[ccp4bb] Postdoctoral position - University of Edinburgh
*** DEADLINE JUNE 27TH *** I would like to draw your attention to a current opening in my laboratory, to work on the electron microscopy of CRISPR complexes with a single particle approach (see Zhang et al., Mol. Cell 2012). The project is really quite exciting. We have plenty of great preliminary data, the facility is fully equipped and runs smoothly, the scientific environment is vibrant, and Edinburgh is a great place to live! Full details are available here: http://www.jobs.ed.ac.uk/vacancies/index.cfm?fuseaction=vacancies.detailvacancy_ref=3015791 Informal enquiries can be sent to my email address: laura.spagn...@ed.ac.uk Laura Dr Laura Spagnolo Institute of Structural Molecular Biology University of Edinburgh Room 506, Darwin Building King's Buildings Campus Edinburgh EH9 3JR United Kingdom T: +44 (0)131 650 7066 F: +44 (0)131 650 8650 http://www.biology.ed.ac.uk/research/institutes/structure/homepage.php?id=lspagnolo laura.spagn...@ed.ac.uk The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.
[ccp4bb] AW: [ccp4bb] Postdoctoral position - University of Edinburgh
Dear colleagues, is there any program enclosed in CCP4 to convert a *.brix file or a *.mrc file into a CCP4.map file? If not, is there any other way to do so?` Alle the best, Chris DIPL.-ING. CHRIS FRÖHLICH Crystallography Dept. | Daumke group Max-Delbrück-Centre for Molecular Medicine (MDC) Robert-Rössle-Str. 10 13125 Berlin-Buch Lab: +49(0)30-9406-3263 Office: +49(0)30-9406-3275 Fax: +49(0)30-9406-3814 www.mdc-berlin.de/daumkehttp://www.mdc-berlin.de/daumke
Re: [ccp4bb] pdb sequence search
Hi Ed, 1. Go to NCBI BLAST and run the sequence against the PDB subset. The resulting list will have identities listed, so manual parsing is doable if there aren't too many hits. Have you looked at using NCBI EUtils for this? This might not solve your problem directly, but it should only take a bit of scripting. Pete
[ccp4bb] brix/mrc to ccp4 (was Re: [ccp4bb] AW: [ccp4bb] Postdoctoral position - University of Edinburgh)
I don't know of anything directly within CCP4 - but mrc format is almost identical to ccp4 map format. You could try using the mrc version as ccp4 and see if it works. If not, upsalla mapman will convert brix to ccp4. Pete Froehlich, Chris wrote: Dear colleagues, is there any program enclosed in CCP4 to convert a *.brix file or a *.mrc file into a CCP4.map file? If not, is there any other way to do so?` Alle the best, Chris DIPL.-ING. CHRIS FRÖHLICH Crystallography Dept. | Daumke group Max-Delbrück-Centre for Molecular Medicine (MDC) Robert-Rössle-Str. 10 13125 Berlin-Buch Lab: +49(0)30-9406-3263 Office: +49(0)30-9406-3275 Fax: +49(0)30-9406-3814 www.mdc-berlin.de/daumkehttp://www.mdc-berlin.de/daumke
Re: [ccp4bb] AW: [ccp4bb] Postdoctoral position - University of Edinburgh
Froehlich, Chris chris.froehl...@mdc-berlin.de writes: is there any program enclosed in CCP4 to convert a *.brix file or a *.mrc file into a CCP4.map file? If not, is there any other way to do so?` MAPMAN from Uppsala (http://xray.bmc.uu.se/usf/) can read BRIX (and write CCP4), not sure about .mrc format though. -- Ian ◎
Re: [ccp4bb] brix/mrc to ccp4 (was Re: [ccp4bb] AW: [ccp4bb] Postdoctoral position - University of Edinburgh)
Indeed, the intention is that .mrc and CCP4 .map are identical. There have been some problems over the years, which I would call bugs, leading to apparent incompatibility. I'd be interested in any specific cases of non-conforming software (i.e. if there are some .mrc files which CCP4 programs can't read (simplest test is mapdump MAPIN foo.mrc), I'd need to know which version of which program generated it). Cheers Martyn -- *** * * * Dr. Martyn Winn * * * * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * * Tel: +44 1925 603455E-mail: martyn.w...@stfc.ac.uk* * Fax: +44 1925 603634Skype name: martyn.winn * * URL: http://www.ccp4.ac.uk/martyn/ * *** -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pete Meyer Sent: 25 June 2012 15:32 To: ccp4bb Subject: [ccp4bb] brix/mrc to ccp4 (was Re: [ccp4bb] AW: [ccp4bb] Postdoctoral position - University of Edinburgh) I don't know of anything directly within CCP4 - but mrc format is almost identical to ccp4 map format. You could try using the mrc version as ccp4 and see if it works. If not, upsalla mapman will convert brix to ccp4. Pete Froehlich, Chris wrote: Dear colleagues, is there any program enclosed in CCP4 to convert a *.brix file or a *.mrc file into a CCP4.map file? If not, is there any other way to do so?` Alle the best, Chris DIPL.-ING. CHRIS FRÖHLICH Crystallography Dept. | Daumke group Max-Delbrück-Centre for Molecular Medicine (MDC) Robert-Rössle-Str. 10 13125 Berlin-Buch Lab: +49(0)30-9406-3263 Office: +49(0)30-9406-3275 Fax: +49(0)30-9406-3814 www.mdc-berlin.de/daumkehttp://www.mdc-berlin.de/daumke
[ccp4bb] Scientific Officer/Higher Scientific Officer Position at ICR, London, UK
