Re: [ccp4bb] Scaling with SCALA high and low resolution data sets
Dear Kyriacos, What kind of high-resolution data do you have? In my experience, while Scala usually produces excellent results, it often fails miserably or even crashes due to too negative intensities in case of low redundancy data. E.g. if one does a second high-resolution scan with a high swing-out angle with little low-resolution data and friedel mates. I have not looked whether Scala could be stabilized by somehow restraining the scale factors or switching off some refinement parameters. Since we process with XDS, in these cases I use XSCALE for scaling, which does produce very good results. My recommendation would be to process with XDS. By using the autoProc procedure from Global Phasing this is very easy, even for people who are normally not able to run a program without a GUI. You will then have to run XSCALE manually, which is again trivial once XDS had run correctly. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kyriacos Petratos Sent: Wednesday, March 20, 2013 6:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Scaling with SCALA high and low resolution data sets Dear All, we have two data sets at about 0.9 and 1.9 Ang. resolution collected from a single crystal. Integration with iMosflm seems to be fine like the scaling within each of the data sets. When we try to merge and scale both of them with 'Scala' we get extremely high scale factors for the lower resolution images varying between approximately 30 and 200! Do we need to pay attention to some particular options for running the program(s)? Thank you, Kyriacos e-mail: petra...@imbb.forth.gr
Re: [ccp4bb] Scaling with SCALA high and low resolution data sets
Dear Kyriacos, Just one thing to add to this suggestion from Herman: I personally view and study the plots generated by the SCALA/TRUNCATE or AIMLESS/TRUNCATE route after XDS processing (in particular the plot of Rmerge values as a function of Batch number) to eliminate deviating batches from processing. Once I have done this, XDS/XSCALE/XDSCONV gives me very adequate results (you will notice that I am not particularly faithful to a single suite of crystallographic software but that I pick what I want from each program suite... :-) ) HTH, Fred. On 21/03/13 08:41, herman.schreu...@sanofi.com wrote: Dear Kyriacos, What kind of high-resolution data do you have? In my experience, while Scala usually produces excellent results, it often fails miserably or even crashes due to too negative intensities in case of low redundancy data. E.g. if one does a second high-resolution scan with a high swing-out angle with little low-resolution data and friedel mates. I have not looked whether Scala could be stabilized by somehow restraining the scale factors or switching off some refinement parameters. Since we process with XDS, in these cases I use XSCALE for scaling, which does produce very good results. My recommendation would be to process with XDS. By using the autoProc procedure from Global Phasing this is very easy, even for people who are normally not able to run a program without a GUI. You will then have to run XSCALE manually, which is again trivial once XDS had run correctly. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kyriacos Petratos Sent: Wednesday, March 20, 2013 6:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Scaling with SCALA high and low resolution data sets Dear All, we have two data sets at about 0.9 and 1.9 Ang. resolution collected from a single crystal. Integration with iMosflm seems to be fine like the scaling within each of the data sets. When we try to merge and scale both of them with 'Scala' we get extremely high scale factors for the lower resolution images varying between approximately 30 and 200! Do we need to pay attention to some particular options for running the program(s)? Thank you, Kyriacos e-mail: petra...@imbb.forth.gr -- Fred. Vellieux (B.Sc., Ph.D., hdr) ouvrier de la recherche IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
Re: [ccp4bb] Scaling with SCALA high and low resolution data sets
