Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner
Dear Artem, Thanks for your reply. You raise a number of points. Immediately, I should comment that the price of the PX Scanner is very considerably less than the $400k that you mention. Whilst - with the proteins and crystallisation conditions that you may be working with, visual inspection may be sufficient to differentiate salt from protein crystals (as you suggest), you will accept that generally this may not be the case. Thus, 'direct' inspection, using X-rays, must surely be the most appropriate way ? As you will be aware, the best looking crystals are seldom the best diffracting. This is well demonstrated through the PX Scanner 'Crystal Challenge', of course. Clearly, that's another prime purpose of the PX Scanner: to identify the 'best' crystals from amongst a multitude of candidates in a single droplet or across a plate, etc. Also, using the PX Scanner, we can check the effect of added cryo-protect. prior to freezing. Therefore, with this range of uses, the PX Scanner is clearly not intended for full 'data-collection' - but rather to most effectively support crystallisation optimisation and as a complement to in-house and central facility data-collection work. From the feedback that we receive, the PX Scanner is much valued by the number of groups which are now using these systems worldwide. However, even the proof of Grandma's apple pie is not until the eating. Accordingly, we most cordially invite you to visit one of our application labs - or perhaps one of our customer sites (by arrangement), with you, hopefully, being able to bring one or more of your crystallisation plates for inspection using the PX Scanner system. Since you are based in North America, I believe, one of my responsible colleagues will take up this invitation with you, off-BB. We look forward to our continuing discussions - with yourself, and all others who may be interested. Many Thanks and Best Regards, Marcus Winter (Agilent Technologies) [cid:image002.gif@01CBFE68.9EE7DB00]http://www.chem.agilent.com/en-US/Products/Instruments/X-raycrystallography/Pages/Crystalchallenge.aspx?cid=4710 From: Artem Evdokimov [mailto:artem.evdoki...@gmail.com] Sent: 19 April 2011 02:50 To: WINTER,MARCUS (A-UnitedKingdom,ex1) Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner Hi, So what's your secret - how did you pack an entire synchrotron into a little box? OK, so I am being facetious a little. However, I cannot help asking myself why would I want to spend so much money on a system that is basically a (vertical) X-ray diffractometer in a box, with fixed distance, and sans the ability to collect data? I can only guess that the system costs in the range of $400K (am I right?) and for that money one could get a pretty nice actual X-ray diffraction set-up... Now, if this thing cost say ... $80K I may be interested, although most of our crystals are so small that this set-up will uniformly score them as 'no idea' because they don't even diffract at home on a 'real' X-ray source with a CCD. Artem P.S. the day I start routinely confusing protein and salt crystals is the day I stop working in the lab :) On Mon, Apr 18, 2011 at 3:00 AM, Marcus Winter marcus.win...@agilent.commailto:marcus.win...@agilent.com wrote: Dear Chris, I'm prompted by your posting just to mention the Agilent Technologies PX Scanner 'Crystal Challenge' at: www.agilent.com/chem/crystalchallengehttp://www.agilent.com/chem/crystalchallenge Thus, the only really useful assessment, or 'score', of objects (putative crystals) - or crystallisation conditions, is by the actual observed diffraction characteristics... and these preferably directly in situ, in the horizontal crystallisation plate, as achieved in the PX Scanner. Many Thanks and Best Regards, Marcus Winter (Agilent Technologies) [https://mail.google.com/mail/html/compose/static_files/blank_quirks.html?ui=2ik=698a5dc8caview=attth=12f67a0673d77925attid=0.1disp=embzw]http://www.chem.agilent.com/en-US/Products/Instruments/X-raycrystallography/Pages/Crystalchallenge.aspx?cid=4710 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Ulens Sent: 18 April 2011 08:24 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] viewing and scoring crystallization drops on the iPad Our laboratory has been developing an application to view and score crystallization drops on the iPad. We would like to know if crystallographers see potential benefits from the functionality of the iPad to swipe and pinch through drops. We are looking for specific comments from Formulatrix users, but other users are also welcome to comment. http://www.youtube.com/watch?v=LezurNhm0pA Specific ideas for future development are: - composition of crystallization buffers on a back-flip of the image drop - back-sync of crystallization scores on the iPad with the image database
Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner
Dear Marcus, I always feel a bit uneasy about the advertisement-like posts that Agilent (and others) place on this BB. Of course, there are interactions between users and suppliers on many fronts, not least the support you guys provide in the form of sponsorship to meetings and conferences. Still, the original purpose of this bulletin board is the exchange of expertise and advice on a particular software package. No doubt, company-based crystallographers make valuable contributions to discussions on the BB. This is, however, different to placing an open sales pitch. I can remember that some in the community were miffed when discussions on non-CCP4 software packages became prominent. I think it is only fair to ask suppliers to minimise marketing of their products here. After all, the infrastructure for the BB is paid for by public money. With the obligatory '2 cents worth', Klaus === Klaus Fütterer, Ph.D. Reader in Structural Biology Undergraduate Admissions School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 19 Apr 2011, at 08:22, Marcus Winter wrote: Dear Artem, Thanks for your reply. You raise a number of points. Immediately, I should comment that the price of the PX Scanner is very considerably less than the $400k that you mention. Whilst - with the proteins and crystallisation conditions that you may be working with, visual inspection may be sufficient to differentiate salt from protein crystals (as you suggest), you will accept that generally this may not be the case. Thus, ‘direct’ inspection, using X-rays, must surely be the most appropriate way ? As you will be aware, the best looking crystals are seldom the best diffracting. This is well demonstrated through the PX Scanner ‘Crystal Challenge’, of course. Clearly, that’s another prime purpose of the PX Scanner: to identify the ‘best’ crystals from amongst a multitude of candidates in a single droplet or across a plate, etc. Also, using the PX Scanner, we can check the effect of added cryo- protect. prior to freezing. Therefore, with this range of uses, the PX Scanner is clearly not intended for full ‘data-collection’ – but rather to most effectively support crystallisation optimisation and as a complement to in-house and central facility data-collection work. From the feedback that we receive, the PX Scanner is much valued by the number of groups which are now using these systems worldwide. However, even the proof of Grandma’s apple pie is not until the eating. Accordingly, we most cordially invite you to visit one of our application labs – or perhaps one of our customer sites (by arrangement), with you, hopefully, being able to bring one or more of your crystallisation plates for inspection using the PX Scanner system. Since you are based in North America, I believe, one of my responsible colleagues will take up this invitation with you, off-BB. We look forward to our continuing discussions – with yourself, and all others who may be interested. Many Thanks and Best Regards, Marcus Winter (Agilent Technologies) image002.gif From: Artem Evdokimov [mailto:artem.evdoki...@gmail.com] Sent: 19 April 2011 02:50 To: WINTER,MARCUS (A-UnitedKingdom,ex1) Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner Hi, So what's your secret - how did you pack an entire synchrotron into a little box? OK, so I am being facetious a little. However, I cannot help asking myself why would I want to spend so much money on a system that is basically a (vertical) X-ray diffractometer in a box, with fixed distance, and sans the ability to collect data? I can only guess that the system costs in the range of $400K (am I right?) and for that money one could get a pretty nice actual X-ray diffraction set- up... Now, if this thing cost say ... $80K I may be interested, although most of our crystals are so small that this set-up will uniformly score them as 'no idea' because they don't even diffract at home on a 'real' X-ray source with a CCD. Artem P.S. the day I start routinely confusing protein and salt crystals is the day I stop working in the lab :) On Mon, Apr 18, 2011 at 3:00 AM, Marcus Winter marcus.win...@agilent.com wrote: Dear Chris, I’m prompted by your posting just to mention the Agilent Technologies PX Scanner ‘Crystal Challenge’ at: www.agilent.com/chem/crystalchallenge Thus, the only really useful
Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner
OK, I took the challenge. I got 7 out of 10. The three I missed were 2 questions about multi-well crystals which would be better (no problem) and the capillary (no problem either, because you can mount it)... I wouldn't be that snipe and braging (pun intended) if I would not agree with Klaus Jens On Tue, 2011-04-19 at 03:02 -0600, Marcus Winter wrote: Dear Klaus, Thanks for your note. Yes: we do understand the point that you make and, sincerely, we are sensitive to this possible criticism. However, we trust that you would agree that this was not a blatant advertisement. Also, my original posting was in direct response to a not unrelated one. Thank you for recognising the contributions made by the manufacturers. No doubt, we're – all of us, dependent upon public funding to some extent - directly or indirectly, and, similarly, we're taxpayers too... Anyway: why not entertain yourself by taking two minutes out for the PX Scanner Crystal Challenge: signature_crystalchall Many Thanks and Very Best Regards, Marcus (Agilent Technologies) -Original Message- From: Klaus Fütterer [mailto:k.futte...