Re: [ccp4bb] Protein aggregation and crystallization
Hi Anita, Won't harm if you put it on crystallization tray. You never know what these proteins might do. Allan Quoting Zheng Zhou : Hi, Anita If you could find a way to test the elute's activity/binding to its' substrat/cofactor, then you will learn much more about your target. If the function assay is elusive, you could try superose column (5KDa-5MKDa). Does your light scattering tell you about the estimated size and MW? Best, Joe On Sat, Aug 27, 2011 at 1:29 AM, anita p wrote: Hi Yury, I have done dynamic light scattering and it shows its polydispersed. Please let me know if it is still ok for setting trays. reg. anita On Fri, Aug 26, 2011 at 10:21 PM, Yuriy Patskovsky < yuriy.patskov...@einstein.yu.edu> wrote: Anita, an assembly may be quite large - I would check it somehow, maybe by light scattering or centrifugation Good luck Yury -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of anita p [crystals...@gmail.com] *Sent:* Friday, August 26, 2011 3:03 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Protein aggregation and crystallization Hi All, I am working on a protein which has a membrane spanning region and as cytosolic domain.I have made various deletion constructs of the protein, so that I can have a crystallizable fragment. There is no homologues mentioned in the pdb for this protein. All of these constructs are purified successfully but when concentrated and loaded on a gel filtration column Superdex-200, they elute in the void volume. But the proteins donot precipitate out !! Is it worth while to go ahead for crystallization trials?? Any other suggestion is most welcome. Thanks Anita -- Allan Pang PhD Student G35 Joseph Priestley Building Queen Mary University of London London E1 4NS Phone number: 02078828480
[ccp4bb] Protein elution in Size Exclusion
Hi there everyone, What does it mean when you have proteins eluting in almost the whole column volume of S200? I ran a gel with fractions from 8ml to 20ml and saw band for my protein all throughout. Judging peaks on chromatogram is not useful as it doesn't have any aromatic rings. Cheers, Allan -- Allan Pang PhD Student G35 Joseph Priestley Building Queen Mary University of London London E1 4NS Phone number: 02078828480
[ccp4bb] Hiro Tsuruta
It is with great sadness that we announce that Dr. Hirotsugu (Hiro) Tsuruta, a senior scientist and biophysicist in the Structural Molecular Biology program at Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC, passed away on August 25, 2011, in Japan, after a courageous fight with cancer. Funeral services took place in Sasebo, Japan, on August 27. Hiro worked at SSRL for more than 20 years, and with great dedication and scientific insight provided leadership for the build-up of the SSRL biological small angle x-ray scattering research program, and a bioSAXS beam line facility that today serves a large user community. He also served the international community in many ways. His contributions were numerous, and he leaves an international legacy in this growing area of science, for which he pursued new developments until the end. SSRL will organize an event to honor his contributions, and information will follow when plans become available. Britt Hedman and Keith Hodgson
Re: [ccp4bb] Protein elution in Size Exclusion
It possibly means: 1. Your sample application volume was too large 2. Total protein quantity was too large 3. Fraction size was too large 4. Sample was too crude (GEC is a finishing step, not a starting step for protein purification. 5. Sample was too concentrated/viscous 6. Flow rate was too fast 7. Ionic strength of buffer too low to suppress ion exchange effects. Roger Rowlett On Aug 28, 2011 5:25 AM, "Allan Pang" wrote: > Hi there everyone, > > What does it mean when you have proteins eluting in almost the whole > column volume of S200? > > I ran a gel with fractions from 8ml to 20ml and saw band for my > protein all throughout. > > Judging peaks on chromatogram is not useful as it doesn't have any > aromatic rings. > > Cheers, > > Allan > > -- > Allan Pang > > PhD Student > > G35 Joseph Priestley Building > Queen Mary University of London > London > E1 4NS > > Phone number: 02078828480
Re: [ccp4bb] Protein elution in Size Exclusion
Following on from Roger's fine suggestions: 8. Your column is knackered. Can you see fine lines or cracks in the column? Good packing is v.important for SEC columns. HTH, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 28 August 2011 10:25, Allan Pang wrote: > Hi there everyone, > > What does it mean when you have proteins eluting in almost the whole column > volume of S200? > > I ran a gel with fractions from 8ml to 20ml and saw band for my protein all > throughout. > > Judging peaks on chromatogram is not useful as it doesn't have any aromatic > rings. > > Cheers, > > Allan > > -- > Allan Pang > > PhD Student > > G35 Joseph Priestley Building > Queen Mary University of London > London > E1 4NS > > Phone number: 02078828480 >
Re: [ccp4bb] Protein elution in Size Exclusion