Or, seeing that this is the CCP4BB smile, you could use the Xia2 pipeline from CCP4i - which will also use XDS if installed on your system. My recommendation would be to process with XDS. By using the autoProc procedure from Global Phasing this is very easy, even for people who are normally not able to run a program without a GUI. You will then have to run XSCALE manually, which is again trivial once XDS had run correctly. Good luck! Herman Tony. On 21 Mar 2013, at 07:41, herman.schreu...@sanofi.com wrote: Dear Kyriacos, -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kyriacos Petratos Sent: Wednesday, March 20, 2013 6:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Scaling with SCALA high and low resolution data sets Dear All, we have two data sets at about 0.9 and 1.9 Ang. resolution collected from a single crystal. Integration with iMosflm seems to be fine like the scaling within each of the data sets. When we try to merge and scale both of them with 'Scala' we get extremely high scale factors for the lower resolution images varying between approximately 30 and 200! Do we need to pay attention to some particular options for running the program(s)? Thank you, Kyriacos e-mail: petra...@imbb.forth.gr
Re: [ccp4bb] Resolution limit of index in XDS
Dear Tim, It could be that COLSPOT does not rely on experimental setup parameters. However, XDS must have reasonably close starting values for distance, direct beam position etc., otherwise the autoindexing would fail, so the information to calculate an approximate TRUSTED_REGION is available. For good data, a limited spot range usually works as well. However, for the weakly diffracting bad crystals with ice rings, salt spots, multiple diffraction patterns etc., one often needs the full range and often needs several tries with different parameters before indexing is successful. Since it is only cpu-time, it is the least of my worries and, as you mention, it is not bad to be forced to think once in a while instead of just clicking buttons in GUI's. Best regards, Herman -Original Message- From: Tim Gruene [mailto:t...@shelx.uni-ac.gwdg.de] Sent: Wednesday, March 20, 2013 11:17 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Herman, the short answer might be that at the stage of COLSPOT the term 'resolution' has a limited meaning because COLSPOT does not rely on the experimental setup like distance and beam direction, so the term 'resolution limit' is conceptually not applicable at this stage. Indexing does often not require the full data set, you can reduce the SPOT_RANGE if you are worried about processing time, or by a multi-CPU machine. One of the great advantages of XDS is that it asks you to think at a level higher than the average MS-Windows user while processing your data, so the effort to figure out the three numbers to set the TRUSTED_REGION is in line with the philosphy of XDS as I understand it. But you are right, I do not have access to the source of XDS and I am not the person to address a request to. Kind regards, Tim On 03/20/2013 10:29 AM, herman.schreu...@sanofi.com wrote: Dear Tim, but probably I should adres this to Kai Diederichs, not including the resolution cutoff in COLSPOT and IDXREF is a feature of XDS I do not understand at all. For most cases, it may not matter since only the strong spots are used, but what are the advantages? In fact there are disadvantages, especially when dealing with poorly diffracting difficult data sets: -when a crystallographer imposes a resolution limit, there are usually good reasons for it. -outside the resolution limit, there may be ice rings or contaminating salt spots, which make the autoindexing fail. -when processing 900 frame Pilatus data sets, running COLSPOT on the complete detector surface takes significantly longer then running it only on the center region. Of course, one could fudge a resolution cutoff by translating resolution into pixels and then calculating a TRUSTED_REGION, or manually editing the SPOT.XDS file, but this is a lot of extra and in my view unneccessary work. I would really consider using the resolution cutoff for COLSPOT as well. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Tuesday, March 19, 2013 11:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS Dear Niu, indexing relies on strong reflections only, that is (in very brieft) why INCLUDE_RESOLUTION_RANGE indeed does not affect the relections collected in COLSPOT which in turn are used by IDXREF. You can work around this, however, by making use of TRUSTED_REGION and set it to e.g. 0.7 or 0.6 (you can use adxv to translate resolution into pixel and then calculate the fraction you need to set the second number in TRUSTED_REGION to (or the first if you want to exclude the inner resolution reflections - I remember one data set where this was essential for indexing - DNA was involved there) Best, Tim On 03/19/2013 08:53 PM, Niu Tou wrote: Dear All, Is there any command can set the resolution limit for index step in XDS? I only found a keyword INCLUDE_RESOLUTION_RANGE, but it looks to be a definition of resolution range after index step as it says: INCLUDE_RESOLUTION_RANGE=20.0 0.0 !Angstroem; used by DEFPIX,INTEGRATE,CORRECT Thanks! Niu - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRSjVtUxlJ7aRr7hoRApZhAJ9RFBs8D9NGjgLY3KOoNHhNtdOWggCgj7U0 zY7jEFDYZfl0Umb9E1Bzs1U= =+HjR -END PGP SIGNATURE-
