[ccp4bb] database-assisted data archive
Dear all, going through some previous lab member's data and trying to make sense of it, I was wondering what kind of solutions exist to simply the archiving and retrieval process. In particular, what I have in mind is a web interface that allows a user who has just returned from the synchrotron or the in-house detector to fill in a few boxes (user, name of protein, mutant, light source, quality of data, number of frames, status of project, etc) and then upload his data from the USB stick, portable hard drive or remote storage. The database application would put the data in a safe place (some file server that's periodically backed up) and let users browse through all the collected data of the lab with minimal effort later. I doesn't seem too hard to implement this, which is why I'm asking if anyone has done so already. Thanks. Andreas -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] database-assisted data archive
Do you want the frames to be accessible too ? If not, then a.wiki would be an easy solution. Alternatively a Filemaker database would do the trick too. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Aug 18, 2010, at 5:52, Andreas Förster docandr...@gmail.com wrote: Dear all, going through some previous lab member's data and trying to make sense of it, I was wondering what kind of solutions exist to simply the archiving and retrieval process. In particular, what I have in mind is a web interface that allows a user who has just returned from the synchrotron or the in-house detector to fill in a few boxes (user, name of protein, mutant, light source, quality of data, number of frames, status of project, etc) and then upload his data from the USB stick, portable hard drive or remote storage. The database application would put the data in a safe place (some file server that's periodically backed up) and let users browse through all the collected data of the lab with minimal effort later. I doesn't seem too hard to implement this, which is why I'm asking if anyone has done so already. Thanks. Andreas -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] database-assisted data archive
I did something like that for plasmids by putting together a web interface, php, and MySQL. It was simple, maybe a little ugly, but worked nicely. The problem was convincing anyone to actually use it was virtually impossible. --paul On 08/18/2010 04:52 AM, Andreas Förster wrote: Dear all, going through some previous lab member's data and trying to make sense of it, I was wondering what kind of solutions exist to simply the archiving and retrieval process. In particular, what I have in mind is a web interface that allows a user who has just returned from the synchrotron or the in-house detector to fill in a few boxes (user, name of protein, mutant, light source, quality of data, number of frames, status of project, etc) and then upload his data from the USB stick, portable hard drive or remote storage. The database application would put the data in a safe place (some file server that's periodically backed up) and let users browse through all the collected data of the lab with minimal effort later. I doesn't seem too hard to implement this, which is why I'm asking if anyone has done so already. Thanks. Andreas -- Paul Paukstelis, Ph.D Assistant Professor University of Maryland Chemistry Biochemistry Dept. Center for Biomolecular Structure Organization pauks...@umd.edu 301-405-9933
Re: [ccp4bb] database-assisted data archive
Dear all As CCP4, we are currently developing the new CCP4i that will include a database application that will store project and job data. The database schema has already been designed but its design is not final and can be modified depending on user feedback. Now, we are in the process of writing the database API. Any suggestions and ideas regarding data storage and retrieval are welcome. George Pelios CCP4 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Andreas Förster Sent: 18 August 2010 10:53 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] database-assisted data archive Dear all, going through some previous lab member's data and trying to make sense of it, I was wondering what kind of solutions exist to simply the archiving and retrieval process. In particular, what I have in mind is a web interface that allows a user who has just returned from the synchrotron or the in-house detector to fill in a few boxes (user, name of protein, mutant, light source, quality of data, number of frames, status of project, etc) and then upload his data from the USB stick, portable hard drive or remote storage. The database application would put the data in a safe place (some file server that's periodically backed up) and let users browse through all the collected data of the lab with minimal effort later. I doesn't seem too hard to implement this, which is why I'm asking if anyone has done so already. Thanks. Andreas -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
[ccp4bb] Refinement/Model-Building Density Modification
