Re: [ccp4bb] Phasing at Low Resolution
OK, here's a concrete case: A 150kDa protein complex, the plate-like crystals can be produced in sufficient number; Se-Met derivatives available, total number of Met around 20, subunits could be marked and combined individually. Diffraction is highly anisotropic, in certain directions up to 3.8A,while in others only 5A. Similarly, the spot quality is very dependent on orientation. Space group I222, a=75 b=150 c=250. Datsets scale well with 3-4% Rsym up to 12 A resolution. At 4.5A Rsym rises above 50% (I/sigma is still at 2.0). The 'sweet' slices of the dataset scale significantly better, but give only 70% (non-anomalous) completness. We hope to improve datasets slightly by orienting the crystals. 65% of the structure would be available as coordinate building blocks from the PDB, however, MR with these components so far did not yield a clear solution. Any suggestions or experiences with similar cases are welcome. Cheers, Clemens Zitat von Clemens Vonrhein vonrh...@globalphasing.com: [Show Quoted Text - 64 lines][Zitattext verstecken] Hi Clemens, maybe re-phrasing your question: What would be the best technique/strategy to phase crystals that [ ] diffract to maximal ___ A [ ] typical diffraction to __ A [ ] are radiation sensitive [ ] easily reproducable [ ] large crystals (up to ___ um) [ ] long needles [ ] thin plates [ ] have ___ mol/asu [ ] spacegroup ___ [ ] nice diffraction pattern [ ] poor diffraction pattern (reason: ___) [ ] anisotropic diffraction (resolution in poorest direction: ___ A) [ ] cell dimensions of roughly ___ ___ ___ ___ ___ ___ [ ] purified from native source [ ] expressed in expression system ___ [ ] anything else: ___ Tick the appropriate boxes and fill out the blanks as much as possible - that should give more important and necessary information. There are consequences to consider for all of those points that would then give some rough guidelines for your particular project/problem. Maybe CCP4 should have an online form to describe a particular crystallographic problem? Cheers Clemens On Thu, May 14, 2009 at 09:35:28AM +0200, Clemens Grimm wrote: Dear all, after the SeMet phasing discussion, what would be -in general- the best technique to phase low resolution data (=4A) of large complexes (=150 kDA) - in terms of - derivatization compounds (is there something like the 'golden five' HA compounds for these cases), - data collection techniques and - phasing methods? Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Na/K Phosphate
The reason for the odd K/Na combination is solubility - mixed phosphate is more soluble than either of the individual ones. I try to avoid high phosphate conditions as the Plague. It's great for molecular biology but horrible for crystallization because phosphate + a variety of other ions = lovely salt crystals. It all usually ends up in tears. If you absolutely must make phosphate buffers - just open up any basic practical biochemistry book, there are standard ratios (both by weight and by volumes of molar solutions) that produce specified pH in a wide range. Artem Dear all, Quite a few crystallisation conditions in the screens feature 'sodium/potassium phosphate'. I'm curious to know why such a Na/K mix is there. As the pH is mostly determined by the (H2PO4)- to (HPO4)2- ratio, is there such a need to have both cations? If so, is the Na to K ratio important? On the more practical side of things, if you want to explore an initial hit based on sodium/potassium phosphate, how would you go about it - e.g. what phosphate buffers would you make and at what ratio would you mix them? Thanks in advance, Geoffrey Kong
Re: [ccp4bb] Glycerol as metal chelator?