Re: [ccp4bb] Resolution limit of index in XDS
Dear Herman, some pros and cons are documented at http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Wishlist#Would_be_nice_to_have , and the workaround is at http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Ice_rings . These XDSwiki articles are old, and nobody has contributed to the discussion since 2007 (after all, is is a Wiki!), so there has not been much reason for a change. Tim is right in that usage of INCLUDE_RESOLUTION_RANGE does not fit well at the COLSPOT stage, since COLSPOT knows nothing about wavelength, distance, pixel size and so on. If there is agreement among XDS users that IDXREF should take INCLUDE_RESOLUTION_RANGE into account, there is a good chance that the next version of XDS will do that. best, Kay On Wed, 20 Mar 2013 09:29:47 +, herman.schreu...@sanofi.com wrote: Dear Tim, but probably I should adres this to Kai Diederichs, not including the resolution cutoff in COLSPOT and IDXREF is a feature of XDS I do not understand at all. For most cases, it may not matter since only the strong spots are used, but what are the advantages? In fact there are disadvantages, especially when dealing with poorly diffracting difficult data sets: -when a crystallographer imposes a resolution limit, there are usually good reasons for it. -outside the resolution limit, there may be ice rings or contaminating salt spots, which make the autoindexing fail. -when processing 900 frame Pilatus data sets, running COLSPOT on the complete detector surface takes significantly longer then running it only on the center region. Of course, one could fudge a resolution cutoff by translating resolution into pixels and then calculating a TRUSTED_REGION, or manually editing the SPOT.XDS file, but this is a lot of extra and in my view unneccessary work. I would really consider using the resolution cutoff for COLSPOT as well. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Tuesday, March 19, 2013 11:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Niu, indexing relies on strong reflections only, that is (in very brieft) why INCLUDE_RESOLUTION_RANGE indeed does not affect the relections collected in COLSPOT which in turn are used by IDXREF. You can work around this, however, by making use of TRUSTED_REGION and set it to e.g. 0.7 or 0.6 (you can use adxv to translate resolution into pixel and then calculate the fraction you need to set the second number in TRUSTED_REGION to (or the first if you want to exclude the inner resolution reflections - I remember one data set where this was essential for indexing - DNA was involved there) Best, Tim On 03/19/2013 08:53 PM, Niu Tou wrote: Dear All, Is there any command can set the resolution limit for index step in XDS? I only found a keyword INCLUDE_RESOLUTION_RANGE, but it looks to be a definition of resolution range after index step as it says: INCLUDE_RESOLUTION_RANGE=20.0 0.0 !Angstroem; used by DEFPIX,INTEGRATE,CORRECT Thanks! Niu - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRSOFJUxlJ7aRr7hoRAo6TAKC+BePgeODbDyngO7N8vCE4CnjxmQCfS5cP srShHNz1sDK0EMHSbE3fDwA= =kAwf -END PGP SIGNATURE-
Re: [ccp4bb] Resolution limit of index in XDS
On Thu, 21 Mar 2013 08:28:27 +, herman.schreu...@sanofi.com wrote: Dear Tim, It could be that COLSPOT does not rely on experimental setup parameters. However, XDS must have reasonably close starting values for distance, direct beam position etc., otherwise the autoindexing would fail, so the information to calculate an approximate TRUSTED_REGION is available. TRUSTED_REGION and INCLUDE_RESOLUTION_RANGE are orthogonal concepts; both are input by the user and not calculated by the program. For good data, a limited spot range usually works as well. However, for the weakly diffracting bad crystals with ice rings, salt spots, multiple diffraction patterns etc., one often needs the full range and often needs several tries with different parameters before indexing is successful. Since it is only cpu-time, it is the least of my worries and, as you mention, it is not bad to be forced to think once in a while instead of just clicking buttons in GUI's. Nevertheless I plan to release a GUI for xds soon; among other things, this enables to visualize and change TRUSTED_REGION and INCLUDE_RESOLUTION_RANGE . best, Kay Best regards, Herman -Original Message- From: Tim Gruene [mailto:t...