@bham.ac.uk] Sent: 19 April 2011 09:40 To: WINTER,MARCUS (A-UnitedKingdom,ex1) Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner Dear Marcus, I always feel a bit uneasy about the advertisement-like posts that Agilent (and others) place on this BB. Of course, there are interactions between users and suppliers on many fronts, not least the support you guys provide in the form of sponsorship to meetings and conferences. Still, the original purpose of this bulletin board is the exchange of expertise and advice on a particular software package. No doubt, company-based crystallographers make valuable contributions to discussions on the BB. This is, however, different to placing an open sales pitch. I can remember that some in the community were miffed when discussions on non-CCP4 software packages became prominent. I think it is only fair to ask suppliers to minimise marketing of their products here. After all, the infrastructure for the BB is paid for by public money. With the obligatory '2 cents worth', Klaus === Klaus Fütterer, Ph.D. Reader in Structural Biology Undergraduate Admissions School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 19 Apr 2011, at 08:22, Marcus Winter wrote: Dear Artem, Thanks for your reply. You raise a number of points. Immediately, I should comment that the price of the PX Scanner is very considerably less than the $400k that you mention. Whilst - with the proteins and crystallisation conditions that you may be working with, visual inspection may be sufficient to differentiate salt from protein crystals (as you suggest), you will accept that generally this may not be the case. Thus, ‘direct’ inspection, using X-rays, must surely be the most appropriate way ? As you will be aware, the best looking crystals are seldom the best diffracting. This is well demonstrated through the PX Scanner ‘Crystal Challenge’, of course. Clearly, that’s another prime purpose of the PX Scanner: to identify the ‘best’ crystals from amongst a multitude of candidates in a single droplet or across a plate, etc. Also, using the PX Scanner, we can check the effect of added cryo- protect. prior to freezing. Therefore, with this range of uses, the PX Scanner is clearly not intended for full ‘data-collection’ – but rather to most effectively support crystallisation optimisation and as a complement to in-house and central facility data-collection work. From the feedback that we receive, the PX Scanner is much valued by the number of groups which are now using these systems worldwide. However, even the proof of Grandma’s apple pie is not until the eating. Accordingly, we most cordially invite you to visit one of our application labs – or perhaps one of our customer
[ccp4bb] Cluster Design
Dear All, Here at NE-CAT, we make extensive use of XDS in a parallel environment. We are looking to purchase some new hardware, so I am soliciting your opinions. Our current cluster is made up of 16 nodes, each with 2 processors that have four cores, running at 2.2 GHz (I believe). We run with hyperthreading on, so 8 physical and 16 virtual cores per node. Our benchmarking with XDS (see https://rapd.nec.aps.anl.gov/wiki/RAPD_NecatStats for an example) shows a diminishing return on increasing the MAXIMUM_NUMBER_OF_PROCESSORS beyond the number of physical cores, and we are wondering if this is due to the test, the processor, the RAM, or XDS. In short, will going to 2 six core processors speed up processing using up to 12 for MAXIMUM_NUMBER_OF_PROCESSORS? Please do not feel the need to constrain the discussion to XDS, as we use our cluster for pretty much all the common crystallographic tasks. Thanks in advance, Frank Murphy Beamline Scientist, NE-CAT
Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner
Hi Marcus, I think you misinterpret my comments :) My first point is not that my specific proteins make it easy to distinguish salt from protein visually - it's rather that I am experienced enough and also know enough chemistry to make an educated conclusion about the contents of my drops. I expect this to be an entirely trainable skill since only very rudimentary inorganic chemistry knowledge is sufficient to knock off about 80% of all salt crystals based simply on the knowledge of drop contents, and the rest is experience that anyone with 1-2 years of crystallization practice can obtain. The crystal challenge while entertaining is nothing like the real world. In the real world I know what's in my drop, and in the real world I yet have to encounter a situation with TWO crystals in one drop of which only ONE is good, and the other will DIE as soon as I harvest the other one. This is too dramatic. Is this some sort of a Shroedinger crystal? Are the two crystals quantum-entangled? Crystallization is not a game, and if I only have one good crystal *ever* then I am not likely to get a structure, even if I know where that crystal is (we're talking new structure here, not a soak of a compound or a close MR, in which case there should not ever be only one crystal). Why don't I have more drops like this so I can harvest a couple of different crystals and get an actual dataset in the process? Come on. Likewise, most of the crystals in the challenge are way too huge - most hits aren't anywhere near that size which makes me wonder