Hi, David, What is the common cause of knackered SEC column? Will equilibrizing a buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause the problem. There was no problem just after packing the column. Nian On Sun, Aug 28, 2011 at 9:24 AM, David Briggs wrote: > Following on from Roger's fine suggestions: > > 8. Your column is knackered. Can you see fine lines or cracks in the > column? Good packing is v.important for SEC columns. > > HTH, > > Dave > > > > David C. Briggs PhD > Father, Structural Biologist and Sceptic > > University of Manchester E-mail: > david.c.bri...@manchester.ac.uk > > http://manchester.academia.edu/DavidBriggs (v.sensible) > http://xtaldave.wordpress.com/ (sensible) > http://xtaldave.posterous.com/ (less sensible) > Twitter: @xtaldave > Skype: DocDCB > > > > > On 28 August 2011 10:25, Allan Pang wrote: > > Hi there everyone, > > > > What does it mean when you have proteins eluting in almost the whole > column > > volume of S200? > > > > I ran a gel with fractions from 8ml to 20ml and saw band for my protein > all > > throughout. > > > > Judging peaks on chromatogram is not useful as it doesn't have any > aromatic > > rings. > > > > Cheers, > > > > Allan > > > > -- > > Allan Pang > > > > PhD Student > > > > G35 Joseph Priestley Building > > Queen Mary University of London > > London > > E1 4NS > > > > Phone number: 02078828480 > > >
Re: [ccp4bb] Protein elution in Size Exclusion
Hi Nian, It can be a number of things - but typically, air getting into the column, or a leaky seal somewhere letting it dry out. Switching from 150mM NaCl to 20% EtOH directly shouldn't cause a problem. The reason I suggest it is that I have had the same situation as described in the original post (protein comes off across entire volume of SEC column elution) and it turned out the column had a leak and had dried out in places. Repacking the column solved the problem. Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 28 August 2011 22:35, Nian Huang wrote: > Hi, David, > What is the common cause of knackered SEC column? Will equilibrizing a > buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause > the problem. There was no problem just after packing the column. > > Nian > > On Sun, Aug 28, 2011 at 9:24 AM, David Briggs > wrote: >> >> Following on from Roger's fine suggestions: >> >> 8. Your column is knackered. Can you see fine lines or cracks in the >> column? Good packing is v.important for SEC columns. >> >> HTH, >> >> Dave >> >> >> >> David C. Briggs PhD >> Father, Structural Biologist and Sceptic >> >> University of Manchester E-mail: >> david.c.bri...@manchester.ac.uk >> >> http://manchester.academia.edu/DavidBriggs (v.sensible) >> http://xtaldave.wordpress.com/ (sensible) >> http://xtaldave.posterous.com/ (less sensible) >> Twitter: @xtaldave >> Skype: DocDCB >> >> >> >> >> On 28 August 2011 10:25, Allan Pang wrote: >> > Hi there everyone, >> > >> > What does it mean when you have proteins eluting in almost the whole >> > column >> > volume of S200? >> > >> > I ran a gel with fractions from 8ml to 20ml and saw band for my protein >> > all >> > throughout. >> > >> > Judging peaks on chromatogram is not useful as it doesn't have any >> > aromatic >> > rings. >> > >> > Cheers, >> > >> > Allan >> > >> > -- >> > Allan Pang >> > >> > PhD Student >> > >> > G35 Joseph Priestley Building >> > Queen Mary University of London >> > London >> > E1 4NS >> > >> > Phone number: 02078828480 >> > > >
Re: [ccp4bb] Protein elution in Size Exclusion
worst-case scenario for crystallization purposes: 9. Your protein runs as a mixture of monomers, dimers, trimers, whatever-mers... Filip On Sun, Aug 28, 2011 at 7:24 AM, David Briggs wrote: > Following on from Roger's fine suggestions: > > 8. Your column is knackered. Can you see fine lines or cracks in the > column? Good packing is v.important for SEC columns. > > HTH, > > Dave > > > > David C. Briggs PhD > Father, Structural Biologist and Sceptic > > University of Manchester E-mail: > david.c.bri...@manchester.ac.uk > > http://manchester.academia.edu/DavidBriggs (v.sensible) > http://xtaldave.wordpress.com/ (sensible) > http://xtaldave.posterous.com/ (less sensible) > Twitter: @xtaldave > Skype: DocDCB > > > > > On 28 August 2011 10:25, Allan Pang wrote: > > Hi there everyone, > > > > What does it mean when you have proteins eluting in almost the whole > column > > volume of S200? > > > > I ran a gel with fractions from 8ml to 20ml and saw band for my protein > all > > throughout. > > > > Judging peaks on chromatogram is not useful as it doesn't have any > aromatic > > rings. > > > > Cheers, > > > > Allan > > > > -- > > Allan Pang > > > > PhD Student > > > > G35 Joseph Priestley Building > > Queen Mary University of London > > London > > E1 4NS > > > > Phone number: 02078828480 > > > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
[ccp4bb] phaser problem
Hi Has anyone a solution to the following problem with Phaser (version 2.1.4)? UNHANDLED ERROR: basic_string::_S_create It seems to be data and model independent – there was an old thread from earlier this year, but I cannot find a follow-up. Thanks! Amir
Re: [ccp4bb] Protein elution in Size Exclusion