@shelx.uni-ac.gwdg.de] Sent: Wednesday, March 20, 2013 11:17 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Herman, the short answer might be that at the stage of COLSPOT the term 'resolution' has a limited meaning because COLSPOT does not rely on the experimental setup like distance and beam direction, so the term 'resolution limit' is conceptually not applicable at this stage. Indexing does often not require the full data set, you can reduce the SPOT_RANGE if you are worried about processing time, or by a multi-CPU machine. One of the great advantages of XDS is that it asks you to think at a level higher than the average MS-Windows user while processing your data, so the effort to figure out the three numbers to set the TRUSTED_REGION is in line with the philosphy of XDS as I understand it. But you are right, I do not have access to the source of XDS and I am not the person to address a request to. Kind regards, Tim On 03/20/2013 10:29 AM, herman.schreu...@sanofi.com wrote: Dear Tim, but probably I should adres this to Kai Diederichs, not including the resolution cutoff in COLSPOT and IDXREF is a feature of XDS I do not understand at all. For most cases, it may not matter since only the strong spots are used, but what are the advantages? In fact there are disadvantages, especially when dealing with poorly diffracting difficult data sets: -when a crystallographer imposes a resolution limit, there are usually good reasons for it. -outside the resolution limit, there may be ice rings or contaminating salt spots, which make the autoindexing fail. -when processing 900 frame Pilatus data sets, running COLSPOT on the complete detector surface takes significantly longer then running it only on the center region. Of course, one could fudge a resolution cutoff by translating resolution into pixels and then calculating a TRUSTED_REGION, or manually editing the SPOT.XDS file, but this is a lot of extra and in my view unneccessary work. I would really consider using the resolution cutoff for COLSPOT as well. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Tuesday, March 19, 2013 11:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS Dear Niu, indexing relies on strong reflections only, that is (in very brieft) why INCLUDE_RESOLUTION_RANGE indeed does not affect the relections collected in COLSPOT which in turn are used by IDXREF. You can work around this, however, by making use of TRUSTED_REGION and set it to e.g. 0.7 or 0.6 (you can use adxv to translate resolution into pixel and then calculate the fraction you need to set the second number in TRUSTED_REGION to (or the first if you want to exclude the inner resolution reflections - I remember one data set where this was essential for indexing - DNA was involved there) Best, Tim On 03/19/2013 08:53 PM, Niu Tou wrote: Dear All, Is there any command can set the resolution limit for index step in XDS? I only found a keyword INCLUDE_RESOLUTION_RANGE, but it looks to be a definition of resolution range after index step as it says: INCLUDE_RESOLUTION_RANGE=20.0 0.0 !Angstroem; used by DEFPIX,INTEGRATE,CORRECT Thanks! Niu - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRSjVtUxlJ7aRr7hoRApZhAJ9RFBs8D9NGjgLY3KOoNHhNtdOWggCgj7U0
Re: [ccp4bb] Resolution limit of index in XDS
I was a little provokative. A GUI with a viewer would actually be an excellent idea since it allows one to see what one is doing, which would be of great help for difficult data sets. Nevertheless, since XDS is part of many automated pipelines, the possibility to run XDS offline with a command file should not be touched. If IDXREF would take the INCLUDE_RESOLUTION_RANGE into account, I am sure this would improve the performance of XDS. Best regards and thank you for the work you put into XDS! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay Diederichs Sent: Thursday, March 21, 2013 10:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS On Thu, 21 Mar 2013 08:28:27 +, herman.schreu...@sanofi.com wrote: Dear Tim, It could be that COLSPOT does not rely on experimental setup parameters. However, XDS must have reasonably close starting values for distance, direct beam position etc., otherwise the autoindexing would fail, so the information to calculate an approximate TRUSTED_REGION is available. TRUSTED_REGION and INCLUDE_RESOLUTION_RANGE are orthogonal concepts; both are input by the user and not calculated by the program. For good data, a limited spot range usually works as well. However, for the weakly diffracting bad crystals with ice rings, salt spots, multiple diffraction patterns etc., one often needs the full range and often needs several tries with different parameters before indexing is successful. Since it is only cpu-time, it is the least of my worries and, as you mention, it is not bad to be forced to think once in a while instead of just clicking buttons in GUI's. Nevertheless I plan to release a GUI for xds soon; among other things, this enables to visualize and change TRUSTED_REGION and INCLUDE_RESOLUTION_RANGE . best, Kay Best regards, Herman -Original Message- From: Tim Gruene [mailto:t...