[ccp4bb] problem in calculation of elbow angle.
Dear all, I am trying to calculate elbow angle of my fab structure using a online software developed by Robyn L. Stanfield *et. al*. but it is giving a solution with the following errors. WARNING: rotation matrix contains significant additional contributions WARNING: and deviates significantly from a pseudo-twofold0.721 WARNING: rotation matrix contains significant additional contributions WARNING: and deviates significantly from a pseudo-twofold :0.726 WARNING: there have been deviations from expected values - please read the log above!) No guarantee that the calulated elbow angle is meaningful The Elbow angle is probably 174.5 deg. *Kindly suggest some other way of accurately, calculating elbow angle.* regards. Tarique khan
[ccp4bb] Synchrotron beam time 2010 at EMBL Hamburg
This is just a gentle reminder that the deadline for beam time 2010 at EMBL Hamburg is today, January 13. With best regards, Victor Call for access to Synchrotron Beamline Facilities 2010 EMBL Hamburg, Germany We announce a call for synchrotron beam time applications in biological small-angle scattering (SAXS) and X-ray crystallography (PX). Up to 32 weeks of beam time will be available at the DORIS storage ring (DESY) from March 2010 to February 2011. EMBL Hamburg will operate the following beamlines: Beamline New featuresScientist responsible X33SAXS Automated and remote operation Dmitri Svergun X11PX MAR555 Flat Panel detector Santosh Panjikar X12PX MAD, S-SAD, fluorescence analysis Andrea Schmidt X13PX Microspec, expert data collection Matthew Groves The deadline for submission of proposals is January 13, 2010. An external Priorities Committee will assess the proposals. Electronic beam proposal forms and a detailed description of the beamline facilities are available at www.embl-hamburg.de (click on 'Access to Infrastructures'). Additional EMBL services can be found at www.embl-hamburg.de/facilities EMBL is constructing three new beamlines for applications in biological X-ray crystallography (PX) and small-angle scattering (SAXS) at the Petra-III synchrotron storage ring, which are scheduled to be available from 2011. See www.embl-hamburg.de/services/petra for further information. For further information tel. +49-40-89902-110, s...@embl-hamburg.de (SAXS) b...@embl-hamburg.de (PX). Access to the EMBL Hamburg facilities will be supported by the European Commission, Research Infrastructure Action under the FP7 ELISA (European Light Sources Activities).
[ccp4bb] Postdoctoral Position at the University of Wuerzburg
A postdoctoral position in Structural Biology of the Spliceosomal Assembly Machinery is available at the Biocenter of the University of Wuerzburg, Germany, within the department of Biochemistry. The successful candidate will participate in all stages of crystallographic structure determination from cloning, protein expression and purification to synchrotron data collection, processing and model building. The following qualifications will be required: - a PhD degree - experience in protein expression and purification - experience in data collection and processing - cumputing skills (scripting languages, linux system administration) would be an advantage. We offer an initial 2 year contract, extendable depending on project success. Payment will be in accordance with German public service positions (TVöD E13), including extensive social security plans. More information about the department and the biocenter is available on the respective web sites at: www.biozentrum.de Please send your application including a letter of interest, a CV and names of two references to: clemens.gr...@biozentrum.uni-wuerzburg.de The University of Wuerzburg is an equal opportunity employer with an affirmative action program for the disabled. -- Dr. Clemens Grimm Institut für Biochemie Biozentrum der Universität Würzburg Am Hubland D-97074 Würzburg Germany e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de phone : +49 0931 888 84031 -
Re: [ccp4bb] problem in calculation of elbow angle.
Inherently you want to calculate: 1. the approximately two-fold relationship between VH and VL 2. the approximately two-fold relationship between CL and CH1 You can use many programs for that (e.g. LSQMAN) but ideally you want a program that will report Direction Cosines for the rotation axis in this superimposition. Particularly wacky CDR conformations could conceivably confuse automatic alignment programs so you could delete those. You should check the superimposed alignments for sanity (e.g. correspondence of the disulfide bonds). Then calculate the elbow angle for the Fab from the dot product of the direction cosines of VL:VH and CL:CH1. Phil Jeffrey Princeton tarique khan wrote: Dear all, I am trying to calculate elbow angle of my fab structure using a online software developed by Robyn L. Stanfield /et. al/. but it is giving a solution with the following errors. WARNING: rotation matrix contains significant additional contributions WARNING: and deviates significantly from a pseudo-twofold0.721 WARNING: rotation matrix contains significant additional contributions WARNING: and deviates significantly from a pseudo-twofold :0.726 WARNING: there have been deviations from expected values - please read the log above!) No guarantee that the calulated elbow angle is meaningful The Elbow angle is probably 174.5 deg. *Kindly suggest some other way of accurately, calculating elbow angle.* regards. Tarique khan
Re: [ccp4bb] problem in calculation of elbow angle.
