[ccp4bb] International PhD Programme at CNIO, Spain
Dear colleagues, We would like to draw your attention to this year’s call for the L*a Caixa-Severo Ochoa International PhD Programme *at the* **Spanish National Cancer Research Centre (CNIO)**, *Madrid, Spain. The programme provides full support for 4-years. The next deadline for applications is *March 15th, 2013*. For more information consult: http://www.cnio.es/ing/cursos/programadoctorado.asp or contact our Training Office at p...@cnio.es. The *CNIO* offers excellent training and research opportunities in cutting edge basic and applied cancer research. The centre is equipped with state of the art facilities for protein expression, crystallisation, X-ray crystallography and NMR (including crystallisation robots, crystal farms, X-ray generators etc.). Several structural biology groups offer research opportunities in various areas of cancer biology. We would greatly appreciate if you could forward this message to potential candidates and distribute it on your local institute networks. Best regards, Daniel Lietha, Group Leader Cell Signalling and Adhesion Group Structural Biology and Biocomputing Programme Spanish National Cancer Research Centre (CNIO) C/ Melchor Fernández Almagro, 3, E-28029 Madrid, Spain Tel: + (34) 917 328 000 ext. 3090 Email: dlie...@cnio.es
[ccp4bb] generating electron density from PDB and structure factor file
Dear All, I would like to know what is the best possible way to generate the density from the published pdb file. thanks, Bhat
Re: [ccp4bb] electron density assignment
Generate an anomalous map and look for peaks. Many metals would generate anomalous. On 02/04/13 07:39, Gang Dong wrote: Dear all, Here are some hexmeric densities we observed in our 1.6-A resolution 2Fo-Fc map. They are located in between two dimers. Although 7 waters would fit nicely in the densities, we are not sure whether they might be something else (metals?). Any suggestions are welcome. Thanks! Gang ... -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] electron density assignment
Hi, I observed a very similar hexagonal arrangement of electron density blobs in one of my structures recentely and asked the CCP4bb community to help me explain it. Unfortunately, noone could come up with some satisfactory explanation, so I gave up on interpreting this rogue density. What was certain beyond doubt is that it was somehow caused by the presence of cobalt ions. Do you, by chance, also have cobalt or similar ions present in your crystallization condition? Regards, Joern ** Address: Joern Krausze Molecular Structural Biology Helmholtz Centre for Infection Research Inhoffenstrasse 7 38124 Braunschweig Germany Email: joern.krau...@helmholtz-hzi.de Phone: +49 (0)531 6181 7023 (office) +49 (0)531 6181 7020 (lab) ** On Mon, 4 Feb 2013, Gang Dong wrote: Dear all, Here are some “hexmeric” densities we observed in our 1.6-A resolution 2Fo-Fc map. They are located in between two dimers. Although 7 waters would fit nicely in the densities, we are not sure whether they might be something else (metals?). Any suggestions are welcome. Thanks! Gang [IMAGE] __ Gang Dong, PhD Junior Group Leader Max F. Perutz Laboratories (MFPL) Dr. Bohrgasse 9/3 A-1030 Vienna, Austria Phone: +43-1-4277-61625 FAX: +43-1-4277-9616 http://www.mfpl.ac.at/mfpl-group/group/dong.html Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 Braunschweig | www.helmholtz-hzi.de Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, Bundesministerium für Bildung und Forschung Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches Ministerium für Wissenschaft und Kultur Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA Gesellschaft mit beschränkter Haftung (GmbH) Sitz der Gesellschaft: Braunschweig Handelsregister: Amtsgericht Braunschweig, HRB 477
[ccp4bb] Frontiers in Neutron Structural Biology Workshop
Frontiers in Neutron Structural Biology, Oak Ridge National Laboratory, Spallation Neutron Source April 16-18, 2013 This meeting will bring together scientists to discuss new opportunities for biomedical research at the two advanced neutron user facilities (SNS and HFIR) at the Department of Energy's (DOE) Oak Ridge National Laboratory (ORNL). Users now have access to some of the world's most intense neutron beamlines for studying the structure, function, and dynamics of complex biological systems. ORNL plans to establish and operate a biomedical neutron technology research center (BNTRC) that will integrate and develop neutron scattering technologies with high performance supercomputing