@shelx.uni-ac.gwdg.de] Sent: Wednesday, March 20, 2013 11:17 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Herman, the short answer might be that at the stage of COLSPOT the term 'resolution' has a limited meaning because COLSPOT does not rely on the experimental setup like distance and beam direction, so the term 'resolution limit' is conceptually not applicable at this stage. Indexing does often not require the full data set, you can reduce the SPOT_RANGE if you are worried about processing time, or by a multi-CPU machine. One of the great advantages of XDS is that it asks you to think at a level higher than the average MS-Windows user while processing your data, so the effort to figure out the three numbers to set the TRUSTED_REGION is in line with the philosphy of XDS as I understand it. But you are right, I do not have access to the source of XDS and I am not the person to address a request to. Kind regards, Tim On 03/20/2013 10:29 AM, herman.schreu...@sanofi.com wrote: Dear Tim, but probably I should adres this to Kai Diederichs, not including the resolution cutoff in COLSPOT and IDXREF is a feature of XDS I do not understand at all. For most cases, it may not matter since only the strong spots are used, but what are the advantages? In fact there are disadvantages, especially when dealing with poorly diffracting difficult data sets: -when a crystallographer imposes a resolution limit, there are usually good reasons for it. -outside the resolution limit, there may be ice rings or contaminating salt spots, which make the autoindexing fail. -when processing 900 frame Pilatus data sets, running COLSPOT on the complete detector surface takes significantly longer then running it only on the center region. Of course, one could fudge a resolution cutoff by translating resolution into pixels and then calculating a TRUSTED_REGION, or manually editing the SPOT.XDS file, but this is a lot of extra and in my view unneccessary work. I would really consider using the resolution cutoff for COLSPOT as well. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Tuesday, March 19, 2013 11:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS Dear Niu, indexing relies on strong reflections only, that is (in very brieft) why INCLUDE_RESOLUTION_RANGE indeed does not affect the relections collected in COLSPOT which in turn are used by IDXREF. You can work around this, however, by making use of TRUSTED_REGION and set it to e.g. 0.7 or 0.6 (you can use adxv to translate resolution into pixel and then calculate the fraction you need to set the second number in TRUSTED_REGION to (or the first if you want to exclude the inner resolution reflections - I remember one data set where this was
Re: [ccp4bb] suitable buffer for CD studies
Respected All, Thanks for your valuable suggestions and inputs. with regards, Harsh On Thu, Mar 21, 2013 at 7:38 AM, Bosch, Juergen jubo...@jhsph.edu wrote: Yep, mostly you should stay away from Tris as this is the worst buffer system when playing with temperature changes. Tris for example has a ∆pKa/10˚C -0.31 Good, N.E. (1986) Biochemistry 5, 467 Jürgen P.S. @Matthew, was this what you meant by the *Good* buffers often not ? or just a coincidence ? On Mar 20, 2013, at 9:57 PM, Matthew Merski wrote: One of the other things you need to be concerned about with thermal melts is the change in buffer pKa as temperature varies (I seem to remember this being called the beta factor). Phosphate is used for CD melts regularly because its pKa is fairly invariant with temperature. (A good reference is Data for Biochemical Research by Dawson, Ch. 18). Acetate also shares this invariance but the Good buffers often do not. This is of course a concern with the Spyro Orange experiment as well. Matthew Merski Shoichet Group UCSF .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
[ccp4bb] homology modeling of dimeric proteins