I wouldn't use a pH of 9 when dealing with metal cations in solution. By and large, metal hydroxides precipitate at alkaline pH. HTH Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Guenter Fritz wrote: Ho, interesting case. Glycerol actually forms weak complexes with metal ions: Journal of Inorganic Biochemistry,60 299-302 (1995) and some references therein. But to be true, I am surprised that glycerol at concentrations of 10-15% (?) as used in cryo conditions can compete with a metal ion binding site in a protein. I remember I myself did titrations with Zn2+ in the presence/absence of 5% glycerol and did not observe any effect on the binding. There might be another effect (?) although I have no idea what it might be. I am working with a metalloprotein that binds cobalt and iron. I was surprised that the solved structures showed the crystals cryoprotected with glycerol are metal free while crystals cryoprotected with ethylene glycol had the metals present. Both cryoprotectant solutions contained metal in the 10 mM range and are buffered at pH 9. I assume glycerol must be a weak chelator otherwise it wouldn't be so ubiquitous in protein biochemistry. Has anyone else experienced this before with glycerol? Ho UC Berkeley
[ccp4bb] MALLS equipment
Dear all, we are interested in the multiangle laser light scatterring (MALLS) methodology but we do not know exactly which is the best system on the market. We plan to use it in a cold room (8°C) combined to an Akta Purifer system. Thanks to all in advance for sharing with us your expertise. Sincerely -- Marc Graille, PhD Yeast Structural Genomics, IBBMC CNRS UMR8619 Bat 430 Universite Paris Sud 91405 Orsay Cedex NEW PHONE NUMBER Tel: (33) 1 69 15 31 57 Fax: (33) 1 69 85 37 15
Re: [ccp4bb] Na/K Phosphate
Hampton Research has a good chart for preparing Na/K phosphate solutions: http://hamptonresearch.com/documents/product/23-000180.pdf Hope this helps! annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) annie.m.hass...@gsk.com Dear all, Quite a few crystallisation conditions in the screens feature 'sodium/potassium phosphate'. I'm curious to know why such a Na/K mix is there. As the pH is mostly determined by the (H2PO4)- to (HPO4)2- ratio, is there such a need to have both cations? If so, is the Na to K ratio important? On the more practical side of things, if you want to explore an initial hit based on sodium/potassium phosphate, how would you go about it - e.g. what phosphate buffers would you make and at what ratio would you mix them? Thanks in advance, Geoffrey Kong
Re: [ccp4bb] Phasing at Low Resolution
My post-doc recently produced a splendid (for its resolution) ~5A map of a medium sized protein-DNA complex using Ta6Br12 clusters. And he's got a good toehold on a ~340kDa complex using the same clusters. So I'm recently converted to these little nuggets. Phoebe Original message Date: Thu, 14 May 2009 09:35:28 +0200 From: Clemens Grimm clemens.gr...@biozentrum.uni-wuerzburg.de Subject: [ccp4bb] Phasing at Low Resolution To: CCP4BB@JISCMAIL.AC.UK Dear all, after the SeMet phasing discussion, what would be -in general- the best technique to phase low resolution data (=4A) of large complexes (=150 kDA) - in terms of - derivatization compounds (is there something like the 'golden five' HA compounds for these cases), - data collection techniques and - phasing methods? Clemens Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
[ccp4bb] Source Ta6Br12 for phasing
Hi, a follow up question: Which supplier offer Ta6Br12? Thanks, Guenter My post-doc recently produced a splendid (for its resolution) ~5A map of a medium sized protein-DNA complex using Ta6Br12 clusters. And he's got a good toehold on a ~340kDa complex using the same clusters. So I'm recently converted to these little nuggets. Phoebe --
Re: [ccp4bb] Source Ta6Br12 for phasing
Try here Jena Scientific or Ryan Scientific in the US http://www.jenabioscience.com/cms/en/1/catalog/1166_tantalum_cluster_derivatization_kit.html Gabriel Birrane Beth Israel Deaconess Medical Center RN325 99 Brookline Ave Boston, MA 02215 USA -Original Message- From: CCP4 bulletin board on behalf of Guenter Fritz Sent: Thu 5/14/2009 1:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Source Ta6Br12 for phasing Hi, a follow up question: Which supplier offer Ta6Br12? Thanks, Guenter My post-doc recently produced a splendid (for its resolution) ~5A map of a medium sized protein-DNA complex using Ta6Br12 clusters. And he's got a good toehold on a ~340kDa complex using the same clusters. So I'm recently converted to these little nuggets. Phoebe --