Hello All, I am currently solving a structure at 2A resolution with phases obtained from molecular replacement. Using the MR solution, I began refinement with Refmac using NCS restraints. I am currently building the parts of the model that were left out of the MR search model, and have just about successfully completed all three NCS-related chains. Obviously I will continue to use the most complete model for refinement, and plan to release the NCS restraints over the parts of the molecule that don't quite seem to obey perfectly. My question is, once I have connected density for all three chains will it still be worthwhile to perform density modification, such as solvent flipping/flattening or histogram matching (implemented through SOLOMON and/or DM) to improve phases? It seems that I have always been told that density modification is typically carried out at the beginning of refinement, prior to any model building, however my understanding of these types of density modification (particularly solvent flipping/flattening) leads me to believe that they would be most effective when more of the molecular envelope can be identified, such as during later stages of refinement. Also, I read something recently that lead me to believe that the solvent flattening procedure may be implicit in the implementation of NCS averaging in refinement software. I understand that the two processes are fundamentally different and independent of one another, but the information I recently read described something like the following (unless I misinterpreted). Because real space NCS averaging requires identification of the molecular envelope in the same fashion as solvent flattening, during NCS averaging the envelope is identified then the map is solvent-flattened and averaged using the NCS operator. I am unfamiliar with the inner-workings of most crystallographic software, so I was wondering if this is how NCS averaging is implemented in Refmac? I suppose another way to ask the question would be: If I have an NCS-averaged map from Refmac, is it already solvent-flattened? Any help would be much appreciated. I am still relatively new to the refinement process. Thanks, Mike Thompson -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Refinement/Model-Building Density Modification
Michael Thompson wrote: Hello All, I am currently solving a structure at 2A resolution with phases obtained from molecular replacement. Using the MR solution, I began refinement with Refmac using NCS restraints. I am currently building the parts of the model that were left out of the MR search model, and have just about successfully completed all three NCS-related chains. Obviously I will continue to use the most complete model for refinement, and plan to release the NCS restraints over the parts of the molecule that don't quite seem to obey perfectly. My question is, once I have connected density for all three chains will it still be worthwhile to perform density modification, such as solvent flipping/flattening or histogram matching (implemented through SOLOMON and/or DM) to improve phases? It seems that I have always been told that density modification is typically carried out at the beginning of refinement, prior to any model building, however my understanding of these types of density modification (particularly solvent flipping/flattening) leads me to believe that they would be most effective when more of the molecular envelope can be identified, such as during later stages of refinement. My feeling is that density modification is most useful when the model is substantially incomplete (or substantially wrong). But I'd use both the unmodified and modified map when trying to interpret the difficult bits. 'dm' is obsolete, I'd use 'parrot' by preference. Also, I read something recently that lead me to believe that the solvent flattening procedure may be implicit in the implementation of NCS averaging in refinement software. I understand that the two processes are fundamentally different and independent of one another, but the information I recently read described something like the following (unless I misinterpreted). Because real space NCS averaging requires identification of the molecular envelope in the same fashion as solvent flattening, during NCS averaging the envelope is identified then the map is solvent-flattened and averaged using the NCS operator. I am unfamiliar with the inner-workings of most crystallographic software, so I was wondering if this is how NCS averaging is implemented in Refmac? I suppose another way to ask the question would be: If I have an NCS-averaged map from Refmac, is it already solvent-flattened? Refinement with NCS restraints essentially imposes the NCS in a way which is different but redundant with density modification, so I wouldn't expect further density modification after refinement with NCS to help much. The same is somewhat true for solvent flattening too. The only thing density modification might get you at this stage is break you away from model bias a little. Again, it's so quick you may as well try it and look at both maps. Kevin -- EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm
Re: [ccp4bb] Refinement/Model-Building Density Modification