Dear All, Could annyone please suggest me any program or server that can build homology based models of dimeric/oligomeric proteins? Previously I have used many software/servers which can build the monomer of my target proteins but not the dimers. Self docking of homology based monomer is not working in my case since at certain domain one monomer is to some extent wrapped around the adjacent monomer in the template structure. I have also tried the project mode of SWISS-MODEL server but the program is not running with an error message of sequence misalignment; although I think the alignment is fine!!! Please suggest something that would be fruitful. Thanking you in advance for your kind cooperation. Regards, Sudipta Bhattacharyya, Department of Biochemistry and Molecular Biology, Colorado State University.
[ccp4bb] Coot question
Dear all, My question concerns the Extension- modelling- Rigid body fit residue ranges function in Coot. Although it works well through the interface, I cannot have it to work in a python script, does somebody know the correct syntax? Thanks in advance, Nicolas
[ccp4bb] Microlytic: Laboratory Sales Representatives (2 US-based positions available immediately)
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[ccp4bb] Oligomerization state
Dear all I have a His-tagged membrane protein with unknown oligomerization state. But I am worried that tag addition may induce different state than in native and affect its crystallizability. Is there a single method that can determine the oligomerization state with nearly 100% accuracy? I have use of AUC and SAXS but there seems to be ambiguity about detergent and lipid effects. Is Thermofluor a right method? Does oligomerization require special assembly proteins, which will mean that tag cleavage is not useful to obtain native state? Thank you. Theresa
Re: [ccp4bb] Off topic: Oligonucleotide purity (Summary)
Dear all I received off-board replies on this subject. Most people use standard desalting for all cloning and mutagenesis experiments regardless of length. One reply recommended desalting for up to 40 bases and HPLC for longer oligos because of larger ratio of (n-1) contaminants. For crystallization, PAGE was suggested. Thank you again for all your suggestions! Theresa
Re: [ccp4bb] Oligomerization state
Dear Theresa, Although I haven't yet done the experiment myself, I am told that microscale thermophoresis (MST) is another useful method for oligomerization measurements, especially since it is designed to work for membrane proteins in presence of detergents etc. If you want to quickly learn more about microscale thermophoresis, you can google the technique for further information and literature. If you end up wanting to try MST experiments and don't have much of a handle on the method yet, contact me off the bulletin board and I'd be happy to give you a few pointers. As far as Thermofluor goes, some of the detergents work better than others in Thermofluor experiments so you may have it to determine the issues in your case empirically. Good luck! Raji On Thu, Mar 21, 2013 at 2:10 PM, Theresa Hsu theresah...@live.com wrote: Dear all I have a His-tagged membrane protein with unknown oligomerization state. But I am worried that tag addition may induce different state than in native and affect its crystallizability. Is there a single method that can determine the oligomerization state with nearly 100% accuracy? I have use of AUC and SAXS but there seems to be ambiguity about detergent and lipid effects. Is Thermofluor a right method? Does oligomerization require special assembly proteins, which will mean that tag cleavage is not useful to obtain native state? Thank you. Theresa -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] homology modeling of dimeric proteins
I have successfully used modeller (standalone software; Sali lab) to generate multimeric complexes up to 24-mers Hope tis helps From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sudipta Bhattacharyya Sent: Friday, 22 March 2013 5:00 a.m. To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] homology modeling of dimeric proteins Dear All, Could annyone please suggest me any program or server that can build homology based models of dimeric/oligomeric proteins? Previously I have used many software/servers which can build the monomer of my target proteins but not the dimers. Self docking of homology based monomer is not working in my case since at certain domain one monomer is to some extent wrapped around the adjacent monomer in the template structure. I have also tried the project mode of SWISS-MODEL server but the program is not running with an error message of sequence misalignment; although I think the alignment is fine!!! Please suggest something that would be fruitful. Thanking you in advance for your kind cooperation. Regards, Sudipta Bhattacharyya, Department of Biochemistry and Molecular Biology, Colorado State University.
