular Biology in Cambridge, to characterise this important family of receptors and develop novel innovative therapeutics for the treatment of disease. We have recently established a state of the art laboratory for membrane protein crystallography. We are now seeking an enthusiastic and motivated scientist with experience in protein crystallisation and structure determination to assist Heptares in obtaining novel GPCRs structures and ligand co-structures for drug discovery. This is an exceptional opportunity to participate in cutting edge science whilst helping to grow an industry-leading drug discovery company. The successful candidate will work closely with the protein engineering and expression team responsible for the generation of pure stabilised GPCRs. In addition the role will include close collaboration with medicinal and computational chemists involved in structure based drug design It is expected that some candidates will have broad experience of protein purification through to structure whilst others may wish to focus on crystallography. The role will include some or all of the following depending on the candidate: • Develop and optimise protocols for receptor purification with a view to crystallisation • Develop protocols for crystallisation of novel stabilised GPCRs • Set up crystallisation screens and optimise conditions for GPCR crystals • Data collection at synchrotrons (Diamond and ESRF) • Structure determination of protein ligand complexes Qualifications The successful candidate should have: • A PhD or equivalent in biophysics, structural biology or related discipline • At least 5 years experience within a research environment either in academia or industry. • Experience in protein purification and crystallisation • Extensive skills in X-ray macromolecular crystallography • Analytical thinking and critical problem solving skills. • Experience of working with membrane proteins would be an advantage as would experience of working in a drug discovery environment where structural studies are used to drive medicinal chemistry • Excellent time management skills and an ability to work independently. • An interest in G protein coupled receptors and drug discovery. Further information can be found on our website (www.heptares.com). Applications should be sent to i...@heptares.com.
Re: [ccp4bb] Protein Protein interactions
Mariah, you may want to try PISA at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html Eugene On Mon, 17 Aug 2009, protein.chemist protein.chemist wrote: Hello All, Can anyone tell me what are the programs used to find out the different interactions in a protein. I am talking about both intra and intermolecular interactions. Thanks in advance. Mariah
Re: [ccp4bb] space groups not supported by Refmac5
Dear Arefeh, Arp/warp works with standard space groups, therefore you have to reindex P21221 to P21212. In this message from Anastassis Perrakis you can find some advice: https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0709L=CCP4BBT=0F=S=P=100954 Michele Arefeh Seyedarabi wrote: Hi, I have a question; recently I have encountered a few space groups which are not supported by Refmac5. Examples include I2 and P21221. Both these space groups have been identified as the best solutions for the two different datasets I am working on using Pointless. However, I am faced with difficulties in Refmac5, and the program fails to complete when I select refinement cycles with Arp-waters...with the message saying 'space group not supported'. Any suggestions on how this problem could be overcome? Regards, Arefeh
Re: [ccp4bb] Protein Protein interactions
Hi, look in our servers list for interaction and contact analysis http://bip.weizmann.ac.il/toolbox/structure/tertiary.htm#contact pay attention to PISA which is very good for intermolecular and PIC for intra. Good luck Rotem On 17 Aug, 2009, at 20:13, protein.chemist protein.chemist wrote: Hello All, Can anyone tell me what are the programs used to find out the different interactions in a protein. I am talking about both intra and intermolecular interactions. Thanks in advance. Mariah -- Mariah Jones Department of Biochemistry University of Florida Rotem Sertchook, Ph.D. Bioinformatics Unit, Biological Services Weizmann Institute of Science, Rehovot 76100, Israel.
Re: [ccp4bb] Scale datasets from different crystals
I tried to scale the two datasets in scala after combining the two unmerged datasets with the sort MTZ program, but scala thinks the two datasets are from the same crystal. The problem is the unit cell constants are not identical. See scala output below. * Number of Datasets = 2 * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength: 1 0409 0409 JL0409 66.7900 69.4500 80.4500 83.1500 70.4700 61.5600 0.97952 2 0609 0409 JL0409 66.9700 69.5600 80.4500 83.0600 70.6700 61.2900 1.03319 Then I get this WARNING: WARNING: output dataset X101/two/JL0609 contains input datasets with different wavelengths WARNING: output dataset X101/two/JL0609 contains input datasets with different cells WARNING: output dataset X101/two/JL0609 contains input datasets with different Crystal Names clearly scala is expecting data from the same crystal. Are there any programs in ccp4 that I can use to combine these two datasets. I know this sort of thing was done a lot before cryoprotection came about. Thanks a lot in advance. -Donald - Donald Raymond Department of Biological Chemistry Janet Smith Lab University of Michigan On Aug 17, 2009, at 10:34 PM, Pete Meyer wrote: If you want to treat the end result as a single dataset, you're probably better off combining unmerged datasets (two different batches in scala, or two different scaling sets in scalepack). As far as I understand it, the data model in the mtz files (project/ crystal/dataset/column) isn't really set up to allow combining two datasets post-merging; so most likely you'd have to cook up a custom procedure (most likely involving some duct tape, or equivalently dumping to ascii after scaling, merging overlapping reflections, and reconversion to mtz). Pete Donald Damian Raymond wrote: Hello all, I have incomplete data from two different crystals. Is there a program in ccp4 that I can use to merge the two scaled datasets? I used CAD and scaleit, but it does not give any scaling statistics like completeness and Rmerge. Any help would be appreciated. -Donald.