about the usefulness of the device in question for small nasty crystals - which are by far more common than the fat shiny ones that feature prominently in the challenge. On the subject of finding 'the best' crystals in a drop, or in a plate: if this was an actual syncrotron-quality beam capable of telling me right away that some of my 10x20x10 micron crystals are better than others then I would be much more curious. I however doubt that the instrument in question is very useful for crystals that are smaller than say 100x50x50 micron (assuming modest unit cell dimensions). As to the price - like you say the proof is in the pudding. The word 'considerable' is subject to *considerable* variance of meaning. For example, the actual price can be $390K which is a price of a cheap car's worth less than my original estimate of $400K -- is this considerable or not and more importantly is it still too much with respect to the value/price ratio? To me personally $10K is a lot, but the price/value ratio is still not favorable, the price would have to be around $80-90K to become interesting to me personally. I understand that it is not likely that the instrument of this sort can cost this little with today's technology. Which is why the cost/value matters :) Suppliers are reluctant to post specific prices of non-commodity items because then everyone would want to negotiate down from the same position - that's understandable, but I am not an equipment manufacturer and therefore I can understand but I do not have to sympathize. Without the actual price you can't really argue. And that's what brings me to the point that several others have made, which is that the etiquette of this board has always been to avoid direct advertisement. In my opinion it would have been more tactful to comment that 'direct X-ray study of crystals in-situ is a cool way to discover interesting things about your crystals' and leave the specific instrument that you sell to be discovered by whoever wants to dig deeper. Cheers, Artem On Tue, Apr 19, 2011 at 2:22 AM, marcus.win...@agilent.com wrote: Dear Artem, Thanks for your reply. You raise a number of points. Immediately, I should comment that the price of the PX Scanner is very considerably less than the $400k that you mention. Whilst - with the proteins and crystallisation conditions that you may be working with, visual inspection may be sufficient to differentiate salt from protein crystals (as you suggest), you will accept that generally this may not be the case. Thus, ‘direct’ inspection, using X-rays, must surely be the most appropriate way ? As you will be aware, the best looking crystals are seldom the best diffracting. This is well demonstrated through the PX Scanner ‘Crystal Challenge’, of course. Clearly, that’s another prime purpose of the PX Scanner: to identify the ‘best’ crystals from amongst a multitude of candidates in a single droplet or across a plate, etc. Also, using the PX Scanner, we can check the effect of added cryo-protect. *prior* to freezing. Therefore, with this range of uses, the PX Scanner is clearly not intended for full ‘data-collection’ – but rather to most effectively support crystallisation optimisation and as a complement to in-house and central facility data-collection work. From the feedback that we receive, the PX Scanner is much valued by the number of groups which
Re: [ccp4bb] Cluster Design
Hi Frank, the following are some recommendation for increasing the processing speed of XDS. You can find them (and add to them !) at http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Performance . Only item 7 is specific for a cluster. In the order of effect: 1. XDS scales well (i.e. the wallclock time for data processing goes down when the number of available cores is increased) in the COLSPOT, IDXREF, INTEGRATE and CORRECT steps when using the MAXIMUM_NUMBER_OF_PROCESSORS keyword. This triggers program-level parallelization, using OpenMP threads. 2. the program scales very well in the COLSPOT and INTEGRATE steps when using the MAXIMUM_NUMBER_OF_JOBS keyword. This triggers a shell-level parallelization. 3. combining these both keywords gives the highest performance in my experience (see [[1]] for an example). As a rough guide, I'd choose them to be approximately equal; an even number for MAXIMUM_NUMBER_OF_PROCESSORS should be chosen because that fits better with usual hardware. 4. some overcommitting of resources (i.e. MAXIMUM_NUMBER_OF_PROCESSORS * MAXIMUM_NUMBER_OF_JOBS number of cores) is beneficial; you'll have to play with these two parameters. 5. the next thing to consider is DELPHI together with OSCILLATION_RANGE: if DELPHI is an integer multiple of MAXIMUM_NUMBER_OF_PROCESSORS * OSCILLATION_RANGE that would be good because it nicely balances the usage of the threads. For this purpose, you may want to change (raise) the value of DELPHI (default is 5 degrees). If you are doing fine-slicing then mis-balancing of threads is not an issue - but for those users who want to collect 1° frames (which I think is not the best way nowadays ...) it should be a consideration. 