Dear CCP4 Community, We are looking for a motivated Scientific Officer / Higher Scientific Officer to join our exciting research programme on the regulation and functions of ADP-ribosyltransferases. Please see the job description and instructions on how to apply below. Best wishes, Sebastian Scientific Officer/Higher Scientific Officer Position at ICR, London, UK Job title: Scientific Officer/Higher Scientific Officer Closing date of vacancy: 20 July 2012 Starting date of position: mid-October / early November 2012 Division: Structural Biology Cancer Biology Team: Guettler Group Type of Contract: fixed term Length of contract: 3 years from mid-October 2012 Salary range: £23,000 to £29,545 p.a. inclusive (depending on skills and experience) Work location: London, UK In the Divisions of Structural and Cancer Biology, our new team led by Dr. Sebastian Guettler has a vacancy for a Scientific Officer or early-career Higher Scientific Officer. The objective of this post will be to provide support within Dr. Guettler's laboratory for research into the structure and function of proteins. The post holder will work as a member of a multidisciplinary team investigating the functions and regulation of ADP-ribosyltransferases. The work will involve the production and purification of proteins and protein complexes, their characterisation, crystallisation and functional analysis. The work in the Divisions of Structural and Cancer Biology is centred on cellular processes that give rise to cancer and on approaches for anti-cancer therapy. Research in the Division of Structural Biology focuses on the structural and biochemical characterisation of proteins and complexes relevant to cancer and employs techniques that include X-ray crystallography, electron microscopy and a variety of biochemical/biophysical methods. Research activities in the Division of Cancer Biology include mammalian genetics, imaging and proteomics. Applicants should minimally possess a BSc. (or equivalent) in molecular biology, biochemistry or cell biology. Experience in molecular cloning, recombinant protein expression in E. coli and large-scale, crystallisation-grade protein purification is essential. Experience with protein production in insect cells (MultiBac) and mammalian cell culture would be an advantage. Appointment will be on a Fixed Term Contract for 3 years in the first instance with a starting salary in the range of £23,000 to £29,545 p.a. inclusive dependent on skills and experience. The earliest expected start date will be mid-October 2012. In addition to pay awards related to annual performance, we offer a generous leave entitlement of 25 days of leave per annum which rises to a maximum of 30 days per annum, related to length of service, as well as 8 bank/public holidays and 3 additional ICR-set privilege days per annum. For further details and instructions on how to apply, please refer to our online vacancy and recruitment site at http://www.icr.ac.uk/jobsearch, quoting job reference number 1260528 for this position. While applications need to be placed via our online system, informal inquiries can be sent to Dr. Sebastian Guettler at sebastian.guett...@icr.ac.uk. The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] High Rfree values and using Bruker detector w/ iMosflm
Hi, We collected data using our in-house Bruker Proteum X8 diffractometer and after using Xprep to integrate the images the output HKL file was converted to an mtz using CCP4's Scalepack2mtz. The prp file generated by Xprep shows good statistics with completeness and good data for 2.4 angstrom resolution. Mean intensity/sigma, Rsym, and Rsigma have good values. Pointless indicates space group is P212121. Later we used molecular replacement and obtained a good model and Refmac to refine the structure. The electron density maps show the correct density for the amino acids, waters, and other metals in the structure and refinement with coot improves the electron density around the atoms. However, after multiple rounds of refinement the R value drops to 0.25 but Rfree does not go below 0.35! Also, the structure factors are very low. We have tried all the obvious reasons as not having the correct space group which is not the case, and have included NCS restraints as well as TLS parameters to improve the refinement. None of this has helped in helping the Rfree value go down. Since we never have this problem with data collected from other detectors (like at SSRL), and this problem repeats with other datasets from collected in-house, we suspect that the original integration of the images using Xprep might be a problem and might be doing something to the reflection data. Does anyone have any experience with this? Also, I have recently tried using iMosflm to refine the data but using the .sfrm images from the Bruker detector is problematic since I get an Application error that reads invalid command name and an Image read error that reads Error reading image header. Message from Mosflm is error reading record 33: check image is correct size. Does anyone have any advice on how to correct this since I understand that iMosflm is supposed to be able to read Bruker .sfrm images? Thanks! Annette
Re: [ccp4bb] High Rfree values and using Bruker detector w/ iMosflm