[ccp4bb] 1-2 October: What is a Macromolecular Complex ? meeting at the NKI, Amsterdam
Dear all, This is a reminder and some news for the meeting: What is a macromolecular Complex? Shades of Meaning Across Cellular, Systems, and Structural Biology http://xtal.nki.nl/Oct2009 - Registration closes on Monday 7th September at 12:00 noon. No exceptions after the deadline. - There will be eight (8) fellowships for attending the meeting, 500 Euro (max) each (*) Best regards on behalf of the organizers, Tassos (*) Only available if your lab is NOT part of the EU programs that support the meeting (SPINE-2-Complexes, TEACH-SG, 3D-Repertoire) and you really wanted to attend but could not for financial reasons, but 500 Euro will make it possible for you to join us, please email a.perrakis.AT.nki.nl with a short motivation and research interests, 10-15 lines max (no attachments please, simple text). Preference will be given to people not yet registered, since we might well assume that people that already registered had the money already sorted. The amount is the MAXimum available per person for ALL expenses, and it will be reimbursed after the meeting, *** based on receipts ***!
Re: [ccp4bb] space groups not supported by Refmac5
Thank you all very much for your advice. I will reindex my data to P21212 in order to use Arp/Warp. Best regards, Arefeh Arefeh Seyedarabi, PhD Postdoctoral research assistant School of Biological and Chemical sciences Queen Mary, University of London Mile End road London E1 4NS Based at Joseph Priestley Building G.35 020 78828480
[ccp4bb] SOMoRe
Dear CC4BB users, Does any one know how to get SOMoRe for molecular replacement? The old link and email to Diane Jamrog no longer functions. Also, does any one have a Mac Intel version? Best regards, Scott * Scott T. R. Walsh, Ph.D. Center for Advanced Research in Biotechnology University of Maryland Biotechnology Institute Rm 3127E CARB II 9600 Gudelsky Drive Rockville, MD 20850 email: wal...@umbi.umd.edu phone: (240) 314-6478 fax: (240) 314-6255 --
Re: [ccp4bb] imosflm with multiple data sets
Hi Hari, This is slightly different, and was indeed a bug in imosflm version 1.0.0. In the new version, released last week (1.0.3) this bug is fixed, so that if images from multiple sectors are used in indexing, the same matrix is defined for all those sectors (ie they will not be marked as Unknown. Best wishes, Andrew On 17 Aug 2009, at 14:53, hari jayaram wrote: I didnt realize the following: You read in images from the two wedges collected with the same crystal orientation. mydata_1_###.img mydata_101_###.img Now when you index ,if you say use images from both datasets mydata_1_###.img use image 1,90 mydata_101_###.img use image 30 , 120 The matrix for the second wedge (mydata_101_###.img) is still marked unknown? Isnt this different from the behaviour in the X- mosflm . SHould the matrixes be the same since the orientation was calculated using images from both. Now , If I did not force the second wedge to have the same matrix , using the save to file and read from file method you just described , does the new imosflm use the last calculated matrix from the running session or calculate a new matrix ?..I guess I have to check some of the data I processed with my erroneous assumption to make sure that the matrixes for the two wedges are the same . Thanks for clarifying this.. hari On Mon, Aug 17, 2009 at 9:13 AM, Andrew Leslieand...@mrc-lmb.cam.ac.uk wrote: Dear Tom, There is a straightforward way to do what you want. It is probably simplest to start by reading in only the images from the first segment (0-180). Then do the indexing, cell refinement and integration in the usual way. Then read in the second segment of data. You will notice that in this second segment, underneath the Sector name, there is a line starting Matrix and this will be Unknown. If you go to the Matrix line of the first segment, the matrix will have a name (based on the image template). Double click on the name of the matrix. A popup window (Matrix properties) will appear. Click on the save matrix file icon (a blue disc) and save the matrix with an appropriate filename. Now go to the Matrix line of the second segment, double click (on Unknown) as before and this time click on the Open matrix file icon (a folder) and read in the matrix that you saved from the first sector. You can now process the second segment using this matrix. It would be even nicer if you could drag and drop the matrix, this is on our to do list. Best wishes, Andrew On 17 Aug 2009, at 13:33, Brett, Thomas wrote: I am an imosflm novice and have a relatively simple question. I have a 360 deg data set collected in two swathes of 180 deg (one with phi=0 and omega going 0-180 and the second with phi = 180 and omega going 0-180). What is the easiest way to process the two datasets using a matching orientation matrix (or one rotated by 180 deg as it were) so that all the data can be merged together. Is there an easy way to do it in imosflm or must one process the two sets separately and then manipulate later with pointless before scalling and merging everything together? Thanks in advance. -Tom Tom J. Brett, PhD Assistant Professor of Medicine Division of Pulmonary and Critical Care Washington University School of Medicine Campus Box 8052, 660 S. Euclid Saint Louis, MO 63110
[ccp4bb] Histogram matching in DM - question
Hi everyone, I am trying to use density modification at rather low resolution (4-5A ) for an RNA structure. My first time ever with RNA. I usually use Histogram matching as part of the density modification scheme in DM. But this method is based on density distribution of protein maps I think. Is histogram matching still valid when it comes to RNA or protein/RNA structures ? And a general question regarding density modification and RNA structure. Can statistical density modification programs (RESOLVE, Pirate) take into consideration the chemical composition of the structure ? Shouldn't this composition affect the expected density distributions ? My gratitude in advance for your comments and advice, Peter.