On 08/18/10 08:25, Michael Thompson wrote: Hello All, I am currently solving a structure at 2A resolution with phases obtained from molecular replacement. Using the MR solution, I began refinement with Refmac using NCS restraints. I am currently building the parts of the model that were left out of the MR search model, and have just about successfully completed all three NCS-related chains. Obviously I will continue to use the most complete model for refinement, and plan to release the NCS restraints over the parts of the molecule that don't quite seem to obey perfectly. My question is, once I have connected density for all three chains will it still be worthwhile to perform density modification, such as solvent flipping/flattening or histogram matching (implemented through SOLOMON and/or DM) to improve phases? It seems that I have always been told that density modification is typically carried out at the beginning of refinement, prior to any model building, however my understanding of these types of density modification (particularly solvent flipping/flattening) leads me to believe that they would be most effective when more of the molecular envelope can be identified, such as during later stages of refinement. As building refinement are a cyclical process, your density-modified phases will keep improving as your improved model provides a better-fitting mask. Once you reach the point where your model phases are better than the density-modified phases, and improvement from the dm maps is no longer apparent, you can quit the density modification. I tend to continue the dm longer than some other people, because it is free of some forms of model bias. Also, I read something recently that lead me to believe that the solvent flattening procedure may be implicit in the implementation of NCS averaging in refinement software. I understand that the two processes are fundamentally different and independent of one another, but the information I recently read described something like the following (unless I misinterpreted). Because real space NCS averaging requires identification of the molecular envelope in the same fashion as solvent flattening, during NCS averaging the envelope is identified then the map is solvent-flattened and averaged using the NCS operator. I am unfamiliar with the inner-workings of most crystallographic software, so I was wondering if this is how NCS averaging is implemented in Refmac? I suppose another way to ask the question would be: If I have an NCS-averaged map from Refmac, is it already solvent-flattened? Different density modification software packages deal with the averaging and solvent-flattening masks in different ways. Some packages solvent-flatten everything outside the NCS mask. Having dealt with situations in which the NCS relationship does not cover the entire molecule, I do not like that, and prefer that NCS and solvent flattening masks be dealt with separately (Schuller (1996) Acta Crystallogr. D52, 425-434.) IIRC, dm allows the option of dealing with the masks separately and I cannot remember which behaviour is the default. REFMAC is not a density modification application, but rather a refinement application. NCS restraints may apply to only part of a molecule if necessary. Any help would be much appreciated. I am still relatively new to the refinement process. Thanks, Mike Thompson -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] database-assisted data archive
Knowing where all the important files are is really all that is needed. Sofistication can come later. I would welcome a CCP4 database-assisted data archive system. Here is my contribution to the discussion: I agree with Paul Paukstelis that getting users to use any database-assisted data archive system is the biggest obstacle. I have had problems with compliance with my system, where all that the student has to do is to provide file and directory names each Friday to keep the database up to date. It is a simple html based access system where through hyperlinks one can access the data anywhere where it is stored. Users need only provide the directories names of where the various pieces of data are stored within the accessible network and the data manager (any HTML competent individual) can then set-up the links to the main control platform (start-up html page). The advantage of such system is that it is platform independent and needs only a well configured browser. It is backward compatible with any old data. George Pelios may want to consider an automated system where mosflm, scala and all subsequent programs contribute to create and update a raw data retrieval file on the basis of the files they have used. When the project is finished a backup program should be able to retrieve all such files to be stored in a consolidated manner for transfer to a long term storage server. A brief description of the system I use for synchrotron data collection: Prior to the synchrotron trip, each sample taken to the synchrotron is entered in a table that represents its position in the puck with hyperlinks to a file describing its position in the crystallization tray (this file will have hyperlinks to crystallization and all prior preparation steps). As data is collected a short comment (resolution and number of frames is included if data has been collected) as the data is transfered in the home lab a link to the directory where the data is stored is then added. To give an idea of data quality Mosflm and gimp screen capture are used to create a jpg of the first data image (with the frame filename added) which is stored in the same directory as the raw data frames. This image is accessed when clicking on the comment. Compliance with the system can be checked by clicking on comments other than not tested. It is all manual but is not very time consuming once the initial html templates have been set up. Still I am looking foward to a simple CCP4 designed system that can do something similar automatically. I would also recommend looking at ispyb implemented at the ESRF which is also web based: www.esrf.eu/UsersAndScience/Experiments/MX/Software/ispyb Enrico. -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] database-assisted data archive