Re: [ccp4bb] Phasing at Low Resolution
You might find Structure 14, 973-982 Jun 2006 of interest. Larger protein, Zn instead of Se (although only 8 sites), roughly comparable to slightly lower resolution. This was mainly testing to confirm that there was enough anomalous signal to phase off of, so the model was essentially known; however 50% completeness was enough to allow for site location by anomalous or dispersive difference fourier (which one sticking point...even if the MR hits don't give a helpful model phased map, they can still be good enough for site location). Pete Clemens Grimm wrote: OK, here's a concrete case: A 150kDa protein complex, the plate-like crystals can be produced in sufficient number; Se-Met derivatives available, total number of Met around 20, subunits could be marked and combined individually. Diffraction is highly anisotropic, in certain directions up to 3.8A,while in others only 5A. Similarly, the spot quality is very dependent on orientation. Space group I222, a=75 b=150 c=250. Datsets scale well with 3-4% Rsym up to 12 A resolution. At 4.5A Rsym rises above 50% (I/sigma is still at 2.0). The 'sweet' slices of the dataset scale significantly better, but give only 70% (non-anomalous) completness. We hope to improve datasets slightly by orienting the crystals. 65% of the structure would be available as coordinate building blocks from the PDB, however, MR with these components so far did not yield a clear solution. Any suggestions or experiences with similar cases are welcome. Cheers, Clemens Zitat von Clemens Vonrhein vonrh...@globalphasing.com: [Show Quoted Text - 64 lines][Zitattext verstecken] Hi Clemens, maybe re-phrasing your question: What would be the best technique/strategy to phase crystals that [ ] diffract to maximal ___ A [ ] typical diffraction to __ A [ ] are radiation sensitive [ ] easily reproducable [ ] large crystals (up to ___ um) [ ] long needles [ ] thin plates [ ] have ___ mol/asu [ ] spacegroup ___ [ ] nice diffraction pattern [ ] poor diffraction pattern (reason: ___) [ ] anisotropic diffraction (resolution in poorest direction: ___ A) [ ] cell dimensions of roughly ___ ___ ___ ___ ___ ___ [ ] purified from native source [ ] expressed in expression system ___ [ ] anything else: ___ Tick the appropriate boxes and fill out the blanks as much as possible - that should give more important and necessary information. There are consequences to consider for all of those points that would then give some rough guidelines for your particular project/problem. Maybe CCP4 should have an online form to describe a particular crystallographic problem? Cheers Clemens On Thu, May 14, 2009 at 09:35:28AM +0200, Clemens Grimm wrote: Dear all, after the SeMet phasing discussion, what would be -in general- the best technique to phase low resolution data (=4A) of large complexes (=150 kDA) - in terms of - derivatization compounds (is there something like the 'golden five' HA compounds for these cases), - data collection techniques and - phasing methods? Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Phasing at Low Resolution
Dear all Now that tantalum clusters have been mentioned I have recently had issues with the solubility of the Ta6Br12 cluster (obtained from Jena Bioscience) in PEG-based crystallisation conditions at pH 8.0-8.5. Has anybody also experienced this? Is there a trick to getting the compound to dissolve? Best regards, Martin
Re: [ccp4bb] Phasing at Low Resolution
Zn and Se have similar f' and f''. Why Zn seems have much better phasing powder in practice? Is it just because Zn always binds tightly on protein but Se might have higher b-factor? Guangyu On 5/14/09 2:31 PM, Pete Meyer pame...