Re: [ccp4bb] Heavy Atom Phasing
Dear Rhys, the humidity control device at Diamond and ESRF (HC1) is apparently especially recommended for crystals that show large variability. You might want to give this a try, collect a few wedges at RT off many crystals and then Blend them. This approach might increase resolution and, a la W. Hendrickson as mentioned already, signal. Andreas On 27/07/2014 10:48, RHYS GRINTER wrote: Hi All, I thought I might put a question to the community, with the hope of getting some tips of the best way to proceed with my heavy atom phasing problem. I'm working on solving the structure of an integral beta-barrel membrane protein of approximately 100 kDa. I've crystallised protein, growing some very flimsy needle like crystals, and collected datasets to around 3.1 A. I then produced selenomet derivative protein and repeated crystallisation trials in the same conditions and also repeated broad screens, however the derivative protein failed to produce crystals that diffracted beyond 10 A (in fact it barely crystallises at all). So I've moved on to heavy atom soaks and have had some success with tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals didn't dissolve (as they did with gold and samarium compounds) and diffracted to some degree. I collected SAD data to around 6.5 A from these crystals and there seems to be anomolous signal. However, while I get a good CC of 0.4 from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps before and after DM are uninterpretable. I'm guessing the quality and resolution of the data I collected just aren't good enough (the data is reasonably anisotropic). I performed the metal soaking, by taking a small amount of the platinate salt and adding it to the crystallisation drop as the crystals are extremely fragile and don't stand up well to handling through a soaking or cryo solution. Leaving the crystals to soak for 48 hours and then, freezing them directly. The solution is on the border of cryoprotection (the conditions has PEG2000MME and PVP and the precipitants), but with native crystals this doesn't seem to be a parameter which affects diffraction. The crystals are very variable in performance, so while I feel that the heavy atom soaking has compromised their diffractability to a degree, inherent variation may play a part. What I was wondering is if some one with more experience than me found themselves in this position, how would they proceed? Questions which spring to mind are, how much heavy atom compound do people add and how long do they soak for? Is there anyway I can squeeze something out of the anomalous data I have, given I have 'reasonable' native data, or will poor quality data give spuriously positive statistics for heavy atom phasing? And are there any tricks people have experienced to improve performance of crystals like these (aside from the usual seeding, additives, different detergents etc which I have spend a fair bit of time on optimization already). Thanks in advance, Rhys -- Andreas Förster Crystallization and X-ray Facility Manager Centre for Structural Biology Imperial College London
Re: [ccp4bb] Heavy Atom Phasing
Rhys, since you can obtain good native crystals, I would try Xenon phasing. I have recently heard several success stories at the Australian Synchrotron, and it just seems to be a straightforward and rational way. Molecular Replacement should also be an option. I did this with beta-barrel proteins quite a bit, and even wrote a program for elliptic distortion of the model, to arrive at models that may be closer to the target. good luck, Kay
[ccp4bb] SCRIPT FOR RUNNING MULTIPLE RUNS OF SHELX C DE FOR RIP
Dear CCPers, I am running multiple runs of shelx c d e for a RIP dataset. Does anyone happen to have a script for doing 100s of runs varying the DSCA parameter for downscaling the 'after' dataset?. It will be of great help to me if you could kindly share it. Also if you have past experience solving structures with RIP in low symmetry sg, eg. P1211 at ~2.9 ang resolution (according to I/sigmaI =1.9 in the highest shell, CC half around 0.85) and would kindly share it that will be great. Thanks a lot, Best Regards, Arka Chakraborty -- *Arka Chakraborty* *ibmb (Institut de Biologia Molecular de Barcelona)* *BARCELONA, SPAIN*
Re: [ccp4bb] SCRIPT FOR RUNNING MULTIPLE RUNS OF SHELX C DE FOR RIP
Dear Arka Chakraborty,
such a task is best done with shell tools. Assuming you have a template
input file shelxc.inp with a line DCSA 0.98, you can create the new
ones with
# for i in $(seq -w 90 99); do
dsca=$(echo scale = 3; $i / 100 | bc);
sed s/DSCA 0.98/DSCA $dsca/ shelxc.inp shelxc_${i}.inp;
done
and run shelxc with
for i in shelxc_*.inp; do
shelxc ${i%.inp} $i | tee ${i%.inp}.log
done
and shelxd with
for i in shelxc_*_fa.ins; do
shelxd ${i%.ins}
done
If you have access to several machines, you can wrap the last,
time-consuming step into a shell script that changes into your working
directory to start shelxd. Then you can add the script to ssh in order
to remotely execute shelxd.