Dear Tarique, The elbow angle in a Fab is just the angle between two pseudo-twofold axes: one relating VL to VH and one relating CL to CH. You can find the pseudo-twofold axes using many different least-squares alignment programs, just pick your favorite. Then, just take the dot product of the two resulting axes/vectors for your elbow angle. You can do this quite accurately with a pencil and paper and maybe a handheld calculator. The error message you report indicates problems calculating the pseudo-two fold axes; you probably either have (a) an unusual Fab where the axes relating light to heavy chains deviate significantly from 180 degrees (b) a glitch or unusual feature in your PDB file that has thrown off the calculation of the pseudo-two folds (thus making them appear to deviate from 180). Bernhard might be able to enlighten you more as he wrote the code and error messages(b...@ruppweb.orgmailto:b...@ruppweb.org) Cheers, Robyn ro...@scripps.edu From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of tarique khan Sent: Wednesday, January 13, 2010 4:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] problem in calculation of elbow angle. Dear all, I am trying to calculate elbow angle of my fab structure using a online software developed by Robyn L. Stanfield et. al. but it is giving a solution with the following errors. WARNING: rotation matrix contains significant additional contributions WARNING: and deviates significantly from a pseudo-twofold0.721 WARNING: rotation matrix contains significant additional contributions WARNING: and deviates significantly from a pseudo-twofold :0.726 WARNING: there have been deviations from expected values - please read the log above!) No guarantee that the calulated elbow angle is meaningful The Elbow angle is probably 174.5 deg. Kindly suggest some other way of accurately, calculating elbow angle. regards. Tarique khan
[ccp4bb] Expression of large proteins in E. coli
Hello All, I apologize for the non-CCP4-related query. I have been working for several weeks now trying, with limited success, to express some very large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli. Limited success means I have expressed enough soluble protein to see on a gel, but not enough to purify. I have tried the obvious tweaks - changing strains (BL21, BL21-star, Rosetta, pLysS), screening induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I am in the process of subcloning into vectors for (1) SUMO fusion and (2) periplasmic expression (pET26b). I get the sense from digging through the literature that high level expression of large proteins depends mostly on the individual protein and I will ultimately have to look for homologs. But, this is my first experience expressing such large proteins and I am curious to know if anyone out there has some magical trick they wouldn't mind sharing. Thanks in advance, Nick - Nicholas R. Silvaggi, Ph.D. University of Wisconsin-Milwaukee Department of Chemistry and Biochemistry 3210 North Cramer Street Milwaukee, WI 53211 Phone: 414-229-2647 Email: silva...@uwm.edu
Re: [ccp4bb] Expression of large proteins in E. coli
Hi Nick, Some success has been reported for large soluble proteins using the C41(DE3) and C43(DE3) *E. coli* strains (see the paper by Miroux Walker). Also you can try another promoter/expression system , the pBAD expression system based on arabinose induction. Hope this helps. Best -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Expression of large proteins in E. coli
Hey Nick, Short answer is that there are no magical tricks, as you probably already expect to hear. Seems there's a lot that you are trying already. I have some limited experience with some 125kDa, 100kDa proteins. 1. I've had some luck with the E. coli C41, C43 strains. Especially if your proteins are inherently toxic to E. coli. There are commercial vendors who sell these strains with the plus or minus pLysS options. 2. Try moving the tags to the other terminus N- to C- or vice versa. Can't do that for SUMO in any easy manner. 3. Try MBP tag as well 4. Try Studier's autoinduction protocol 5. Try expression with chaperone kit, trigger factor (Takara) 6. You don't mention whether the protein is human etc., but you may have to move to yeast or insect cells, in the worst case. DISCLAIMER: I am not paid by Takara to mention their kit. Good luck! Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Jan 13, 2010, at 4:53 PM, n...@silvaggi.com wrote: Hello All, I apologize for the non-CCP4-related query. I have been working for several weeks now trying, with limited success, to express some very large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli. Limited success means I have expressed enough soluble protein to see on a gel, but not enough to purify. I have tried the obvious tweaks - changing strains (BL21, BL21-star, Rosetta, pLysS), screening induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I am in the process of subcloning into vectors for (1) SUMO fusion and (2) periplasmic expression (pET26b). I get the sense from digging through the literature that high level expression of large proteins depends mostly on the individual protein and I will ultimately have to look for homologs. But, this is my first experience expressing such large proteins and I am curious to know if anyone out there has some magical trick they wouldn't mind sharing. Thanks in advance, Nick - Nicholas R. Silvaggi, Ph.D. University of Wisconsin-Milwaukee Department of Chemistry and Biochemistry 3210 North Cramer Street Milwaukee, WI 53211 Phone: 414-229-2647 Email: silva...@uwm.edu
Re: [ccp4bb] Reindexing P21.. the process?