@mcw.edu wrote: You might find Structure 14, 973-982 Jun 2006 of interest. Larger protein, Zn instead of Se (although only 8 sites), roughly comparable to slightly lower resolution. This was mainly testing to confirm that there was enough anomalous signal to phase off of, so the model was essentially known; however 50% completeness was enough to allow for site location by anomalous or dispersive difference fourier (which one sticking point...even if the MR hits don't give a helpful model phased map, they can still be good enough for site location). Pete Clemens Grimm wrote: OK, here's a concrete case: A 150kDa protein complex, the plate-like crystals can be produced in sufficient number; Se-Met derivatives available, total number of Met around 20, subunits could be marked and combined individually. Diffraction is highly anisotropic, in certain directions up to 3.8A,while in others only 5A. Similarly, the spot quality is very dependent on orientation. Space group I222, a=75 b=150 c=250. Datsets scale well with 3-4% Rsym up to 12 A resolution. At 4.5A Rsym rises above 50% (I/sigma is still at 2.0). The 'sweet' slices of the dataset scale significantly better, but give only 70% (non-anomalous) completness. We hope to improve datasets slightly by orienting the crystals. 65% of the structure would be available as coordinate building blocks from the PDB, however, MR with these components so far did not yield a clear solution. Any suggestions or experiences with similar cases are welcome. Cheers, Clemens Zitat von Clemens Vonrhein vonrh...@globalphasing.com: [Show Quoted Text - 64 lines][Zitattext verstecken] Hi Clemens, maybe re-phrasing your question: What would be the best technique/strategy to phase crystals that [ ] diffract to maximal ___ A [ ] typical diffraction to __ A [ ] are radiation sensitive [ ] easily reproducable [ ] large crystals (up to ___ um) [ ] long needles [ ] thin plates [ ] have ___ mol/asu [ ] spacegroup ___ [ ] nice diffraction pattern [ ] poor diffraction pattern (reason: ___) [ ] anisotropic diffraction (resolution in poorest direction: ___ A) [ ] cell dimensions of roughly ___ ___ ___ ___ ___ ___ [ ] purified from native source [ ] expressed in expression system ___ [ ] anything else: ___ Tick the appropriate boxes and fill out the blanks as much as possible - that should give more important and necessary information. There are consequences to consider for all of those points that would then give some rough guidelines for your particular project/problem. Maybe CCP4 should have an online form to describe a particular crystallographic problem? Cheers Clemens On Thu, May 14, 2009 at 09:35:28AM +0200, Clemens Grimm wrote: Dear all, after the SeMet phasing discussion, what would be -in general- the best technique to phase low resolution data (=4A) of large complexes (=150 kDA) - in terms of - derivatization compounds (is there something like the 'golden five' HA compounds for these cases), - data collection techniques and - phasing methods? Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Na/K Phosphate
On a side note, I have created a web server that, based on your sample buffer, makes a crude attempt at prediciting conditions within a screen that may form salt crystals (though this probably should be taken with a grain of. salt, ahem!). http://www.pageforaday.com/xtalwizard/salt.php Date: Thu, 14 May 2009 09:37:05 -0400 From: ar...@xtals.org Subject: Re: [ccp4bb] Na/K Phosphate To: CCP4BB@JISCMAIL.AC.UK The reason for the odd K/Na combination is solubility - mixed phosphate is more soluble than either of the individual ones. I try to avoid high phosphate conditions as the Plague. It's great for molecular biology but horrible for crystallization because phosphate + a variety of other ions = lovely salt crystals. It all usually ends up in tears. If you absolutely must make phosphate buffers - just open up any basic practical biochemistry book, there are standard ratios (both by weight and by volumes of molar solutions) that produce specified pH in a wide range. Artem Dear all, Quite a few crystallisation conditions in the screens feature 'sodium/potassium phosphate'. I'm curious to know why such a Na/K mix is there. As the pH is mostly determined by the (H2PO4)- to (HPO4)2- ratio, is there such a need to have both cations? If so, is the Na to K ratio important? On the more practical side of things, if you want to explore an initial hit based on sodium/potassium phosphate, how would you go about it - e.g. what phosphate buffers would you make and at what ratio would you mix them? Thanks in advance, Geoffrey Kong _ Insert movie times and more without leaving Hotmail®. http://windowslive.com/Tutorial/Hotmail/QuickAdd?ocid=TXT_TAGLM_WL_HM_Tutorial_QuickAdd1_052009
[ccp4bb] March 15, 2009 deadline- User proposal submission for Collaborative Crystallography at BCSB