Dear All, I would just like to add to Enrico's mention of ISPyB. This LIMS system will log all your data collected at the beamline (experimental parameters, screening images, data sets, edge scans, xrf spectra, crystal snapshots etc) automatically and is stored indefinitely. Your colleagues can also follow data collections in real time by logging on from their home labs. In addition, you can upload large amounts of information on your samples (acronym, space group, pin barcode etc) to the data base that can be recovered at the beamline through MXCuBE and the sample changer, tying all data collections to this information. You can also track your dewars to and from the ESRF using it - even receiving an email when it reaches the beamline. It has recently delved into the world of data analysis, as you can rank crystals against each other using a number of criteria. For those not in an exclusive relationship with the ESRF, you will be glad to hear it is also available at Diamond and I believe will be at PETRAIII. Cheers, Matt Some links: ISPyB: http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/ISPYB Sample tracking: http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/ISPYB/ispyb-dewar-tracking Ranking: http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/ISPYB/ispyb-sample-ranking Enrico Stura wrote: Knowing where all the important files are is really all that is needed. Sofistication can come later. I would welcome a CCP4 database-assisted data archive system. Here is my contribution to the discussion: I agree with Paul Paukstelis that getting users to use any database-assisted data archive system is the biggest obstacle. I have had problems with compliance with my system, where all that the student has to do is to provide file and directory names each Friday to keep the database up to date. It is a simple html based access system where through hyperlinks one can access the data anywhere where it is stored. Users need only provide the directories names of where the various pieces of data are stored within the accessible network and the data manager (any HTML competent individual) can then set-up the links to the main control platform (start-up html page). The advantage of such system is that it is platform independent and needs only a well configured browser. It is backward compatible with any old data. George Pelios may want to consider an automated system where mosflm, scala and all subsequent programs contribute to create and update a raw data retrieval file on the basis of the files they have used. When the project is finished a backup program should be able to retrieve all such files to be stored in a consolidated manner for transfer to a long term storage server. A brief description of the system I use for synchrotron data collection: Prior to the synchrotron trip, each sample taken to the synchrotron is entered in a table that represents its position in the puck with hyperlinks to a file describing its position in the crystallization tray (this file will have hyperlinks to crystallization and all prior preparation steps). As data is collected a short comment (resolution and number of frames is included if data has been collected) as the data is transfered in the home lab a link to the directory where the data is stored is then added. To give an idea of data quality Mosflm and gimp screen capture are used to create a jpg of the first data image (with the frame filename added) which is stored in the same directory as the raw data frames. This image is accessed when clicking on the comment. Compliance with the system can be checked by clicking on comments other than not tested. It is all manual but is not very time consuming once the initial html templates have been set up. Still I am looking foward to a simple CCP4 designed system that can do something similar automatically. I would also recommend looking at ispyb implemented at the ESRF which is also web based: www.esrf.eu/UsersAndScience/Experiments/MX/Software/ispyb Enrico. -- Matthew Bowler Structural Biology Group European Synchrotron Radiation Facility B.P. 220, 6 rue Jules Horowitz F-38043 GRENOBLE CEDEX FRANCE === Tel: +33 (0) 4.76.88.29.28 Fax: +33 (0) 4.76.88.29.04 http://www.esrf.fr/UsersAndScience/Experiments/MX/ ===
Re: [ccp4bb] autoBuster--Rfree_falg