6. performance-wise, I/O also plays a role because as soon as you run 24 or so processes then a single GB ethernet connection may be limiting. OTOH shell-level parallelization smoothes the load. 7. XDS with the MAXIMUM_NUMBER_OF_JOBS keyword can use several machines. This requires some setup as explained at the bottom of http://www.mpimf-heidelberg.mpg.de/~kabsch/xds/html_doc/downloading.html . 8. Hyperthreading (SMT), if available on Intel CPUs, is beneficial. A virtual core has only about 20% performance of a physical core but it comes at no cost - you just have to switch it on in the BIOS of the machine. 9. The 64-bit binaries generally are a bit faster than the 32-bit binaries (but that's not specific for XDS). HTH, Kay On 04/19/2011 02:06 PM, Frank Murphy wrote: Dear All, Here at NE-CAT, we make extensive use of XDS in a parallel environment. We are looking to purchase some new hardware, so I am soliciting your opinions. Our current cluster is made up of 16 nodes, each with 2 processors that have four cores, running at 2.2 GHz (I believe). We run with hyperthreading on, so 8 physical and 16 virtual cores per node. Our benchmarking with XDS (see https://rapd.nec.aps.anl.gov/wiki/RAPD_NecatStats for an example) shows a diminishing return on increasing the MAXIMUM_NUMBER_OF_PROCESSORS beyond the number of physical cores, and we are wondering if this is due to the test, the processor, the RAM, or XDS. In short, will going to 2 six core processors speed up processing using up to 12 for MAXIMUM_NUMBER_OF_PROCESSORS? Please do not feel the need to constrain the discussion to XDS, as we use our cluster for pretty much all the common crystallographic tasks. Thanks in advance, Frank Murphy Beamline Scientist, NE-CAT -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature smime.p7s. smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] Cluster Design
Hi Frank, sorry, my first response was not very specific for your situation! I studied your list and graphs and would just like to point out that in 2.1 Testing Filesystem Performance you may be severely overcommitting the CPU resources, if this was for one machine only with 16 (=8+8) cores. (but maybe your were really only interested in filesystem performance; we are generally happy with NFSv4 but haven't tried anything else) If testing with one machine, I'd try for XDS JOBS PROCESSORS 1 16 28 44 82 16 1 and then - if 4 4 was best, for example 54 64 We use several 48-core AMD machines (4*12-core 6176SE 2.3GHz CPUs), and we are happy with them for general work. We also use Intel (2*6-core X5670 2.93GHz Xeon, plus Hyperthreading) machines, which give a higher single-CPU performance, but of course 48 cores are nice in some situations (for XDS, e.g. 6 to 8 JOBS of 8 PROCESSORS each). OTOH more machines may mean more rack space, and more administration. HTH, Kay On 04/19/2011 02:06 PM, Frank Murphy wrote: Dear All, Here at NE-CAT, we make extensive use of XDS in a parallel environment. We are looking to purchase some new hardware, so I am soliciting your opinions. Our current cluster is made up of 16 nodes, each with 2 processors that have four cores, running at 2.2 GHz (I believe). We run with hyperthreading on, so 8 physical and 16 virtual cores per node. Our benchmarking with XDS (see https://rapd.nec.aps.anl.gov/wiki/RAPD_NecatStats for an example) shows a diminishing return on increasing the MAXIMUM_NUMBER_OF_PROCESSORS beyond the number of physical cores, and we are wondering if this is due to the test, the processor, the RAM, or XDS. In short, will going to 2 six core processors speed up processing using up to 12 for MAXIMUM_NUMBER_OF_PROCESSORS? Please do not feel the need to constrain the discussion to XDS, as we use our cluster for pretty much all the common crystallographic tasks. Thanks in advance, Frank Murphy Beamline Scientist, NE-CAT -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature smime.p7s. smime.p7s Description: S/MIME Cryptographic Signature
[ccp4bb] viewing and scoring diffraction using the PX Scanner
Dear Artem, Thanks for your very comprehensive reply. At the risk of reaping further criticism, I will now do little more than just reiterate my earlier comment: that from the feedback that we receive, the PX Scanner is much valued by the number and range of groups that are now using these systems.. and even for some very small crystals !! Also, I am pleased to confirm our invitation for you to collect data with us, wherever and whenever might be mutually convenient. Alternatively, you may be quite content that only your own Grandma's apple pie ever need be tasted. Many Thanks and Best Regards, Marcus Winter (Agilent Technologies) From: Artem Evdokimov [mailto:artem.evdoki...