Dear Users, The deadline for July/August 2009 Collaborative Crystallography proposals will be May 15, 2009. = Thumbnail Description: Through the Collaborative Crystallography Program (CC) at the Advanced Light Source (ALS), scientists can send protein crystals to Berkeley Center for Structural Biology (BCSB) staff researchers for data collection and analysis. The CC Program can provide a number of benefits to researchers: * Obtain high quality data and analysis through collaborating with expert beamline researchers; * Rapid turn around on projects; and * Reduced travel costs. To apply, please submit a proposal through the ALS General User proposal review process for beamtime allocation. Proposals are reviewed and ranked by the Proposal Study Panel, and beamtime is allocated accordingly. BCSB staff schedule the CC projects on Beamlines 5.0.1 and 5.0.2 to fit into the available resources. Only non-proprietary projects will be accepted. As a condition of participation, BCSB staff researchers who participate in data collection and/or analysis must be appropriately acknowledged - typically being included as authors on publications and in PDB depositions. Please consult the website for additional information at: http://bcsb.als.lbl.gov/wiki/index.php/Collaborative_Crystallography = How To Apply: To submit a proposal, go to: http://www-als.lbl.gov/als/quickguide/independinvest.html Scroll down to Structural Biology and SAXS and click on New Proposal. Enter your proposal information, with attention to the following details: * For question 3/First choice, select 5.0.1 (Monochromatic); for question 3/Second choice, select 5.0.2 (MAD). * Check box (yes) in response to question Do you want collaborative crystallography (beamline 5.0.1 or 5.0.2 only ?) * In question 9, please describe a specific research project with a clear end point. In order to request CC time for July/August 2009allocation period, proposals must be submitted by *May 15, 2009*. Please note that the ALS will be shut down in September, 2009. The deadline for CC proposals for the time period October/November/December 2009 will be August 15, 2009. Regards, Banumathi Sankaran
Re: [ccp4bb] Phasing at Low Resolution
I have two questions regarding derivatization: (i) Is there a pH dependence to making derivatives ? i.e. effectiveness of getting derivatives at low vs high pH ? (ii) Any thoughts on the effectiveness of Barium for phasing low resolution structures (especially for magnesium binding proteins ) ??? Thanks
[ccp4bb] Crystallizing Fluorescent Molecules
Dear Crystallographers, I have exactly two spherulite crystals of a protein-peptide complex which have a fluorescently-labelled peptide in them, and are therefore nicely colorful in both the light and fluorescence microscopes, making it easier to know that at least the peptide is in the crystal. However, they are not reproducible. Having gone through the usual list of possible variations which might account for the irreproducibility, I have hit the bottom of the barrel. I was thinking that since the original crystals grew in utter darkness, undisturbed for two weeks while I was away, they were able to nucleate. Is it possible that light exciting the fluorophores is detrimental to crystallization? Or perhaps the complete uniformity of temperature? Even microseeding from one of the spherulites produced nothing (except in the original well.) Any brilliant suggestions welcome... Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Phasing at Low Resolution
Regarding question 1, see: Acta Cryst. (2008). D64, 354-367 Towards a rational approach for heavy-atom derivative screening in protein crystallography J. Agniswamy, M. G. Joyce, C. H. Hammer and P. D. Sun Cheers. riya doreen wrote: I have two questions regarding derivatization: (i) Is there a pH dependence to making derivatives ? i.e. effectiveness of getting derivatives at low vs high pH ? (ii) Any thoughts on theeffectiveness of Barium for phasing low resolution structures (especially formagnesium bindingproteins) ??? Thanks -- Roger S. Rowlett Professor Colgate University Presidential Scholar 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...@mail.colgate.edu
Re: [ccp4bb] Phasing at Low Resolution