Hi Annette Mosflm can only read Bruker images correctly after they have been converted with frm2frm (available from Bruker) - this program unwarps them and writes them in a format that Mosflm can understand. On 25 Jun 2012, at 17:25, Annette Medina Morales wrote: Also, I have recently tried using iMosflm to refine the data but using the .sfrm images from the Bruker detector is problematic since I get an Application error that reads invalid command name and an Image read error that reads Error reading image header. Message from Mosflm is error reading record 33: check image is correct size. Does anyone have any advice on how to correct this since I understand that iMosflm is supposed to be able to read Bruker .sfrm images? Thanks! Annette Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
[ccp4bb] Diffractive Imaging Postdoctoral Scientist Opportunity
I'd like to alert subscribers to an exciting postdoctoral scientist opportunity, using diffractive imaging to examine organelle-scale biological structures at the Linac Coherent Light Source. The job posting is listed at: http://academicemployment.chance.berkeley.edu/DetailsJobSearch.cfm?recordID=1252 The advertised position is strongly oriented toward the development of computational methods. The successful candidate will join the group of Cheryl Kerfeld at UC Berkeley, and collaborate daily with scientists at LBNL. Informal inquiries may be directed to Nick Sauter, nksauter at lbl.gov Peter Zwart, phzwart at lbl.gov -- Nicholas K. Sauter, Ph. D. Computer Staff Scientist, Physical Biosciences Division Lawrence Berkeley National Laboratory 1 Cyclotron Rd., Bldg. 64R0121 Berkeley, CA 94720-8118 (510) 486-5713
Re: [ccp4bb] High Rfree values and using Bruker detector w/ iMosflm
Dear Annette, xprep (at least the xprep written by George Sheldrick, which comes with Bruker machines) is not a data integration program! Given that you collected data on a Bruker machine, presumably used saint/ the apex software for data integration, then xprep, possibly after scaling the data with sadabs, and finally pointless (which does - very roughly speaking - about the same as xprep), chances are that your data were also run through scala and maybe truncate. This means that your data were scaled twice and maybe even truncated twice. In the first case you only messed up your standard deviations, which would not have such a severe impact on poor R/Rfree, but the latter case would explain the low amplitudes. The default in scalepack2mtz, if I remember correctly, is to run ctruncate, hence the amplitude. Maybe you could carefully check all the steps you did. There is no need to run pointless after converting the xprep output sca-file with scalepack2mtz. Regard, Tim On Mon, Jun 25, 2012 at 09:25:23AM -0700, Annette Medina Morales wrote: Hi, We collected data using our in-house Bruker Proteum X8 diffractometer and after using Xprep to integrate the images the output HKL file was converted to an mtz using CCP4's Scalepack2mtz. The prp file generated by Xprep shows good statistics with completeness and good data for 2.4 angstrom resolution. Mean intensity/sigma, Rsym, and Rsigma have good values. Pointless indicates space group is P212121. Later we used molecular replacement and obtained a good model and Refmac to refine the structure. The electron density maps show the correct density for the amino acids, waters, and other metals in the structure and refinement with coot improves the electron density around the atoms. However, after multiple rounds of refinement the R value drops to 0.25 but Rfree does not go below 0.35! Also, the structure factors are very low. We have tried all the obvious reasons as not having the correct space group which is not the case, and have included NCS restraints as well as TLS parameters to improve the refinement. None of this has helped in helping the Rfree value go down. Since we never have this problem with data collected from other detectors (like at SSRL), and this problem repeats with other datasets from collected in-house, we suspect that the original integration of the images using Xprep might be a problem and might be doing something to the reflection data. Does anyone have any experience with this? Also, I have recently tried using iMosflm to refine the data but using the .sfrm images from the Bruker detector is problematic since I get an Application error that reads invalid command name and an Image read error that reads Error reading image header. Message from Mosflm is error reading record 33: check image is correct size. Does anyone have any advice on how to correct this since I understand that iMosflm is supposed to be able to read Bruker .sfrm images? Thanks! Annette -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] High Rfree values and using Bruker detector w/ iMosflm