Re: [ccp4bb] Oligomerization state
Hello Theresa, Maybe a functional assay (if possible) is better than determining the oligomeric state? Why is finding out the oligomeric state (which is unknown anyway and the answer can be compared to nothing), the answer to is having a tag ok? will the protein crystallize? What if the analysis shows a mixture of different protein oligomeric states in solution? For the general analysis, try SEC-MALLS. I guess the accuracy is 90-95 % for membrane proteins. You might have however to try different detergents or try different columns depending on the empty micelle size and how close it is to the protein-micelle peak etc (please ask if you need a detailed explanation). You will still need to do a func assay, because again you don't know if a particular detergent is harsh from the beginning affecting the oligomeric state, and it is not the tag. Best wishes toufic PS: (1) 100 % accuracy is a complicated term (even crystallography has a R-factor). The error value might be a different oligomeric state or simply an error of the method or experiment. (2) there is always a problem with detergents and lipids with memb proteins :) (3) Thermofluor is just a method, might need fluorescence background optimization, also trying different hydrophobic compounds/detergents etc ** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2, Ireland ** On Thu, Mar 21, 2013 at 6:10 PM, Theresa Hsu theresah...@live.com wrote: Dear all I have a His-tagged membrane protein with unknown oligomerization state. But I am worried that tag addition may induce different state than in native and affect its crystallizability. Is there a single method that can determine the oligomerization state with nearly 100% accuracy? I have use of AUC and SAXS but there seems to be ambiguity about detergent and lipid effects. Is Thermofluor a right method? Does oligomerization require special assembly proteins, which will mean that tag cleavage is not useful to obtain native state? Thank you. Theresa
[ccp4bb] Map Alignment
Dear all, Does anybody know some softwares for aligning electron density maps? I tried transforming map by SQL model fit extension in COOT, which turned out to be not working: the map it transformed is the one supposed to be fixed. If I switch the moving model with the reference model, I only got some error messages. I also tried the Superpose maps utility in PHENIX, however, since they are nucleic acid structures, it seems that the sequences cannot be recognized. Thank you very much! Best, Chen
Re: [ccp4bb] Map Alignment
If I understand your question correctly, you have a couple of atoms which you could align to get the rotation translation then you can use these values with maprot (CCP4) or mama (USF) to actually superimpose maps. Jürgen On Mar 21, 2013, at 11:29 PM, Chen Zhao wrote: Dear all, Does anybody know some softwares for aligning electron density maps? I tried transforming map by SQL model fit extension in COOT, which turned out to be not working: the map it transformed is the one supposed to be fixed. If I switch the moving model with the reference model, I only got some error messages. I also tried the Superpose maps utility in PHENIX, however, since they are nucleic acid structures, it seems that the sequences cannot be recognized. Thank you very much! Best, Chen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Map Alignment
Hi Chen, I also tried the Superpose maps utility in PHENIX, however, since they are nucleic acid structures, it seems that the sequences cannot be recognized. I'm not aware of such problem. If it does exist and you want me to fix it please send me the files and I'll have a look. Pavel