Dear Jerry, On Tue, Aug 17, 2010 at 04:26:14PM -0700, Jerry McCully wrote: Dear All: I am currently using autoBuster to refine my structure. I notice that autobuster generates a new column in the MTZ data file with the label of FreeR_flag. Because my MTZ file has already had the FreeRflag, I am wondering whether autoBuster generated a new set of Rfreeflags of just renamed the existing FreeRflags. Can anyone give some comments? Yes, it created a new column because the existing one has a label different from the supported ones - see: http://www.globalphasing.com/buster/manual/autobuster/manual/appendix1.html#SetvarParameter_ColumnName_FreeR_flag_allowed What you could do is to add the following line to a ~/.autoBUSTER file: ColumnName_FreeR_flag_allowed= I FreeR_flag| I FREE| I FreeRflag Or use this on the command-line: % refine ColumnName_FreeR_flag_allowed= I FreeRflag \ -p some.pdb -m other.mtz ... Both should work. Alternatively you might want to use the more common (?) labels FREE or FreeR_flag when you create your MTZ file. Cheers Clemens PS: for BUSTER questions you can also use buster-deve...@globalphasing.com -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
[ccp4bb] Scaling up from an Intelliplate to Linbro Plate
Hi all, I know scaling up from a hit found from a high throughput screen is an empirical process, but does anyone have a good rule of thumb as a starting point when it comes to scaling up from a hit observed in an Intelliplate to a Linbro plate (i.e., change in volume ratios, amount to add to reservoir, etc)? I've Googled around but haven't seen anything which either suggests I shouldn't be asking this question, I've not looked hard enough, or it really is a case of try and see. Thanks
Re: [ccp4bb] database-assisted data archive
There is an image archiving system called TARDIS (http://tardis.edu.au/) that sounds more-or-less exactly like what you describe. I agree that it would be nice if you can get your synchrotron to do it for you, but since every single beamline and home-source setup in the world has already been providing you with a database that is more commonly called the image header, I don't think it is too hard to imagine how accurate the data in your database is going to be. If I may interject my two cents, I have found that when a user is asked to fill out a form, compliance is inversely proportional to the number of fields on the form. But far more important than that: if you ask them to answer a question that they simply don't know the answer to, they will likely skip the whole thing. An excellent example (I think) is asking for the space group BEFORE they have even taken their first snapshot of a brand new crystal. This datum is simply not known until AFTER the structure is solved! For example, is it P41 or P43? You don't really know that until after you see a helix in the map. What is the molecular weight? That depends on whether or not it is a complex. (if I had a nickel for every user who was certain they had a protein-DNA complex with a very low solvent content, I would be quite rich). All that said, I don't think it is unreasonable to expect an image header (or any other database) to contain motor positions, detector type, wavelength, beam center etc. Clearly this is not always the case, and this problem still needs a lot of work, but my point is that we should try to write down things that we really know (observations) and not try to muddle the database with derived quantities (interpretations). When it comes to what you really know about the sample, all you can realistically hope to be sure of is the list of chemicals that went into the drop: macromolecule sequence, salts, PEGs, and their respective concentrations. Sometimes you don't even kow that! (i.e. proteolysis). However, the macromolecule sequence is INCREDIBLY useful for deriving (or at least guessing) a great many other things (such as the molecular weight, solvent content, number of heavy atom sites). The list of salts is also absolutely critical for doing radiation damage predictions. So, as my rant comes to an end, I would strongly suggest focusing on trying to capture the important things that we actually do know, rather than confusing our poor users further by asking them to write down a lot of things that they don't. -James Holton MAD Scientist Andreas Förster wrote: Dear all, going through some previous lab member's data and trying to make sense of it, I was wondering what kind of solutions exist to simply the archiving and retrieval process. In particular, what I have in mind is a web interface that allows a user who has just returned from the synchrotron or the in-house detector to fill in a few boxes (user, name of protein, mutant, light source, quality of data, number of frames, status of project, etc) and then upload his data from the USB stick, portable hard drive or remote storage. The database application would put the data in a safe place (some file server that's periodically backed up) and let users browse through all the collected data of the lab with minimal effort later. I doesn't seem too hard to implement this, which is why I'm asking if anyone has done so already. Thanks. Andreas
Re: [ccp4bb] database-assisted data archive