Dear Martin You can either add the cluster as a powder, just dip the tip of a needle in the powder and gently approach the drop with the needle, the Ta6Br12 crystals will spray itself onto the drop. Then you just wait for the crystal to turn green, I usually marked the position where the powder had hit the drop, then mounted crystals lying with various distance from the powder site. This was how we derivatized the Na,K ATPase crystals (pH 7, 14 % PEG2kmme), with the H-ATPase crystal it was a bit more tricky, the cluster would not dissolve that readily, and one way to get around this was to dissolve the Ta6Br12 in water (approx 5 mM) then use a speed vac to increase the concentration even further. The highly concentrated Ta6Br12 will form a small round green ball, that you can break with a needle and transfer a tiny droplet to your crystal drop. The same approach was used with orange Pt ( Pt(II)- terpyridine chloride) best of luck Preben On 14/05/2009, at 20.29, Martin Jinek wrote: Dear all Now that tantalum clusters have been mentioned I have recently had issues with the solubility of the Ta6Br12 cluster (obtained from Jena Bioscience) in PEG-based crystallisation conditions at pH 8.0-8.5. Has anybody also experienced this? Is there a trick to getting the compound to dissolve? Best regards, Martin Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds Vej 10 C DK - 8000 Aarhus C Tel. +45 8942 5257, Fax. +45 8612 3178 j...@mb.au.dk website: http://person.au.dk/da/j...@mb
Re: [ccp4bb] Phasing at Low Resolution
Guangyu Zhu wrote: Zn and Se have similar f' and f''. Why Zn seems have much better phasing powder in practice? Is it just because Zn always binds tightly on protein but Se might have higher b-factor? I don't have a better explanation than tighter binding for Zn, although I suppose that it's possible partial Se incorporation could be a factor as well. Guangyu On 5/14/09 2:31 PM, Pete Meyer pame...@mcw.edu wrote: You might find Structure 14, 973-982 Jun 2006 of interest. Larger protein, Zn instead of Se (although only 8 sites), roughly comparable to slightly lower resolution. This was mainly testing to confirm that there was enough anomalous signal to phase off of, so the model was essentially known; however 50% completeness was enough to allow for site location by anomalous or dispersive difference fourier (which one sticking point...even if the MR hits don't give a helpful model phased map, they can still be good enough for site location). Pete Clemens Grimm wrote: OK, here's a concrete case: A 150kDa protein complex, the plate-like crystals can be produced in sufficient number; Se-Met derivatives available, total number of Met around 20, subunits could be marked and combined individually. Diffraction is highly anisotropic, in certain directions up to 3.8A,while in others only 5A. Similarly, the spot quality is very dependent on orientation. Space group I222, a=75 b=150 c=250. Datsets scale well with 3-4% Rsym up to 12 A resolution. At 4.5A Rsym rises above 50% (I/sigma is still at 2.0). The 'sweet' slices of the dataset scale significantly better, but give only 70% (non-anomalous) completness. We hope to improve datasets slightly by orienting the crystals. 65% of the structure would be available as coordinate building blocks from the PDB, however, MR with these components so far did not yield a clear solution. Any suggestions or experiences with similar cases are welcome. Cheers, Clemens Zitat von Clemens Vonrhein vonrh...@globalphasing.com: [Show Quoted Text - 64 lines][Zitattext verstecken] Hi Clemens, maybe re-phrasing your question: What would be the best technique/strategy to phase crystals that [ ] diffract to maximal ___ A [ ] typical diffraction to __ A [ ] are radiation sensitive [ ] easily reproducable [ ] large crystals (up to ___ um) [ ] long needles [ ] thin plates [ ] have ___ mol/asu [ ] spacegroup ___ [ ] nice diffraction pattern [ ] poor diffraction pattern (reason: ___) [ ] anisotropic diffraction (resolution in poorest direction: ___ A) [ ] cell dimensions of roughly ___ ___ ___ ___ ___ ___ [ ] purified from native source [ ] expressed in expression system ___ [ ] anything else: ___ Tick the appropriate boxes and fill out the blanks as much as possible - that should give more important and necessary information. There are consequences to consider for all of those points that would then give some rough guidelines for your particular project/problem. Maybe CCP4 should have an online form to describe a particular crystallographic problem? Cheers Clemens On Thu, May 14, 2009 at 09:35:28AM +0200, Clemens Grimm wrote: Dear all, after the SeMet phasing discussion, what would be -in general- the best technique to phase low resolution data (=4A) of large complexes (=150 kDA) - in terms of - derivatization compounds (is there something like the 'golden five' HA compounds for these cases), - data collection techniques and - phasing methods? Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Phasing at Low Resolution