and biomolecular synthesis and deuterium-labeling. Participants will identify new scientific challenges and lines of biomedical inquiry that will drive the development and integration of these leading-edge technologies. An important function of the BNTRC will be to provide training, access, and expert assistance in these technologies to biomedical researchers. In a satellite meeting, the developers of the comprehensive software suite Phenix (http://www.phenix-online.org/)http://www.phenix-online.org/ for macromolecular structure determination will give a one-day workshop on the use of the software for macromolecular neutron crystallography (MNC). Participants are encouraged to bring a laptop for the afternoon tutorial session. MNC is in a period of expansion at the moment; in North America the number of beamlines suitable for MNC is expected to quadruple over the next year. This workshop will introduce current and future MNC users to the new experimental beamlines at ORNL and provide a tutorial for structure refinement using PHENIX and nCNS. New developments will be described that greatly enhance structure refinement. Participants will be given real X-ray and neutron (XN) data and will be taken through joint XN structure refinement of proteins. Scholarships for attendance are available from: * Joint Institute for Neutron Scienceshttp://jins.tennessee.edu/ for those attendees from EPSCOR states (Alabama, Alaska, Arkansas, Delaware, Hawaii, Idaho, Iowa, Kansas, Kentucky, Louisiana, Maine, Mississippi, Montana, Nebraska, Nevada, New Mexico, North Dakota, Oklahoma, Rhode Island, South Carolina, South Dakota, Tennessee, Utah, Vermont, West Virginia, Wyoming, the Commonwealth of Puerto Rico, and the Virgin Islands). For details, please visit http://jins.tennessee.edu/epscor/index.html for the application form and contact Hope Moore at hmoo...@utk.edumailto:hmoo...@utk.edu. * The ORAU Travel Grants Programhttp://www.orau.org/university-partnerships/faculty-student-programs/default.aspx provides up to $800 to facilitate travel by a faculty member from an ORAU Sponsoring or Associate Institution or Branch Campus. Visits can be to collaborate with researchers at ORNL, Y-12, ORAU laboratories or work sites, or another ORAU institution. To apply, visit the ORAU Members Universities pagehttp://www.orau.org/university-partnerships/members.aspx, find your school, and contact your member councilor, who can submit a proposal for travel funding to the ORAU University Partnerships Office through the Members Onlyhttp://www.orau.org/university-partnerships/members-only.aspx section of that site. More information can be found at this link https://neutrons.ornl.gov/conf/frontier2013.
[ccp4bb] need some suggestions for crystallization
So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave
Re: [ccp4bb] need some suggestions for crystallization
Hi David try going back to the one that started it all,* myoglobin, a recipe is at http://www.rigaku.com/products/protein/recipes (* feel free to argue about this) On 4 Feb 2013, at Mon4 Feb 16:03, David Roberts wrote: So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
Re: [ccp4bb] generating electron density from PDB and structure factor file
One more option: phenix.fetch_pdb 1akg --maps will fetch structure and reflection data files from PDB and generate 2mFo-DFc and mFo-DFc maps (as well as anomalous difference map if reflection data is anomalous). Pavel On Mon, Feb 4, 2013 at 5:52 AM, Robbie Joosten robbie_joos...@hotmail.comwrote: Hi Bhat, You can run Refmac for 0-cycles to make the maps. If you use unrestrained refinement, you don't have to mess with restraint files. There is also an option in Coot to do this. But if you are in Coot anyway, you might as well get the maps from EDS directly or (after installing a plugin: http://www.cmbi.ru.nl/pdb_redo/coot.html) get a re-refined model and map form PDB_REDO. Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of bhat Sent: Monday, February 04, 2013 14:16 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] generating electron density from PDB and structure factor file Dear All, I would like to know what is the best possible way to generate the density from the published pdb file. thanks, Bhat
Re: [ccp4bb] electron density assignment
*** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** Dear Gang, By chance, P222 is your space group? It seems to be three perpendicular two-fold axes are passing through your central atom which makes them all fall in the same plane. From the density it looks more like a water in the center but anomalous density map should help. Good luck. Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: Gang Dong gang.d...@univie.ac.at To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 4 Feb 2013 13:39:39 +0100 Subject: [ccp4bb] electron density assignment *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** Dear all, Here are some hexmeric densities we observed in our 1.6-A resolution 2Fo-Fc map. They are located in between two dimers. Although 7 waters would fit nicely in the densities, we are not sure whether they might be something else (metals?). Any suggestions are welcome. Thanks! Gang __ Gang Dong, PhD Junior Group Leader Max F. Perutz Laboratories (MFPL) Dr. Bohrgasse 9/3 A-1030 Vienna, Austria Phone: +43-1-4277-61625 FAX: +43-1-4277-9616 http://www.mfpl.ac.at/mfpl-group/group/dong.html --- End of Original Message --- This Mail Scanned by ClamAV and Spammassassin
Re: [ccp4bb] need some suggestions for crystallization
Dear Powell, Isn't it there a way to data mine the PDB or the other repository source for the time/duration/days of the crystals obtained. Dr. Jayashankar Selvadurai Hannover Germany On Mon, Feb 4, 2013 at 5:10 PM, Harry Powell ha...@mrc-lmb.cam.ac.ukwrote: Hi David try going back to the one that started it all,* myoglobin, a recipe is at http://www.rigaku.com/products/protein/recipes (* feel free to argue about this) On 4 Feb 2013, at Mon4 Feb 16:03, David Roberts wrote: So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
Re: [ccp4bb] need some suggestions for crystallization
I don't know. Is there? On 4 Feb 2013, at Mon4 Feb 16:15, Jayashankar wrote: Dear Powell, Isn't it there a way to data mine the PDB or the other repository source for the time/duration/days of the crystals obtained. Dr. Jayashankar Selvadurai Hannover Germany On Mon, Feb 4, 2013 at 5:10 PM, Harry Powell harry@mrc- lmb.cam.ac.uk wrote: Hi David try going back to the one that started it all,* myoglobin, a recipe is at http://www.rigaku.com/products/protein/recipes (* feel free to argue about this) On 4 Feb 2013, at Mon4 Feb 16:03, David Roberts wrote: So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing) Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
Re: [ccp4bb] need some suggestions for crystallization
Hi David, You could try the Glucose Isomerase supplied by Hampton. It crystallizes under a number of conditions, details of which you can find in their manual. http://hamptonresearch.com/product_detail.aspx?cid=28sid=56pid=56 Ganesh Le 04/02/13 17:03, David Roberts a écrit : So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave
Re: [ccp4bb] need some suggestions for crystallization
*** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** Dave, You can try any or all of these proteins commercially available and all conditions for crystallization and freezing established. LysozymeFerritinGlucose isomerase Myoglobin Proteinase KThaumatin Trypsin You can follow this link. We used some of the conditions mentioned in their successfully. http://www.rigaku.com/products/protein/recipes Disclaimer: I do not have any commercial interest with Rigaku. Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: Harry Powell ha...@mrc-lmb.cam.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 4 Feb 2013 16:10:54 + Subject: Re: [ccp4bb] need some suggestions for crystallization *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** Hi David try going back to the one that started it all,* myoglobin, a recipe is at http://www.rigaku.com/products/protein/recipes (* feel free to argue about this) On 4 Feb 2013, at Mon4 Feb 16:03, David Roberts wrote: So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing) --- End of Original Message --- This Mail Scanned by ClamAV and Spammassassin
Re: [ccp4bb] need some suggestions for crystallization
My suggestions would be to look up citations for thaumatin and glucose isomerase. If I remember correctly, both of them form well diffracting crystals within a short period of time. I think you can also buy the purified protein from a vendor. Perhaps you could also try the good old lysozyme. Cheers, Raji On Mon, Feb 4, 2013 at 11:03 AM, David Roberts drobe...@depauw.edu wrote: So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] need some suggestions for crystallization
