Re: [ccp4bb] Phaser Fatal runtime error.
Dear all, Thanks for the many of responses, the data from Crysalis is scaled, but unmerged so needed to be fed through scala/truncate before running Phaser. Phaser is now running with no problems and I am looking at some nice maps. Bestr wishes and thanks again for your help, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717 From: Roger Rowlett [rrowl...@colgate.edu] Sent: 27 June 2012 13:07 To: Carr, Stephen (MRC,RAL,RCAH) Cc: ccp4bb Subject: Re: [ccp4bb] Phaser Fatal runtime error. We have an in-house Agilent (Oxford) system and routinely use data with CCP4. You will need to run sortmtz, scala (w/constant scale), and truncate to prep the data properly. This can be done via batch file or GUI.You may also have to reset/reassign the space group for some space groups due to an apparent bug in the CrysalisPro MTZ conversion routine. You can find details at capsicum.colgate.edu/chwikihttp://capsicum.colgate.edu/chwiki in our crystallography pages. Roger Rowlett On Jun 26, 2012 1:34 PM, lt;Stephen Carrgt; stephen.c...@rc-harwell.ac.ukmailto:stephen.c...@rc-harwell.ac.uk wrote: Dear CCP4bb I have collected a data-set using the supernova x-ray generator from Agilent and taken the mtz file generated by the data processing software in crysalis pro forward for structure solution. The data collection was straight forward and the software seemingly processed the data successfully - space-group P2221, overall Rmerge 9%, I/sigmaI 11, redundancy 4.5 etc. Truncate converted the intensities to structure factors with no problems, but when I tried to use the data for molecular replacement with Phaser it produced the following error: FATAL RUNTIME ERROR: Reflections are not a unique set by symmetry I'm not sure how to proceed from here as other programs in the suite do not seem to detect this problem. Also when this error has been mentioned in the past on the bb it was with a data set collected on a Bruker home source and the data processed with Denzo/scalepack, and the suggested solution was to use the Bruker software to process the data. I am currently attempting to reprocess the data with mosflm, but that is likely to be the subject of another post! Any suggestions will be gratefully received. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.ukmailto:stephen.c...@rc-harwell.ac.uk tel 01235 567717
[ccp4bb] PDRA position at Diamond
Dear all, Can I draw your attention to the below post doctoral scientist opportunity at Diamond. I24 is the microfocus MX beamline at Diamond. We are looking for a PDRA to build on recent exciting results in the fields of radiation damage, fast data collection and in-situ spectroscopy. The PDRA will exploit the state of the art detector and beamline technology available at I24 to develop new methods and instrumentation, and further our understanding in these fields. For full details and information on how to apply see http://www.diamond.ac.uk/Home/Jobs/Current/DIA0739_CG.html Please feel free to contact me for more information. All the best, Robin Dr Robin Owen Senior Beamline Scientist Microfocus Macromolecular Crystallography Diamond Light Source, UK Tel: +44 1235 77 8522 email robin.o...@diamond.ac.ukmailto:robin.o...@diamond.ac.uk web http://www.diamond.ac.ukhttp://www.diamond.ac.uk/
[ccp4bb] off topic: protein peptide interaction using DSF
Hi all, Has anyone performed protein peptide interaction experiments using flurophore on a real time PCR machine? Please advice me on how the experiment is performed. Can I use ANS? with regards -- rashmi
Re: [ccp4bb] off topic: protein peptide interaction using DSF
Yes add peptide in different concentrations to your protein and run the assay* Yes, or Sypro Orange *Rule of thumb 20 kDa protein use 1 mg/ml 50 kDa protein use 0.5 mg/ml also have a peptide which should not interact with your protein as control Jürgen On Jun 28, 2012, at 7:34 AM, rashmi panigrahi wrote: Hi all, Has anyone performed protein peptide interaction experiments using flurophore on a real time PCR machine? Please advice me on how the experiment is performed. Can I use ANS? with regards -- rashmi .. 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://web.mac.com/bosch_lab/
Re: [ccp4bb] off topic: protein peptide interaction using DSF
I can second this. Whether or not you get a signal with DSF depends on your protein, not on the type of ligand. We also use Sypro orange. Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bosch, Juergen Sent: Thursday, June 28, 2012 1:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: protein peptide interaction using DSF Yes add peptide in different concentrations to your protein and run the assay* Yes, or Sypro Orange *Rule of thumb 20 kDa protein use 1 mg/ml 50 kDa protein use 0.5 mg/ml also have a peptide which should not interact with your protein as control Jürgen On Jun 28, 2012, at 7:34 AM, rashmi panigrahi wrote: Hi all, Has anyone performed protein peptide interaction experiments using flurophore on a real time PCR machine? Please advice me on how the experiment is performed. Can I use ANS? with regards -- rashmi .. 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://web.mac.com/bosch_lab/
[ccp4bb] Crystallization with antibody
Dear crystallographers Trying to crystallize a membrane protein complex of 100 kDa with a soluble protein of 20 kDa which is interact with the membrane protein. So far, no co-crystals in 200 conditions. Some conditions gave crystals but mass spec of crystals show only either one protein present. I am thinking of antibody but don't know where to start. Can I use the anti-His tag antibody? Thank you.