@gmail.com] Sent: 19 April 2011 13:30 To: WINTER,MARCUS (A-UnitedKingdom,ex1) Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner Hi Marcus, I think you misinterpret my comments :) My first point is not that my specific proteins make it easy to distinguish salt from protein visually - it's rather that I am experienced enough and also know enough chemistry to make an educated conclusion about the contents of my drops. I expect this to be an entirely trainable skill since only very rudimentary inorganic chemistry knowledge is sufficient to knock off about 80% of all salt crystals based simply on the knowledge of drop contents, and the rest is experience that anyone with 1-2 years of crystallization practice can obtain. The crystal challenge while entertaining is nothing like the real world. In the real world I know what's in my drop, and in the real world I yet have to encounter a situation with TWO crystals in one drop of which only ONE is good, and the other will DIE as soon as I harvest the other one. This is too dramatic. Is this some sort of a Shroedinger crystal? Are the two crystals quantum-entangled? Crystallization is not a game, and if I only have one good crystal *ever* then I am not likely to get a structure, even if I know where that crystal is (we're talking new structure here, not a soak of a compound or a close MR, in which case there should not ever be only one crystal). Why don't I have more drops like this so I can harvest a couple of different crystals and get an actual dataset in the process? Come on. Likewise, most of the crystals in the challenge are way too huge - most hits aren't anywhere near that size which makes me wonder about the usefulness of the device in question for small nasty crystals - which are by far more common than the fat shiny ones that feature prominently in the challenge. On the subject of finding 'the best' crystals in a drop, or in a plate: if this was an actual syncrotron-quality beam capable of telling me right away that some of my 10x20x10 micron crystals are better than others then I would be much more curious. I however doubt that the instrument in question is very useful for crystals that are smaller than say 100x50x50 micron (assuming modest unit cell dimensions). As to the price - like you say the proof is in the pudding. The word 'considerable' is subject to *considerable* variance of meaning. For example, the actual price can be $390K which is a price of a cheap car's worth less than my original estimate of $400K -- is this considerable or not and more importantly is it still too much with respect to the value/price ratio? To me personally $10K is a lot, but the price/value ratio is still not favorable, the price would have to be around $80-90K to become interesting to me personally. I understand that it is not likely that the instrument of this sort can cost this little with today's technology. Which is why the cost/value matters :) Suppliers are reluctant to post specific prices of non-commodity items because then everyone would want to negotiate down from the same position - that's understandable, but I am not an equipment manufacturer and therefore I can understand but I do not have to sympathize. Without the actual price you can't really argue. And that's what brings me to the point that several others have made, which is that the etiquette of this board has always been to avoid direct advertisement. In my opinion it would have been more tactful to comment that 'direct X-ray study of crystals in-situ is a cool way to discover interesting things about your crystals' and leave the specific instrument that you sell to be discovered by whoever wants to dig deeper. Cheers, Artem On Tue, Apr 19, 2011 at 2:22 AM, marcus.win...@agilent.commailto:marcus.win...@agilent.com wrote: Dear Artem, Thanks for your reply. You raise a number of points. Immediately, I should comment that the price of the PX Scanner is very considerably less than the $400k that you mention. Whilst - with the proteins and crystallisation conditions that you may be working with, visual inspection may be sufficient to differentiate salt from protein crystals (as you suggest), you will
[ccp4bb] Research Scientist – Structural Biology (Vertex)
Posted on behalf of Vertex Pharmaceuticals (please do not reply directly to me!) Research Scientist – Structural Biology Vertex Pharmaceuticals Incorporated is a global biotechnology company committed to the discovery and development of breakthrough small-molecule drugs for serious diseases. Our product pipeline is focused on viral diseases, inflammation, autoimmune diseases, cancer, pain and bacterial infection. A vacancy exists for a structural biologist in an expanding structural biology group in a dynamic, multi-disciplinary research environment. Working in a small team, you will play a critical role in providing insight into structure and function of proteins of therapeutic interest and understanding their interactions with ligands/inhibitors. As a creative thinker who can work independently to find innovative solutions, you will have every opportunity to draw on your proven track record in structural biology and your passion for its role in guiding structure-driven drug design. You will have a PhD (or equivalent) in physics, biophysics, biochemistry or related subject with evidence of productive post-doctoral/industrial employment experience in protein crystallography. Excellent analytical, organisational, communication and interpersonal skills are essential. Closing date: 13th May 2011. To apply, please visit our websitehttps://ukcareers.vrtx.com/1033/asp/tg/cim_jobdetail.asp?jobId=445798PartnerId=25119SiteId=5143type=mailJobReqLang=1recordstart=1JobSiteId=5143JobSiteInfo=445798_5143gqid=0
[ccp4bb] Flag tag vs. his-tag