Negating the DANO columns is the correct thing to do if the reindex operator changes hkl to h,-k,-h-l which will then be put back into the asymmetric unit with Friedel's law + symmetry eg hkl (1,2,3) - (1,-2,-4) -Operator[-h,-k,-l] - -1,+2, +4 The Friedel's law operator -h,-k,-l interchanges I+ I-, ie negates DANO Phil On 13 Jan 2010, at 21:18, Francis E Reyes wrote: Hi all I have data integrated and scaled to P21 via denzo/scalepack. I'm concerned about the workflow to obtain the alternate indexing arrangement (h,k,l) - (h,-k,-h-l). I was thinking .sca ( not specifying NO MERGE) - .mtz - reindex but the documentation for reindex says all my DANO columns are 'negated' ( I do not specify NOREDUCE). So I must run truncate on the reindexed intensities to recalculate F(+/-) and DANO/SIGDANO) ? Thanks FR
Re: [ccp4bb] Reindexing P21.. the process?
+1 phenix FTW FR On Jan 13, 2010, at 3:28 PM, Peter Zwart phzw...@lbl.gov wrote: try using phenix.reflection_file_converter data.sca --change_of_basis=h,-k,-h-l --sca=reindex.sca or something like that HTH Peter 2010/1/13 Francis E Reyes francis.re...@colorado.edu: Hi all I have data integrated and scaled to P21 via denzo/scalepack. I'm concerned about the workflow to obtain the alternate indexing arrangement (h,k,l) - (h,-k,-h-l). I was thinking .sca ( not specifying NO MERGE) - .mtz - reindex but the documentation for reindex says all my DANO columns are 'negated' ( I do not specify NOREDUCE). So I must run truncate on the reindexed intensities to recalculate F(+/-) and DANO/SIGDANO) ? Thanks FR -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -
Re: [ccp4bb] Expression of large proteins in E. coli
Hi Nick, I work with a 120kDa protein and using IPTG 50uM was crucial to get some soluble protein. Try to reduce the amount of IPTG and temperature, increase the induction time (24h,18°C) and volume of culture. It works fine for my protein and may be suitable for yours as well. Best, Ricardo On Wed, 13 Jan 2010 15:53:22 -0600, nick wrote Hello All, I apologize for the non-CCP4-related query. I have been working for several weeks now trying, with limited success, to express some very large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli. Limited success means I have expressed enough soluble protein to see on a gel, but not enough to purify. I have tried the obvious tweaks - changing strains (BL21, BL21-star, Rosetta, pLysS), screening induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I am in the process of subcloning into vectors for (1) SUMO fusion and (2) periplasmic expression (pET26b). I get the sense from digging through the literature that high level expression of large proteins depends mostly on the individual protein and I will ultimately have to look for homologs. But, this is my first experience expressing such large proteins and I am curious to know if anyone out there has some magical trick they wouldn't mind sharing. Thanks in advance, Nick - Nicholas R. Silvaggi, Ph.D. University of Wisconsin-Milwaukee Department of Chemistry and Biochemistry 3210 North Cramer Street Milwaukee, WI 53211 Phone: 414-229-2647 Email: silva...@uwm.edu -- Ricardo Augusto Pereira de Pádua Laboratory of Protein Crystallography of Ribeirão Preto Faculty of Pharmaceutical Sciences of Ribeirão Preto University of São Paulo - Brazil
Re: [ccp4bb] CCP4BB Digest - 12 Jan 2010 to 13 Jan 2010 (#2010-12)
Hi Nick, In the past, I've successfully expressed a 130 kd eukaryotic protein in large quantities in plain BL21. I don't think size per se is a major problem for expression although I've heard others say they had problems with heterogeneous termination. I'd try the standard repertoire of techniques to improve expression. What has worked best for me in the past were 1) lowering the temperature of induction/expression and 2) using different tags. It sounds like you've already tried many different parameters. Consider different expression hosts. ho
Re: [ccp4bb] Expression of large proteins in E. coli