Testing for cracks in the column is quite easy. Take whichever standard you happen to have (lisozyme, GST, etc) and do a run. If it comes out in a single peak the problem is with your prep. If it comes out throughout the run the problem is with your column. Mario Sanches On Sun, Aug 28, 2011 at 5:35 PM, Nian Huang wrote: > Hi, David, > What is the common cause of knackered SEC column? Will equilibrizing a > buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause > the problem. There was no problem just after packing the column. > > Nian > > > On Sun, Aug 28, 2011 at 9:24 AM, David Briggs wrote: > >> Following on from Roger's fine suggestions: >> >> 8. Your column is knackered. Can you see fine lines or cracks in the >> column? Good packing is v.important for SEC columns. >> >> HTH, >> >> Dave >> >> >> >> David C. Briggs PhD >> Father, Structural Biologist and Sceptic >> >> University of Manchester E-mail: >> david.c.bri...@manchester.ac.uk >> >> http://manchester.academia.edu/DavidBriggs (v.sensible) >> http://xtaldave.wordpress.com/ (sensible) >> http://xtaldave.posterous.com/ (less sensible) >> Twitter: @xtaldave >> Skype: DocDCB >> >> >> >> >> On 28 August 2011 10:25, Allan Pang wrote: >> > Hi there everyone, >> > >> > What does it mean when you have proteins eluting in almost the whole >> column >> > volume of S200? >> > >> > I ran a gel with fractions from 8ml to 20ml and saw band for my protein >> all >> > throughout. >> > >> > Judging peaks on chromatogram is not useful as it doesn't have any >> aromatic >> > rings. >> > >> > Cheers, >> > >> > Allan >> > >> > -- >> > Allan Pang >> > >> > PhD Student >> > >> > G35 Joseph Priestley Building >> > Queen Mary University of London >> > London >> > E1 4NS >> > >> > Phone number: 02078828480 >> > >> > > -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
[ccp4bb] Windows 7 and Xtal Software
Dear Crystallographers, are there any additional problems or known issues running ccp4 or other xtal software on windows 7 (beyond those of Vista, etc.?) Your input would be really appreciate before I sink my own personal $$$ into a new laptop Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Methods for dehydrating crystals
Dear Andrea, as others have suggested there are many ways to dehydrate crystals by increasing the concentration of salt/precipitant or adding glycerol/EG/PEG 200-400 to the solutions surrounding your crystals. I have always found controlled dehydration using a specific device to be much better. The most common are the FMS and the HC1, the great advantage is that any changes that are induced by dehydration can been observed immediately by diffraction and the whole process can be thoroughly characterised. You never know if it is going to work but it is always worth a try, good indications are relatively large solvent content/solvent channels or that you have already observed variation in unit cell parameters after cryocooling. In Europe the HC1 is available at the ESRF (http://go.esrf.eu/HC1b - you can apply for rolling access here, http://www.esrf.fr/UsersAndScience/UserGuide/Applying/ProposalGuidelines/MXnon-BAGproposal), Diamond, MAXLab and I think BESSY. I believe the FMS is available to use at Proteros. Here are some links to papers describing the use of these devices, good luck! Matt. FMS - http://journals.iucr.org/j/issues/2000/05/00/he0257/index.html HC1 - http://scripts.iucr.org/cgi-bin/paper?S0907444909037822 and http://www.sciencedirect.com/science/article/pii/S1047847711000499 On 26/08/2011 22:53, Andrea L Edwards wrote: Hi all, What are the most successful methods you know of for dehydrating a crystal prior to freezing it? I am trying to push the resolution of my crystals. Thanks, Andrea -- Matthew Bowler Structural Biology Group European Synchrotron Radiation Facility B.P. 220, 6 rue Jules Horowitz F-38043 GRENOBLE CEDEX FRANCE === Tel: +33 (0) 4.76.88.29.28 Fax: +33 (0) 4.76.88.29.04 http://go.esrf.eu/MX http://go.esrf.eu/Bowler ===
[ccp4bb] Self-rotation Patterson maps
Hello, I wish to learn about self-rotation Patterson maps such as how to interpret a map and find out non-crystallographic symmetry. Could you suggest some good papers (for dummies), web-sites etc. Thanks a lot for your help. John
Re: [ccp4bb] HTP ligand screening
Dear Jacob, Globalphasing has a new programm called Pipedream that should do just what you want. I am not sure if it is available to non consortium members. Best regards, Alexander -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Friday, August 26, 2011 10:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HTP ligand screening Dear Crystallographers, I have ~30 data sets from ligand soaks of my protein of known structure (all approximately the same cell. Can anyone suggest a high throughput method which would do molecular replacement, refine, then output new blobs, perhaps as a water pdb file? I am sure they do this or better in pharma every day... Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Windows 7 and Xtal Software
On Sun, Aug 28, 2011 at 7:23 PM, Jacob Keller < j-kell...@fsm.northwestern.edu> wrote: > are there any additional problems or known issues running ccp4 or > other xtal software on windows 7 (beyond those of Vista, etc.?) Phenix, ARP/wARP, and HKL2000 do not run on Windows. I'm pretty sure none of Global Phasing's software does either (aside from web interfaces). -Nat