Hi all, I am trying to decide between using a N-term his-tag or an N-term flag tag to be expressed on a protein that I will eventually want to try and crystallize. I usually don't cleave my tags unless absolutely necessary and I want to avoid double tagging. I am leaning towards the flag since from my experience this tag is good for protein interactions (pulldowns), but I don't know the effects of a flag tag in crystallization trials. Does anyone have experience with crystallizing protein with flag tags? Thanks, Christine
[ccp4bb] General question about Heme Binding Proteins
Dear CCP4 community, I have a question a bit off CCP4 topic, but that could use so expert input. While discussing about a bacterial secreted hemophore (heme scavenging protein) for which the apo form has been solved, it seems that attempts to obtain the Heme bound form are failing; in fact during data collection on a supposed Heme bound form, not only the Iron state changes (reduction), but the Heme position seems to be affected too. Would anybody have some experience with such a system and care to send some inputs (off line is fine as we are here a bit off topic)? Thank you in advance for your help. Best regards and Happy Building, Steph -- Stephane B. Richard, Ph.D., VP Business Development MEDIT US, 7985 Dunbrook Rd., Ste A, San Diego, CA 92126, USA MEDIT SA, 2 rue du Belvedere, 91120 Palaiseau, France http://www.linkedin.com/in/stephanebrichard Web: http://www.medit-pharma.com/ Email: srich...@medit.fr Cell: +1(858)342.6807 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nian Huang Sent: Monday, April 11, 2011 2:20 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] immobilized DNA resin Heparin simulates the structure of DNA and RNA, so it has nonspecific affinity towards DNA or RNA binding protein. It has also been used as DNAse or RNase inhibitor but it is not very good one. Nian Huang, Ph.D. UT Southwestern Medical Center On Sat, Apr 9, 2011 at 7:44 PM, Alexandra Deaconescu deac...@brandeis.edu wrote: Hello ccp4 enthusiasts: I am afraid this is a non-ccp4 related question. Can anyone recommend an immobilized dsDNA chromatographic resin for purification of DNA-binding proteins? GE seems to have something - I was wondering if people have other recommendations? In the age of GST and His tags etc., these are not very much used, but I do not have a tag in this case... Thanks a lot, Alex
Re: [ccp4bb] Flag tag vs. his-tag
His-tag is close to neutral but flag-tag is acidic. Using flag-tag for affinity purification usually gives cleaner protein but it costs a fortune due to low binding capacity of commercially available anti-flag resins. --Chun From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Harman, Christine Sent: Tuesday, April 19, 2011 11:11 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Flag tag vs. his-tag Hi all, I am trying to decide between using a N-term his-tag or an N-term flag tag to be expressed on a protein that I will eventually want to try and crystallize. I usually don't cleave my tags unless absolutely necessary and I want to avoid double tagging. I am leaning towards the flag since from my experience this tag is good for protein interactions (pulldowns), but I don't know the effects of a flag tag in crystallization trials. Does anyone have experience with crystallizing protein with flag tags? Thanks, Christine
Re: [ccp4bb] General question about Heme Binding Proteins
On 04/19/11 14:18, Stephane Richard wrote: Dear CCP4 community, I have a question a bit off CCP4 topic, but that could use so expert input. While discussing about a bacterial secreted hemophore (heme scavenging protein) for which the apo form has been solved, it seems that attempts to obtain the Heme bound form are failing; in fact during data collection on a supposed Heme bound form, not only the Iron state changes (reduction), but the Heme position seems to be affected too. Would anybody have some experience with such a system and care to send some inputs (off line is fine as we are here a bit off topic)? Thank you in advance for your help. Best regards and Happy Building, Steph 1) supposed heme bound form - I would imagine the colour difference should be quite clear. 2) Reduction - this is a well-known phenomenon. If it causes problems, you could pursue the strategy of Berglund, Carlsson, Smith, Szoke, Henriksen and Hajdu (23 May 2002), Nature 417, 463-468. Briefly, they collected data from multiple crystals and binned the images by X-ray exposure, thus obtaining a series of structures with different degrees of X-ray induced reduction. They collected their data at 100 K. 3) Heme position affected - A) At what temperature were data collected? B) How would you determine that the heme position changed w/o solving the structure? Cheers, -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Flag tag vs. his-tag