Re: [ccp4bb] Crystallization with antibody
I would advise against using anti-His FaBs for that particular purpose. Even if you get them to bind to your complex, the inherent flexibility of the terminal regions were His-tags are placed might ruin the final result, and they're not cheap to begin with. You can further characterize your system to spot if there are certain experimental conditions that dissociate the complex, in order to avoid designing crystallization trials that include those. There are a lot of things to be tested before jumping into the Antibody train, and 200 conditions don't seem enough test ground to me for discarding traditional screening. Good luck, Jon 2012/6/28 Theresa Hsu theresah...@live.com Dear crystallographers Trying to crystallize a membrane protein complex of 100 kDa with a soluble protein of 20 kDa which is interact with the membrane protein. So far, no co-crystals in 200 conditions. Some conditions gave crystals but mass spec of crystals show only either one protein present. I am thinking of antibody but don't know where to start. Can I use the anti-His tag antibody? Thank you. -- Dr. Jon Agirre Biophysics Unit (CSIC-UPV/EHU) http://www.ehu.es/jon.agirre +0034946013357
Re: [ccp4bb] Crystallization with antibody
Try seeding the complex with the crystals that you have. If you have crystals of both proteins, crush them and mix them together. Suspend the seeds in whichever reservoir solution has the least salt in it* (or suspend in 50% PEG**) . Use random Microseed Matrix Screening (rMMS) i.e. microseeding into random screens***. rMMS with crystals of one component of a complex has successfully given crystals of the complex in the past. It's very quick and easy to try with a robot or by hand. *Radaev and Sun, J. Appl. Cryst. (2002). 35, 674-676 ** Cryst. Growth Design (2011) 11(8), 3432-3441 *** D’Arcy et al. Acta Cryst. (2007). D63 550-554 http://www.douglas.co.uk/MMS_proc.htm On 28 June 2012 13:25, Theresa Hsu theresah...@live.com wrote: Dear crystallographers Trying to crystallize a membrane protein complex of 100 kDa with a soluble protein of 20 kDa which is interact with the membrane protein. So far, no co-crystals in 200 conditions. Some conditions gave crystals but mass spec of crystals show only either one protein present. I am thinking of antibody but don't know where to start. Can I use the anti-His tag antibody? Thank you. -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] Postdoctoral research position at the University of Edinburgh
Postdoctoral Research Associate--Biochemistry and Structural Biology of Microtubule Nucleation A post-doctoral position is available in Dr. Ken Sawin’s laboratory, in the Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK to study the molecular mechanisms of microtubule nucleation. The project will involve in vitro functional reconstitution and structural analysis of the fission yeast gamma-tubulin complex and the associated Mto1/2 complex, using purified/recombinant proteins and protein complexes. Applicants should have a PhD, or will shortly obtain a PhD, and a strong background in protein purification, as demonstrated by publications. Internal motivation, enthusiasm and communication skills are essential, as is the desire to learn new methods (e.g., single-molecule EM, mass spectrometry). A background in cytoskeleton and/or structural biology is helpful but not essential. Although the laboratory works primarily with fission yeast, a background in yeast cell biology or genetics is not important for this position. This post may be ideal for someone who has gained protein biochemistry experience via a PhD in crystallography but wants to broaden his/her perspective in cell biology. Our preliminary data suggest that this will be both an exciting and complex project! To apply, visit www.jobs.ed.ac.uk and enter vacancy reference number 3015902. More details about the position and the materials required for submitting an application can then be found by clicking on “further information” on the resulting webpage. Informal enquiries about the position and research in the laboratory can be made to me at ken.sa...@ed.ac.uk. But please note that any formal application must be made via the University website. Applications received after the deadline (8 August 2012) may or may not be considered. Further information about the Wellcome Trust Centre for Cell Biology can be found at http://www.wcb.ed.ac.uk. Ken Kenneth E. Sawin, Ph.D. The Wellcome Trust Centre for Cell Biology School of Biological Sciences, University of Edinburgh Swann Building, Mayfield Road Edinburgh EH9 3JR United Kingdom tel: 44-131-650-7064 fax: 44-131-650-7360 email: ken.sa...@ed.ac.uk The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.
Re: [ccp4bb] The effect of His-tag location on crystallization
In trying to reproduce a very nice public structure a cloning mis-hap put a -GS- prior to the C-term 6His tag. The resultant crystals had a 500Ang C dimension and 16 molecules in the au. Even a single amino acid could make all the difference David Hargreaves Associate Principal Scientist _ AstraZeneca DECS, CPSS Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of weliu Sent: 27 June 2012 02:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] The effect of His-tag location on crystallization Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
Re: [ccp4bb] Crystallization with antibody
There are plenty of reports (some even successful) where small concentrations of proteases have been used to enable crystallisation (with soluble proteins). Having put the effort into getting your material in the first place it's a small extra step to try. The professional way is to run a time coarse proteolysis and analyse the protein fragment you get or you can just add a bit of you favourite protease to the drop. I guess if your complex is stable enough it might survive this treatment. There are companies that will raise specific antibodies, scFvs or other immunoglobulin (and possibly non-immunoglobulin) like molecules to your components or complex using technologies like phage/ribosome display. From experience this option could be very time consuming and expensive requiring much iteration. Generating entities with sufficient affinity that bind a desirable epitope and producing them on a crystallisation scale is not easy. You could screen more conditions but there is a law of diminishing returns (redundancy in crystallisation conditions) Try the simple things first... David Hargreaves Associate Principal Scientist _ AstraZeneca DECS, CPSS Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa Hsu Sent: 28 June 2012 13:26 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystallization with antibody Dear crystallographers Trying to crystallize a membrane protein complex of 100 kDa with a soluble protein of 20 kDa which is interact with the membrane protein. So far, no co-crystals in 200 conditions. Some conditions gave crystals but mass spec of crystals show only either one protein present. I am thinking of antibody but don't know where to start. Can I use the anti-His tag antibody? Thank you.
Re: [ccp4bb] Off-topic His-Antibody
Dear All, Thanks for all the replies on and off-board. I received around twenty replies and the majority have spoken in favor of the QIAgen BSA-free anti-5His mAb from QIAGEN. Not to be bias, a couple of people recommended the one from Abcam as well. Thanks again, Dan