Maybe I'm wrong. Myself solved structure with 1Zn/400aa. In Pete's case 1Zn/570 aa structure was solved at low resolution. I also heard some other cases of 1Zn for 400 aa. But for Se, I never heard 1se for 300aa. Se could be partially incorporated or have mixed oxidation state. This could affect phasing, but could be avoided in most cases. Guangyu On 5/14/09 4:48 PM, Tommi Kajander tommi.kajan...@helsinki.fi wrote: ...Based on what? i doubt thats true. Quoting Guangyu Zhu g...@hwi.buffalo.edu: Zn and Se have similar f' and f''. Why Zn seems have much better phasing powder in practice? Is it just because Zn always binds tightly on protein but Se might have higher b-factor? Guangyu -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Crystallizing Fluorescent Molecules
CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote on 05/14/2009 03:42:05 PM: Dear Crystallographers, I have exactly two spherulite crystals of a protein-peptide complex which have a fluorescently-labelled peptide in them, and are therefore nicely colorful in both the light and fluorescence microscopes, making it easier to know that at least the peptide is in the crystal. However, they are not reproducible. Having gone through the usual list of possible variations which might account for the irreproducibility, I have hit the bottom of the barrel. I was thinking that since the original crystals grew in utter darkness, undisturbed for two weeks while I was away, they were able to nucleate. Is it possible that light exciting the fluorophores is detrimental to crystallization? Or perhaps the complete uniformity of temperature? Even microseeding from one of the spherulites produced nothing (except in the original well.) Any brilliant suggestions welcome... Jacob Keller Hi Jacob - I just want to raise a warning, since a very similar situation bit me back in grad school. I was trying to crystallize RecA with a fluorescently-labeled oligo, and just like you, I got green-glowing crystals, along with some glowing precipitate. I got extremely excited, and a couple of months later had the structure done. There was no DNA in the structure. I had crystallized unliganded RecA, and the fluorescent DNA had precipitated all over the outside of the crystal, painting it and making it look green! Are you certain that your peptide doesn't precipitate under your crystallization conditions? I hope for your sake that my problem is not yours... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] Phasing at Low Resolution
I like PIP (di-mu-iodo-bis-ethylenediamine-di-platinum (II) nitrate). At low resolution it offers you 4 heavy atoms (I2Pt2), a choice of anomalous signal, and at higher resolution if you can resolve the 4 heavy atoms you then instantly get higher resolution phases. It likes binding as a low-res ball in active sites and with more order at -S-S- (disulfide) and -S- (methionine thioether) positions. I've also seen it rather arbitrarily located in a solvent channel Cheers, Charlie Phoebe Rice wrote: My post-doc recently produced a splendid (for its resolution) ~5A map of a medium sized protein-DNA complex using Ta6Br12 clusters. And he's got a good toehold on a ~340kDa complex using the same clusters. So I'm recently converted to these little nuggets. Phoebe Original message Date: Thu, 14 May 2009 09:35:28 +0200 From: Clemens Grimm clemens.gr...@biozentrum.uni-wuerzburg.de Subject: [ccp4bb] Phasing at Low Resolution To: CCP4BB@JISCMAIL.AC.UK Dear all, after the SeMet phasing discussion, what would be -in general- the best technique to phase low resolution data (=4A) of large complexes (=150 kDA) - in terms of - derivatization compounds (is there something like the 'golden five' HA compounds for these cases), - data collection techniques and - phasing methods? Clemens Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310 35 Stirling Highway Crawley WA 6009 Australia charles.b...@uwa.edu.au +61 8 6488 4406