Thanks everyone for your comments. The overall opinion appears to be that large-scale purification of flag-tag protein is expensive. I 100% agree and have thought about that plus the dilemma with the flag-tag elution difficulty (low pH etc) which some of you also pointed out. So I will probably go with His. Thanks everyone for keeping me frugal. Cheers, Christine From: Chun Luo [mailto:c...@accelagen.com] Sent: Tuesday, April 19, 2011 3:18 PM To: Harman, Christine; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Flag tag vs. his-tag His-tag is close to neutral but flag-tag is acidic. Using flag-tag for affinity purification usually gives cleaner protein but it costs a fortune due to low binding capacity of commercially available anti-flag resins. --Chun From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Harman, Christine Sent: Tuesday, April 19, 2011 11:11 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Flag tag vs. his-tag Hi all, I am trying to decide between using a N-term his-tag or an N-term flag tag to be expressed on a protein that I will eventually want to try and crystallize. I usually don't cleave my tags unless absolutely necessary and I want to avoid double tagging. I am leaning towards the flag since from my experience this tag is good for protein interactions (pulldowns), but I don't know the effects of a flag tag in crystallization trials. Does anyone have experience with crystallizing protein with flag tags? Thanks, Christine
[ccp4bb] off topic: problematic protein
Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
Re: [ccp4bb] off topic: problematic protein
I recently followed a protocol from Stephen Sligar's lab for the purification of his nanodisc protein, which has strong hydrophobic character as it associates with phospholipids. His protocol includes washes with 1% Triton X-100 and then with 50 mM cholate (both at pH 8 in the presence of 300 mM NaCl). Worked great, and I saw stuff coming off the column in both washes. The reference is: Bayburt et al. (2002) Nano Letters, vol. 2, pp 853-856. http://pubs.acs.org/doi/abs/10.1021/nl025623k Good luck, Arthur Arthur Glasfeld Department of Chemistry Reed College 3203 SE Woodstock Blvd. Portland, OR 97202 USA On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
Re: [ccp4bb] off topic: problematic protein
HI Savvas, We recently had a protein that showed two overlapping peaks on the disposable fast flow Q columns, so we decided to see if we could resolve them with a higher resolution Q media. It ended up having 7 distinct peaks, only one of which was free of contaminants. We have also noticed that the presence of heat shock protein bound to our favorite protein is highly dependent on the induction time/temp, and also varies between bacterial strains. It is also effected by the media. Yo might try osmotic shock or other additives in the media. We have also had luck by adding 1-3 molar urea in the lysis buffer. We get lots more protein out, with better purity, but some of it crashes, which I think is purifying out the misfolded protein. Lastly, you might try a fusion protein to something that has chaperone activity, like MBP, which may mask the binding epitopes for the other proteins. Best regards, Kendall Nettles On Apr 19, 2011, at 3:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
Re: [ccp4bb] Flag tag vs. his-tag
My vote is for His-tag *unless* your protein is of the same general class as Zn-fingers, B-boxes, etc. (weak/multisite/uncertain metal binders) - these proteins often do not fare too well with His-tags because a) the tag residues can participate in artefactual metal-binding sites and b) the use of chelators (imidazole, histidine) can adversely affect the end product. We have had an example fairly recently where his-tag was forming an artefactual half-site with two other residues from a real zinc site, and the resulting protein preparation was brown/red due to the (artefact!) binding of iron. There is also I believe a recent paper highlighting a similar problem with a B-box protein (or a RING finger, don't recall the specifics, sorry). Artem On Tue, Apr 19, 2011 at 2:28 PM, Harman, Christine christine.har...@fda.hhs.gov wrote: Thanks everyone for your comments. The overall opinion appears to be that large-scale purification of flag-tag protein is expensive. I 100% agree and have thought about that plus the dilemma with the flag-tag elution difficulty (low pH etc) which some of you also pointed out. So I will probably go with His. Thanks everyone for keeping me frugal. Cheers, Christine -- *From:* Chun Luo [mailto:c...@accelagen.com] *Sent:* Tuesday, April 19, 2011 3:18 PM *To:* Harman, Christine; CCP4BB@JISCMAIL.AC.UK *Subject:* RE: [ccp4bb] Flag tag vs. his-tag His-tag is close to neutral but flag-tag is acidic. Using flag-tag for affinity purification usually gives cleaner protein but it costs a fortune due to low binding capacity of commercially available anti-flag resins. --Chun *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Harman, Christine *Sent:* Tuesday, April 19, 2011 11:11 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Flag tag vs. his-tag Hi all, I am trying to decide between using a N-term his-tag or an N-term flag tag to be expressed on a protein that I will eventually want to try and crystallize. I usually don't cleave my tags unless absolutely necessary and I want to avoid double tagging. I am leaning towards the flag since from my experience this tag is good for protein interactions (pulldowns), but I don't know the effects of a flag tag in crystallization trials. Does anyone have experience with crystallizing protein with flag tags? Thanks, Christine