What about XTrack? http://xray.bmc.uu.se/xtrack/ -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of James Holton Sent: 18 August 2010 16:54 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] database-assisted data archive There is an image archiving system called TARDIS (http://tardis.edu.au/) that sounds more-or-less exactly like what you describe. I agree that it would be nice if you can get your synchrotron to do it for you, but since every single beamline and home-source setup in the world has already been providing you with a database that is more commonly called the image header, I don't think it is too hard to imagine how accurate the data in your database is going to be. If I may interject my two cents, I have found that when a user is asked to fill out a form, compliance is inversely proportional to the number of fields on the form. But far more important than that: if you ask them to answer a question that they simply don't know the answer to, they will likely skip the whole thing. An excellent example (I think) is asking for the space group BEFORE they have even taken their first snapshot of a brand new crystal. This datum is simply not known until AFTER the structure is solved! For example, is it P41 or P43? You don't really know that until after you see a helix in the map. What is the molecular weight? That depends on whether or not it is a complex. (if I had a nickel for every user who was certain they had a protein-DNA complex with a very low solvent content, I would be quite rich). All that said, I don't think it is unreasonable to expect an image header (or any other database) to contain motor positions, detector type, wavelength, beam center etc. Clearly this is not always the case, and this problem still needs a lot of work, but my point is that we should try to write down things that we really know (observations) and not try to muddle the database with derived quantities (interpretations). When it comes to what you really know about the sample, all you can realistically hope to be sure of is the list of chemicals that went into the drop: macromolecule sequence, salts, PEGs, and their respective concentrations. Sometimes you don't even kow that! (i.e. proteolysis). However, the macromolecule sequence is INCREDIBLY useful for deriving (or at least guessing) a great many other things (such as the molecular weight, solvent content, number of heavy atom sites). The list of salts is also absolutely critical for doing radiation damage predictions. So, as my rant comes to an end, I would strongly suggest focusing on trying to capture the important things that we actually do know, rather than confusing our poor users further by asking them to write down a lot of things that they don't. -James Holton MAD Scientist Andreas Förster wrote: Dear all, going through some previous lab member's data and trying to make sense of it, I was wondering what kind of solutions exist to simply the archiving and retrieval process. In particular, what I have in mind is a web interface that allows a user who has just returned from the synchrotron or the in-house detector to fill in a few boxes (user, name of protein, mutant, light source, quality of data, number of frames, status of project, etc) and then upload his data from the USB stick, portable hard drive or remote storage. The database application would put the data in a safe place (some file server that's periodically backed up) and let users browse through all the collected data of the lab with minimal effort later. I doesn't seem too hard to implement this, which is why I'm asking if anyone has done so already. Thanks. Andreas Evotec (UK) Ltd is a limited company registered in England and Wales. Registration number:2674265. Registered office: 114 Milton Park, Abingdon, Oxfordshire, OX14 4SA, United Kingdom.
Re: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate
Hi Mo What you need to remember is that a relatively large amount of protein is lost from smaller drops. The ratio of surface area to volume is greater. With 100 + 100 nl drops about half of the protein is lost, either as skin on the drops or on the plastic of the plate. So when you scale up you need to reduce the protein by about half. (Another approach, suggested by Heather Ringrose, is to put extra protein into the drops at the screening stage, e.g. 200 nl protein + 100 nl reservoir solution. The hits found can usually be scaled up by dispensing 1 + 1 microlitre drops.) This is counterintuitive because people expect the small drops to dry out more quickly - so they expect, if anything, to get more precipitation in the small drops. Instead they get precipitation when they scale up, assuming they keep the ratio of protein to reservoir constant. It can also help, when you scale up, to increase the salt by 50 to 100% - this is indicated by data mining but I'm not sure what the mechanism is Hope that's helpful Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mo Wong Sent: 18 August 2010 16:18 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate Hi all, I know scaling up from a hit found from a high throughput screen is an empirical process, but does anyone have a good rule of thumb as a starting point when it comes to scaling up from a hit observed in an Intelliplate to a Linbro plate (i.e., change in volume ratios, amount to add to reservoir, etc)? I've Googled around but haven't seen anything which either suggests I shouldn't be asking this question, I've not looked hard enough, or it really is a case of try and see. Thanks