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Dave, many methods articles mention a small set of commonly used proteins. E.g. Mueller et al, Optimal fine phi-slicing for single-photon-counting pixel detectors, Acta Cryst D68, p42-56 list Insulin, Lysozyme, Thaumatin, and Thermolysin; Nanao et al, Improving radiation-damage substructures for RIP, Acta D61, 1227-1237, list Elastase, Insulin, Lysozyme, Ribonuclease A, Thaumatin, and Trypsin. Elastase, though, decided not to crystallise any longer about ten'ish years ago. Attached is a different condition for Lysozyme which I received from Prof. Susana Andrade and which I usually use for teaching. - the crystals grow during lunch time. They can be picked directly and you can show the cryo-protecting effect of Ethylene glycol with increasing concentration. Best, Tim On 02/04/2013 05:03 PM, David Roberts wrote: So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave - -- - -- 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/ iD8DBQFRD+M2UxlJ7aRr7hoRAnDhAJ0bE6xl0XDxV6qoKslThPyyVd3CwACgpjnA sIjr3296ejuwby0ZyXLw5po= =JgwT -END PGP SIGNATURE- xtalization_lysozyme.ods Description: application/vnd.oasis.opendocument.spreadsheet
[ccp4bb] Fwd: Strange Density
Hello all, I have recently solved a 2.0 angstrom resolution structure. The structure is near complete but I have some unusual density at the crystallographic interface between two chains of different asymmetric units. The linked photos show the density at with a Fo-Fc at 3 sigma and 2Fo-Fc at 1 sigma. http://s1359.beta.photobucket.com/user/jatmp06/media/asp210b3_zpsfc64f0cb.png.html?sort=3o=1 http://s1359.beta.photobucket.com/user/jatmp06/media/asp310b2_zps5a52816a.png.html?sort=3o=0 The center of the density is approximately 2.1 angtroms from both the Asp sidechain and the carbonyl oxygen on the other chain. I have attempted to fit in either calcium or sodium atom and waters, but these attempts don't satisfy the positive density while maintaining proper coordination. My buffer components are PIPES, NaCl, TCEP and the crystallization conditions are PEG 1K, Calcium acetate, Imidazole pH 8.0, and glycerol. Any insight as to what this density could be would be greatly appreciated. Thanks, Jared Pitts Jared Pitts Doctoral Student Department of Molecular Biology and Microbiology Tufts University School of Medicine 136 Harrison Avenue Arnold Building Room 611 Boston, MA 02139
Re: [ccp4bb] need some suggestions for crystallization
Bovine trypsin works well. You can buy it pretty cheap from Sigma and it crystallizes without further purification, within a week. Crystals diffract to 1.1-1.3 A and are quite robust to handling and soaking. Conditions that I used are described in this ref: http://www.pnas.org/content/103/18/6835.long I would avoid commercial preps of porcine elastase; I messed around with that one around 10 years ago using many reported crystallization conditions and suppliers but never got crystals. Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David Roberts Sent: Monday, February 04, 2013 11:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] need some suggestions for crystallization So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave
Re: [ccp4bb] need some suggestions for crystallization
I second that. Gently swirling the bottle (very important) before pipetting a few microliters (say 100 microliters or whatever). Dialyse overnight vs 10 mM HEPES 2 mM MgCl2 ph7, then get the protein concentration to 10 mg/ml. Set up the drops (sitting drops) versus the same buffer with 30% (v/v) MPD. Direct cryo conditions. Depending on the material (plastic) used for the sitting drop trays you may find it difficult to get crystals out - you'll break up a few - but then no messing up to try to find cryo-conditions. Fred. On 04/02/13 17:27, Ganesh Natrajan wrote: Hi David, You could try the Glucose Isomerase supplied by Hampton. It crystallizes under a number of conditions, details of which you can find in their manual. http://hamptonresearch.com/product_detail.aspx?cid=28sid=56pid=56 Ganesh -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
Re: [ccp4bb] generating electron density from PDB and structure factor file
Dear Bhat, You could use the molmap command within UCSF-Chimera (which implements the routine pdb2mrc from the EM analysis package EMAN). Best Hernando From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of bhat Sent: Monday, February 04, 2013 8:16 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] generating electron density from PDB and structure factor file Dear All, I would like to know what is the best possible way to generate the density from the published pdb file. thanks, Bhat
[ccp4bb] Post-Doctoral Associate in Structural Biology