I did not explicitly test the scripts, but except for typos which should
be easy to correct, they should work.
Regards,
Tim
On 07/28/2014 01:10 PM, Arka Chakraborty wrote:
Dear CCPers,
I am running multiple runs of shelx c d e for a RIP dataset. Does anyone
happen to have a script for doing 100s of runs varying the DSCA parameter
for downscaling the 'after' dataset?. It will be of great help to me if you
could kindly share it.
Also if you have past experience solving structures with RIP in low
symmetry sg, eg. P1211 at ~2.9 ang resolution (according to I/sigmaI =1.9
in the highest shell, CC half around 0.85) and would kindly share it that
will be great.
Thanks a lot,
Best Regards,
Arka Chakraborty
--
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
signature.asc
Description: OpenPGP digital signature
[ccp4bb] areaimol and hydrogens
BSD Dear All, I understood from the areaimol documentation that hydrogens are not included as one of the default atoms. But one can add atoms, and so I added an ATOM line for hydrogen. The program quite happily accepted my input line, but later on stated explicitly that it was ignoring the hydrogens. Is there no way to include hydrogens? Thanks Harry - Harry M. Greenblatt Associate Staff Scientist Dept of Structural Biology Weizmann Institute of SciencePhone: 972-8-934-3625 234 Herzl St.Facsimile: 972-8-934-4159 Rehovot, 76100 Israel harry.greenbl...@weizmann.ac.ilmailto:harry.greenbl...@weizmann.ac.il
Re: [ccp4bb] SCRIPT FOR RUNNING MULTIPLE RUNS OF SHELX C DE FOR RIP
Dear Dr. Tim Gruene,
Thanks a lot for helping me out. My limited knowledge of shell scripting
failed me. I will try it out.
Best Regards,
Arko
On Mon, Jul 28, 2014 at 1:49 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:
Dear Arka Chakraborty,
such a task is best done with shell tools. Assuming you have a template
input file shelxc.inp with a line DCSA 0.98, you can create the new
ones with
# for i in $(seq -w 90 99); do
dsca=$(echo scale = 3; $i / 100 | bc);
sed s/DSCA 0.98/DSCA $dsca/ shelxc.inp shelxc_${i}.inp;
done
and run shelxc with
for i in shelxc_*.inp; do
shelxc ${i%.inp} $i | tee ${i%.inp}.log
done
and shelxd with
for i in shelxc_*_fa.ins; do
shelxd ${i%.ins}
done
If you have access to several machines, you can wrap the last,
time-consuming step into a shell script that changes into your working
directory to start shelxd. Then you can add the script to ssh in order
to remotely execute shelxd.
I did not explicitly test the scripts, but except for typos which should
be easy to correct, they should work.
Regards,
Tim
On 07/28/2014 01:10 PM, Arka Chakraborty wrote:
Dear CCPers,
I am running multiple runs of shelx c d e for a RIP dataset. Does anyone
happen to have a script for doing 100s of runs varying the DSCA parameter
for downscaling the 'after' dataset?. It will be of great help to me if
you
could kindly share it.
Also if you have past experience solving structures with RIP in low
symmetry sg, eg. P1211 at ~2.9 ang resolution (according to I/sigmaI =1.9
in the highest shell, CC half around 0.85) and would kindly share it that
will be great.