Re: [ccp4bb] Histogram matching in DM - question
Hi Peter, Histogram matching works well for RNA structure, but be aware that density modification in general can be very tricky at low resolution. If by any means you can exploit averaging, e.g. multicrystal averaging of non-isomorphous crystals, it will probably be very helpful. It will also depend on the source of your phases Poul On 18/08/2009, at 21.43, Peter Grey wrote: Hi everyone, I am trying to use density modification at rather low resolution (4-5A ) for an RNA structure. My first time ever with RNA. I usually use Histogram matching as part of the density modification scheme in DM. But this method is based on density distribution of protein maps I think. Is histogram matching still valid when it comes to RNA or protein/ RNA structures ? And a general question regarding density modification and RNA structure. Can statistical density modification programs (RESOLVE, Pirate) take into consideration the chemical composition of the structure ? Shouldn't this composition affect the expected density distributions ? My gratitude in advance for your comments and advice, Peter.
Re: [ccp4bb] Histogram matching in DM - question
Peter Grey wrote: Hi everyone, I am trying to use density modification at rather low resolution (4-5A ) for an RNA structure. My first time ever with RNA. I usually use Histogram matching as part of the density modification scheme in DM. But this method is based on density distribution of protein maps I think. Is histogram matching still valid when it comes to RNA or protein/RNA structures ? I have the same question with respect to metalloproteins. Presumably the heavier metal atoms make spikes that are completely off the scale of a normal protein histogram. Is it then a bad idea to use histogram matching? Do the metals get flattened down to the highest density expected for protein on every cycle? Ed
[ccp4bb] selenomethionine labeled protein expression in insect cells
Hi everyone, I am looking for a method for expressing selenomethionine labeled protein in insect cells. Can anyone point me to a suitable protocol ? Additionally, is anyone aware of companies that may provide this service for a fee ? Thanks
Re: [ccp4bb] selenomethionine labeled protein expression in insect cells
Hi Riya- Expression Systems has a protocol for SeMet incorporation (Click on the Selenomethionine Incorporation pdf file at http://www.expressionsystems.com/?mvcTask=documents) and they advertise that they do just about anything you would want to do with insect cells (see http://www.expressionsystems.com/?mvcTask=contractProduction), including expression of recombinant protein in insect cells. I promise that I am not an employee or a shareholder of this company. Just used their products and I was looking for a SeMet protocol recently, too, and happen to find theirs online. You can contact me off the board if you want more details. HTH- Brad On Tue, Aug 18, 2009 at 5:09 PM, riya doreen driy...@gmail.com wrote: Hi everyone, I am looking for a method for expressing selenomethionine labeled protein in insect cells. Can anyone point me to a suitable protocol ? Additionally, is anyone aware of companies that may provide this service for a fee ? Thanks
Re: [ccp4bb] selenomethionine labeled protein expression in insect cells
We have unsuccessfully used in the past (by that I mean less than stellar incorporation) the Gibco/Invitrogen Sf900-SFM w/o Met/Cys media. Then we saw Karin Reinisch's paper (Dong et al, Immunity, 2009) and they used Expression Systems' ESF921. This protocol works for us. Engin On 8/18/09 2:49 PM, Brad Bennett wrote: Hi Riya- Expression Systems has a protocol for SeMet incorporation (Click on the Selenomethionine Incorporation pdf file at http://www.expressionsystems.com/?mvcTask=documents) and they advertise that they do just about anything you would want to do with insect cells (see http://www.expressionsystems.com/?mvcTask=contractProduction), including expression of recombinant protein in insect cells. I promise that I am not an employee or a shareholder of this company. Just used their products and I was looking for a SeMet protocol recently, too, and happen to find theirs online. You can contact me off the board if you want more details. HTH- Brad On Tue, Aug 18, 2009 at 5:09 PM, riya doreen driy...@gmail.com mailto:driy...@gmail.com wrote: Hi everyone, I am looking for a method for expressing selenomethionine labeled protein in insect cells. Can anyone point me to a suitable protocol ? Additionally, is anyone aware of companies that may provide this service for a fee ? Thanks -- Engin Özkan Post-doctoral Scholar Laboratory of K. Christopher Garcia Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111