The Neutron Sciences Directorate (NScD) at Oak Ridge National Laboratory operates the High Flux Isotope Reactor (HFIR), the United States' highest flux reactor based neutron source, and the Spallation Neutron Source (SNS), the world's most intense pulsed accelerator based neutron source. Together these facilities operate 24 instruments for neutron scattering research, each year carrying out in excess of 1,000 experiments in the physical, chemical, materials, biological and medical sciences for more than 3,000 visiting scientists. HFIR also provides unique facilities for isotope production and neutron irradiation. To learn more about Neutron Sciences at ORNL go to: http://neutrons.ornl.gov. The Biology and Soft Matter Division at Oak Ridge National Laboratory (ORNL) invites applications for a post-doctoral structural biologist or macromolecular crystallographer to join its Biology and Biomedical Science group. The successful candidate will join a multi-disciplinary research team investigating the structure and function of biological complexes that are involved in cell-signaling, DNA repair and light harvesting systems. These projects involve multi-task efforts in molecular biology, protein crystallography, small-angle scattering and computational modeling. The position represents an excellent opportunity for researchers to develop their careers and interact with leading scientists from around the world. As an ORNL postdoctoral fellow you will have access to leading small angle neutron scattering and neutron diffraction facilities, biofermentation laboratories, and biophysical characterization laboratories, in addition to in house small angle X-ray scattering and X-ray diffraction instrumentation. A Ph.D. degree in structural biology or a related field is required. The candidates should have a proven track record in molecular biology techniques including cloning, protein expression and purification, crystallographic analysis data and structure determination and refinement. Additional experience in small angle scattering and/or other biophysical characterization techniques would be a distinct advantage. The candidate should be self-motivated, have good interpersonal, communication and presentational skills and demonstrated ability to interact effectively with staff at all levels and to work within a multi-disciplinary team. To find out more information and apply, please visit http://1.usa.gov/WJLxXO Questions regarding this position can be directed to Dr. Dean Myles (myle...@ornl.gov).
Re: [ccp4bb] need some suggestions for crystallization
Human carbonic anhdyrase II can be easily crystallized from 1.3 M sodium citrate/0.1 M TrisCl pH 8.5 at 10 mg/mL protein concentration. Crystals are P21 and easily diffract to beyond 2.0 A on a home source. We cryopreserve in ML + 30% glucose. Sulfonamide ligands are easy to soak into the crystals in a few minutes during cryopreservation, and provide teaching opportunities for protein-ligand model building and refinement. Cheers, ___ Roger Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 02/04/2013 12:31 PM, Jim Pflugrath wrote: A number of protein crystal recipes are available on the Rigaku web site: http://www.rigaku.com/products/protein/recipes Lysozyme is nice because it is so cheap, grows quickly, cryoprotectants in a straightforward way, and the unit cells are not large nor are the crystals fragile. One can get triclinic, monoclinic, orthorhombic, and tetragonal crystals relatively quickly. With hen egg white lysozyme if you are not up for sulfur-SAD phasing, then co-crystallizing or quick-soaking in iodide (say 100 mM) gives a very nice anomalous signal at just about any wavelength. That is, no need to worry about going to a so-called edge. While there are many other easy-to-go crystals, I have found that none of them combine all the properties of hen egg white lysozyme has a good teaching tool. Jim == Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave
Re: [ccp4bb] Fwd: Strange Density