Dear Annette, I suspect that a trivial error like confusing F and intensity is the cause of your problem. You would probably get a better response if you sent your message to the very informative Bruker mailing list or asked one of the Bruker applications scientists. If you wish to send me your data in confidence I would be happy to try to sort it out for you. I suggest that you send me the .raw files produced by the Bruker integration program SAINT after gzipping them, please do not send the frames(!). Note that the Bruker control software can also write 'unwarped' frames if you really need them, however we get excellent data with the normal Bruker procedure (SAINT, SADABS, XPREP and then F2mtz etc.). As Tim says, XPREP is not an integration program but it does determine the space group and provide a lot of useful statistics. My other strong recommendation, irrespective of which beamline or in-house system you are using, is to collect a quick cubic insulin or lysozyme dataset from time to time. This will tell you if all the hardware and software is functioning correctly, and incidentally provides you with a accurate X,Y beam position which can be invaluable for indexing problem datasets. Best wishes, George On 06/25/2012 06:25 PM, Annette Medina Morales wrote: Hi, We collected data using our in-house Bruker Proteum X8 diffractometer and after using Xprep to integrate the images the output HKL file was converted to an mtz using CCP4's Scalepack2mtz. The prp file generated by Xprep shows good statistics with completeness and good data for 2.4 angstrom resolution. Mean intensity/sigma, Rsym, and Rsigma have good values. Pointless indicates space group is P212121. Later we used molecular replacement and obtained a good model and Refmac to refine the structure. The electron density maps show the correct density for the amino acids, waters, and other metals in the structure and refinement with coot improves the electron density around the atoms. However, after multiple rounds of refinement the R value drops to 0.25 but Rfree does not go below 0.35! Also, the structure factors are very low. We have tried all the obvious reasons as not having the correct space group which is not the case, and have included NCS restraints as well as TLS parameters to improve the refinement. None of this has helped in helping the Rfree value go down. Since we never have this problem with data collected from other detectors (like at SSRL), and this problem repeats with other datasets from collected in-house, we suspect that the original integration of the images using Xprep might be a problem and might be doing something to the reflection data. Does anyone have any experience with this? Also, I have recently tried using iMosflm to refine the data but using the .sfrm images from the Bruker detector is problematic since I get an Application error that reads invalid command name and an Image read error that reads Error reading image header. Message from Mosflm is error reading record 33: check image is correct size. Does anyone have any advice on how to correct this since I understand that iMosflm is supposed to be able to read Bruker .sfrm images? Thanks! Annette -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
[ccp4bb] Off-topic His-Antibody
Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish.
Re: [ccp4bb] Off-topic His-Antibody
The one we have is rubbish. I can tell a vendor name if you want off-list. A lot of non-specific bands + high background level makes it almost impossible to use against cell lysate. It sort of works against purified His-tag protein, but not perfect either. On Mon, Jun 25, 2012 at 4:33 PM, D Bonsor dbon...@ihv.umaryland.edu wrote: Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish.
Re: [ccp4bb] Off-topic His-Antibody
We routinely use BSA-free anti-5His mAb from QIAGEN, and it works just fine (at least using media and lysates of both insect and mammalian cells): http://www.qiagen.com/products/protein/detection/qiaexpressdetectionsystem/penta-hisantibody.aspx Good luck, Luca Luca Jovine, Ph.D. Assistant Professor EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 83 Huddinge, Sweden Voice: +46.(0)8.524-81136 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of D Bonsor [dbon...@ihv.umaryland.edu] Sent: Monday, June 25, 2012 23:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic His-Antibody Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish.
Re: [ccp4bb] Off-topic His-Antibody
We also use that same antibody, and are satisfied overall. On 6/25/12 8:59 PM, Luca Jovine wrote: We routinely use BSA-free anti-5His mAb from QIAGEN, and it works just fine (at least using media and lysates of both insect and mammalian cells): http://www.qiagen.com/products/protein/detection/qiaexpressdetectionsystem/penta-hisantibody.aspx Good luck, Luca Luca Jovine, Ph.D. Assistant Professor EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 83 Huddinge, Sweden Voice: +46.(0)8.524-81136 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of D Bonsor [dbon...@ihv.umaryland.edu] Sent: Monday, June 25, 2012 23:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic His-Antibody Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish. -- Engin Özkan Post-doctoral Scholar Laboratory of K. Christopher Garcia Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 w ph: (650)-498-7111 cell: (650)-862-8563