[ccp4bb] Job opportunities at the Protein Data Bank in Europe
There are three more vacancies coming up at the Protein Data Bank in Europe (PDBe; pdbe.org): - Head of PDBe Deposition and Annotation http://ig14.i-grasp.com/fe/tpl_embl01.asp?s=MbkMjPUrEcTFkHhTczjobid=40182,2388233441 - 50% Oracle DBA/50% Senior Software Engineer http://ig14.i-grasp.com/fe/tpl_embl01.asp?s=AdmOlRWtGeVHmJjVebjobid=40179,2133526947 - Scientific Programmer http://ig14.i-grasp.com/fe/tpl_embl01.asp?s=PyAxDIfSqHTyVvHqnjobid=40183,4715140288 For other job opportunities at the EBI, see http://www.embl.de/aboutus/jobs/searchjobs/index.php?searchregion=669 --Gerard --- Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK ger...@ebi.ac.uk . pdbe.org Secretary: Pauline Haslam pdbe_ad...@ebi.ac.uk
Re: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate
Thank you Patrick for your reply. As a note to others who might be interested, I found a few comments about scaling up interwoven in a long thread about which robot to buy that was posted on this bb a few years ago. The most salient link is probably: http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg04387.html Also, I found Patrick has a more detailed description about what should be of primary consideration during scale-up written in the following post: http://groups.google.com/group/oryx_group/browse_thread/thread/b04a2d7736d5974d?pli=1 Regards On Wed, Aug 18, 2010 at 12:56 PM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: Hi Mo What you need to remember is that a relatively large amount of protein is lost from smaller drops. The ratio of surface area to volume is greater. With 100 + 100 nl drops about half of the protein is lost, either as skin on the drops or on the plastic of the plate. So when you scale up you need to reduce the protein by about half. (Another approach, suggested by Heather Ringrose, is to put extra protein into the drops at the screening stage, e.g. 200 nl protein + 100 nl reservoir solution. The hits found can usually be scaled up by dispensing 1 + 1 microlitre drops.) This is counterintuitive because people expect the small drops to dry out more quickly - so they expect, if anything, to get more precipitation in the small drops. Instead they get precipitation when they scale up, assuming they keep the ratio of protein to reservoir constant. It can also help, when you scale up, to increase the salt by 50 to 100% - this is indicated by data mining but I’m not sure what the mechanism is Hope that’s helpful Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Mo Wong *Sent:* 18 August 2010 16:18 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Scaling up from an Intelliplate to Linbro Plate Hi all, I know scaling up from a hit found from a high throughput screen is an empirical process, but does anyone have a good rule of thumb as a starting point when it comes to scaling up from a hit observed in an Intelliplate to a Linbro plate (i.e., change in volume ratios, amount to add to reservoir, etc)? I've Googled around but haven't seen anything which either suggests I shouldn't be asking this question, I've not looked hard enough, or it really is a case of try and see. Thanks
[ccp4bb] PDBePISA assembly, summary and XML data files available from the Protein Data Bank in Europe
The Protein Data Bank in Europe (PDBe; http://pdbe.org/) is pleased to announce the availability of PISA assembly files, summaries and associated XML descriptors for all relevant entries in the Protein Data Bank (PDB) archive for download and in-house analysis. PDBePISA (http://pdbe.org/pisa/) is an advanced interactive tool for the prediction of probable quaternary structures (assemblies), analysis of macromolecular interfaces and surfaces, database searches for similar interfaces and assemblies, and retrieval of results based on various search criteria. PDBePISA also allows the upload of your own structure in PDB or mmCIF format for interface analysis or quaternary structure prediction. PDBePISA-predicted stable assembly files in PDB format are now available for download from the PDBe FTP area for all structures determined by diffraction methods. In addition, interface parameters (interface contacts, symmetry operators, hydrogen and disulphide bonds, salt bridges etc.) and assembly parameters (overall and buried surface areas, dissociation energies, contact interface characteristics) are available for every entry in XML format for further analysis. A summary index file is also provided in the FTP area containing a one-line summary for every stable assembly predicted by PDBePISA. This file provides an at-a-glance summary of the salient features of every stable assembly predicted by the program. This area is updated every Wednesday to coincide with the public update of the PDB archive. DATA ACCESS --- Please point your browser to: ftp://ftp.ebi.ac.uk/pub/databases/msd/pisa/ The file ftp://ftp.ebi.ac.uk/pub/databases/msd/pisa/index.txt contains the one-line summary for each assembly in column delimited format. Individual entry data may be found in a path like this: ftp://ftp.ebi.ac.uk/pub/databases/msd/pisa/data/xx/1xxx/ where 'xx' are the second and third characters of the PDB id code and 1xxx is the actual PDB id code. For example, information for PDB entry 1cbr may be found under ftp://ftp.ebi.ac.uk/pub/databases/msd/pisa/data/cb/1cbr/ The files in this directory will have names like - 1xxx.pdb.gz or 1xxx_n.pdb.gz for the assembly files (where 'n' is the assembly number when PDBePISA predicts multiple assemblies). - 1xxx_assembly.xml.gz : The assembly description in XML. - 1xxx_interface.xml.gz: The interface description in XML. As always, we welcome comments and suggestions on new features (preferably using the big, fat FEEDBACK button on the PDBe web pages). --Gerard --- Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK ger...