It's possibly a transition metal ion. Zinc is a common adventitious contaminant of solutions. Typical Zn-O distances (tetrahedral or pseudo-tetrahedral coordination) are 2.0 A. ICP-OES or ICP-MS of the protein solution might offer a clue to the possible identity of the metal ion, since it appears to be nearly stoichiometric with your protein. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/4/2013 11:33 AM, Jared Pitts wrote: Hello all, I have recently solved a 2.0 angstrom resolution structure. The structure is near complete but I have some unusual density at the crystallographic interface between two chains of different asymmetric units. The linked photos show the density at with a Fo-Fc at 3 sigma and 2Fo-Fc at 1 sigma. http://s1359.beta.photobucket.com/user/jatmp06/media/asp210b3_zpsfc64f0cb.png.html?sort=3o=1 http://s1359.beta.photobucket.com/user/jatmp06/media/asp310b2_zps5a52816a.png.html?sort=3o=0 The center of the density is approximately 2.1 angtroms from both the Asp sidechain and the carbonyl oxygen on the other chain. I have attempted to fit in either calcium or sodium atom and waters, but these attempts don't satisfy the positive density while maintaining proper coordination. My buffer components are PIPES, NaCl, TCEP and the crystallization conditions are PEG 1K, Calcium acetate, Imidazole pH 8.0, and glycerol. Any insight as to what this density could be would be greatly appreciated. Thanks, Jared Pitts Jared Pitts Doctoral Student Department of Molecular Biology and Microbiology Tufts University School of Medicine 136 Harrison Avenue Arnold Building Room 611 Boston, MA 02139
Re: [ccp4bb] Fwd: Strange Density
On Mon, Feb 4, 2013 at 12:24 PM, Roger Rowlett rrowl...@colgate.edu wrote: It's possibly a transition metal ion. Zinc is a common adventitious contaminant of solutions. Typical Zn-O distances (tetrahedral or pseudo-tetrahedral coordination) are 2.0 A. ICP-OES or ICP-MS of the protein solution might offer a clue to the possible identity of the metal ion, since it appears to be nearly stoichiometric with your protein. Zinc tends not to bind carbonyl oxygens, however, but calcium does quite frequently. Also, the presence of calcium acetate in the crystallization solution strongly suggests that this is what is actually bound (especially if it's at a concentration around 200mM, as is common in many crystallization screens). Jared: what do you mean by proper coordination? Surface ions bound non-specifically frequently don't have recognizable coordination, and they become even more vague as resolution decreases. As always, it would be worth looking at the anomalous difference map, although whether you'll actually see anything depends on the element and on wavelength, data quality, anomalous completeness, etc. -Nat
Re: [ccp4bb] need some suggestions for crystallization
Hi Dave, ProteinaseK is also a good one. Crystallizes rapidly, big crystals, and relatively high resolution data (1.0-1.5A) usually. You can also buy the lyophilized powder from sigma and prepare the sample directly from the commercial material. We use proK for a course here at UCLA, so if you are interested I can send you more details about the protocol. Mike - Original Message - From: David Roberts drobe...@depauw.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, February 4, 2013 8:03:09 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] need some suggestions for crystallization So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] CCP4BB Digest - 3 Feb 2013 to 4 Feb 2013 (#2013-35)
Hi Dave, My experience is that students learn much better when they work with colored proteins or crystals. Myoglobin sounds good, but I haven't worked with it before. I worked previously with a GFP variant, and that was challenging to grow good crystals. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
[ccp4bb] SSRL Workshop on Small-Angle Scattering and Diffraction Studies, March 18 - 20, 2013
The SSRL Structural Molecular Biology Group will host a 3-day comprehensive workshop on the use of non-crystalline small-angle x-ray scattering and diffraction techniques in structural biology research. The workshop will focus on solution x-ray scattering studies on biological macromolecules and macromolecular complexes. The first half of the workshop will have lectures on basic theoretical and experimental aspects of solution scattering as well as recent applications of solution SAXS in structural biology research. Computational approaches to data analysis and structural modeling will also be covered in the lectures. The second half of the workshop will be devoted to hands-on experimental tutorials on solution x-ray scattering at the SSRL Beam Line 4-2 and extensive software tutorial sessions covering all aspects from basic analysis to advanced modeling methods. Participants are encouraged to bring their own solution samples for the experimental tutorials. More details can be found here: http://www-conf.slac.stanford.edu/smbsax/2013/ Registration is $100. Lester Carter BL4-2 - Biological Small Angle X-ray Scattering/Diffraction Beam line SSRL/SLAC Bldg. 120 2575 Sand Hill Road Menlo Park, CA 94025 US Tel: (650) 926-8653 Email: lgcar...@slac.stanford.edu