Hi Nick, A couple of other suggestions: (1) Try an E. coli K strain (e.g. JM109). They're different in glucose metabolism (JN Phue et al, 2005, Biotechnol Bioeng 90:805820) and in my experience tend to grow more slowly - which seems to help improve yields of large constructs. (2) Optimal oxygenation also seems to be very important in producing large constructs. Not sure what vessels you're using but Fernback flasks work well for me. Or a fermenter might solve all you problems. Good luck, Will. Hello All, I apologize for the non-CCP4-related query. I have been working for several weeks now trying, with limited success, to express some very large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli. Limited success means I have expressed enough soluble protein to see on a gel, but not enough to purify. I have tried the obvious tweaks - changing strains (BL21, BL21-star, Rosetta, pLysS), screening induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I am in the process of subcloning into vectors for (1) SUMO fusion and (2) periplasmic expression (pET26b). I get the sense from digging through the literature that high level expression of large proteins depends mostly on the individual protein and I will ultimately have to look for homologs. But, this is my first experience expressing such large proteins and I am curious to know if anyone out there has some magical trick they wouldn't mind sharing. Thanks in advance, Nick - Nicholas R. Silvaggi, Ph.D. University of Wisconsin-Milwaukee Department of Chemistry and Biochemistry 3210 North Cramer Street Milwaukee, WI 53211 Phone: 414-229-2647 Email: silva...@uwm.edu
Re: [ccp4bb] Expression of large proteins in E. coli
Nick, We have recently got much improved expression (both total amount and in soluble form) of 72 kDa protein by using the EnBase Flo cell cultivation kit from Biosilta (www.biosilta.com). The method is based on enzymatic slow release of glucose from a starch(?)-like polymer - thus mimicking a fed-batch principle of bioreactor cultivations, but in shake flasks. Induction is done at a much higher cell density than conventionally, and long induction periods (24 h) can be used. Consequently the yields become higher and the folding/refolding machinery has time to make more correctly folded protein. Smaller cultivation volumes can be used, yet results in more cells and product. We also tried with a 100 kDa protein without great improvement yet. But the kit is certainly worth a go. Tuomo Glumoff == Tuomo Glumoff, FT Tuomo Glumoff, Ph.D. lehtori, dosentti lecturer and docent Oulun yliopisto University of Oulu Biokemian laitos Dept. Biochemistry PL 3000 BOX 3000 90014 OULUN YLIOPISTO FI-90014 UNIVERSITY OF OULU Finland == Kontaktitiedot - Contact information: www.biochem.oulu.fi/henkilokunta/glumoff/ == matkapuh. / mobile: +358-(0)50-5226136 == -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Will Stanley Sent: 14. tammikuuta 2010 3:18 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Expression of large proteins in E. coli Hi Nick, A couple of other suggestions: (1) Try an E. coli K strain (e.g. JM109). They're different in glucose metabolism (JN Phue et al, 2005, Biotechnol Bioeng 90:805-820) and in my experience tend to grow more slowly - which seems to help improve yields of large constructs. (2) Optimal oxygenation also seems to be very important in producing large constructs. Not sure what vessels you're using but Fernback flasks work well for me. Or a fermenter might solve all you problems. Good luck, Will. Hello All, I apologize for the non-CCP4-related query. I have been working for several weeks now trying, with limited success, to express some very large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli. Limited success means I have expressed enough soluble protein to see on a gel, but not enough to purify. I have tried the obvious tweaks - changing strains (BL21, BL21-star, Rosetta, pLysS), screening induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I am in the process of subcloning into vectors for (1) SUMO fusion and (2) periplasmic expression (pET26b). I get the sense from digging through the literature that high level expression of large proteins depends mostly on the individual protein and I will ultimately have to look for homologs. But, this is my first experience expressing such large proteins and I am curious to know if anyone out there has some magical trick they wouldn't mind sharing. Thanks in advance, Nick - Nicholas R. Silvaggi, Ph.D. University of Wisconsin-Milwaukee Department of Chemistry and Biochemistry 3210 North Cramer Street Milwaukee, WI 53211 Phone: 414-229-2647 Email: silva...@uwm.edu