@ebi.ac.uk . pdbe.org Secretary: Pauline Haslam pdbe_ad...@ebi.ac.uk
Re: [ccp4bb] database-assisted data archive
Dear Andreas, If you really want to do this, and want to define what is the data is, it's not _so_ difficult to do it yourself, with Ruby on Rails ( http://rubyonrails.org/) You have to know how to script a bit, and know a bit about Model/View/Controller frameworks. http://www.youtube.com/watch?v=Gzj723LkRJY That's not what you asked, but if you want to define what is the data to be input, you end up being unhappy with someone else's implementation. Mark 2010/8/18 Andreas Förster docandr...@gmail.com Dear all, going through some previous lab member's data and trying to make sense of it, I was wondering what kind of solutions exist to simply the archiving and retrieval process. In particular, what I have in mind is a web interface that allows a user who has just returned from the synchrotron or the in-house detector to fill in a few boxes (user, name of protein, mutant, light source, quality of data, number of frames, status of project, etc) and then upload his data from the USB stick, portable hard drive or remote storage. The database application would put the data in a safe place (some file server that's periodically backed up) and let users browse through all the collected data of the lab with minimal effort later. I doesn't seem too hard to implement this, which is why I'm asking if anyone has done so already. Thanks. Andreas -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- Skype: markabrooks
Re: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate
I assume you're sure you even *need* to scale up? Most of our structures come from crystals from small (150-300nl) drops, we consider a 100um crystal already huge. And if a smaller crystal doesn't diffract far enough on a modern beamline, chances are a large one won't either (quite apart from the trouble you'll have cryo-protecting it). (And yes, of course there *are* a few cases where larger = better.) phx On 18/08/2010 19:39, Mo Wong wrote: Thank you Patrick for your reply. As a note to others who might be interested, I found a few comments about scaling up interwoven in a long thread about which robot to buy that was posted on this bb a few years ago. The most salient link is probably: http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg04387.html Also, I found Patrick has a more detailed description about what should be of primary consideration during scale-up written in the following post: http://groups.google.com/group/oryx_group/browse_thread/thread/b04a2d7736d5974d?pli=1 Regards On Wed, Aug 18, 2010 at 12:56 PM, Patrick Shaw Stewart patr...@douglas.co.uk mailto:patr...@douglas.co.uk wrote: Hi Mo What you need to remember is that a relatively large amount of protein is lost from smaller drops. The ratio of surface area to volume is greater. With 100 + 100 nl drops about half of the protein is lost, either as skin on the drops or on the plastic of the plate. So when you scale up you need to reduce the protein by about half. (Another approach, suggested by Heather Ringrose, is to put extra protein into the drops at the screening stage, e.g. 200 nl protein + 100 nl reservoir solution. The hits found can usually be scaled up by dispensing 1 + 1 microlitre drops.) This is counterintuitive because people expect the small drops to dry out more quickly - so they expect, if anything, to get more precipitation in the small drops. Instead they get precipitation when they scale up, assuming they keep the ratio of protein to reservoir constant. It can also help, when you scale up, to increase the salt by 50 to 100% - this is indicated by data mining but I’m not sure what the mechanism is Hope that’s helpful Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.uk mailto:patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Mo Wong *Sent:* 18 August 2010 16:18 *To:* CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Scaling up from an Intelliplate to Linbro Plate Hi all, I know scaling up from a hit found from a high throughput screen is an empirical process, but does anyone have a good rule of thumb as a starting point when it comes to scaling up from a hit observed in an Intelliplate to a Linbro plate (i.e., change in volume ratios, amount to add to reservoir, etc)? I've Googled around but haven't seen anything which either suggests I shouldn't be asking this question, I've not looked hard enough, or it really is a case of try and see. Thanks