Thanks a lot,
Best Regards,
Arka Chakraborty
--
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
--
*Arka Chakraborty*
*ibmb (Institut de Biologia Molecular de Barcelona)*
*BARCELONA, SPAIN*
Re: [ccp4bb] areaimol and hydrogens
I believe that ignoring hydrogen atoms is the default behaviour of any software calculating ASA values. Normally the VdW radii values of the heavy atoms already include the hydrogens implicitly. If your input structure has hydrogens and they were included in the calculation it would result in over-estimating the ASA values. I'm not familiar with AREAIMOL but I guess it must behave in a similar way. Jose On 28/07/14 14:02, Harry Mark Greenblatt wrote: BSD Dear All, I understood from the areaimol documentation that hydrogens are not included as one of the default atoms. But one can add atoms, and so I added an ATOM line for hydrogen. The program quite happily accepted my input line, but later on stated explicitly that it was ignoring the hydrogens. Is there no way to include hydrogens? Thanks Harry - Harry M. Greenblatt Associate Staff Scientist Dept of Structural Biology Weizmann Institute of SciencePhone:972-8-934-3625 234 Herzl St. Facsimile: 972-8-934-4159 Rehovot, 76100 Israel harry.greenbl...@weizmann.ac.il mailto:harry.greenbl...@weizmann.ac.il
Re: [ccp4bb] equivalent osmotic pressures for xtal transfer
Frank, It is not just osmotic factors that need balancing, but the fact that polymers and salts don't always mix well. PEG and high salt can form 2-phase systems that don't disperse well, particularly in a crystal. Check out Ray et al. (Biochemistry 1991, 30, 6866-6875) where Bill Ray and and others from Purdue needed to exchange ~2.1 M ammonium sulfate in phosphoglucomutase crystals to various PEGs just to do the kind of experiments you suggest. It can be a pain, but osmolarity matching was not the big issue. As Bill was an excellent, old-time physical biochemist, he developed the system based on first-principles. You may also look up the work of Pierre Douzou and the cryoenzymology. Douzou collaborated with Petsko on some protein crystal work doing the same kind of exchange to reduce the crystal's freezing point. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jul 26, 2014, at 3:42 PM, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Hi all - The Google fails me, so I'll the CCP4BBoogle: Can anybody conjure up some of the references that describe the details of how to get a (say) PEG solution to have the same osmotic pressure as a (say) salt solution - in particular, this is for transferring a crystal from it's original salt condition into a more ligand-friendly PEG condition. I know there was something from the late nineties, where specific lookup tables were either shown or mentioned; but I've not been able to track it down. Thanks! phx
Re: [ccp4bb] Heavy Atom Phasing
Rhys, If crystals grow reproducibly and within a reasonable timeframe, I would always do co-crystallization, particularly if the HA is a good anomalous scatterer. We have had good success with this method, including a recent membrane protein structure. Even if you get crystals that are not isomorphous with the native, SIRAS is easy to do these days. Also broaden your soaking screens of HAs (if you haven't already), to include mercury compounds, which don't always need a Cys to bind well and love hydrophobic nooks and crannies, and metal clusters (like tantalum bromide). As this is a beta-barrel membrane protein, iodine might also be a way to go. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jul 27, 2014, at 5:48 PM, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi All, I thought I might put a question to the community, with the hope of getting some tips of the best way to proceed with my heavy atom phasing problem. I'm working on solving the structure of an integral beta-barrel membrane protein of approximately 100 kDa. I've crystallised protein, growing some very flimsy needle like crystals, and collected datasets to around 3.1 A. I then produced selenomet derivative protein and repeated crystallisation trials in the same conditions and also repeated broad screens, however the derivative protein failed to produce crystals that diffracted beyond 10 A (in fact it barely crystallises at all). So I've moved on to heavy atom soaks and have had some success with tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals didn't dissolve (as they did with gold and samarium compounds) and diffracted to some degree. I collected SAD data to around 6.5 A from these crystals and there seems to be anomolous signal. However, while I get a good CC of 0.4 from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps before and after DM are uninterpretable. I'm guessing the quality and resolution of the data I collected just aren't good enough (the data is reasonably anisotropic). I performed the metal soaking, by taking a small amount of the platinate salt and adding it to the crystallisation drop as the crystals are extremely fragile and don't stand up well to handling through a soaking or cryo solution. Leaving the crystals to soak for 48 hours and then, freezing them directly. The solution is on the border of cryoprotection (the conditions has PEG2000MME and PVP and the precipitants), but with native crystals this doesn't seem to be a parameter which affects diffraction. The crystals are very variable in performance, so while I feel that the heavy atom soaking has compromised their diffractability to a degree, inherent variation may play a part. What I was wondering is if some one with more experience than me found themselves in this position, how would they proceed? Questions which spring to mind are, how much heavy atom compound do people add and how long do they soak for? Is there anyway I can squeeze something out of the anomalous data I have, given I have 'reasonable' native data, or will poor quality data give spuriously positive statistics for heavy atom phasing? And are there any tricks people have experienced to improve performance of crystals like these (aside from the usual seeding, additives, different detergents etc which I have spend a fair bit of time on optimization already). Thanks in advance, Rhys
Re: [ccp4bb] areaimol and hydrogens
Jose, Your are most probably right. Atoms used for ASA calculations are unified atoms as their vdW radii incorporate light atoms (hydrogens) which, by and large, crystallographers don't see. Adding extra H atoms is likely to end up in miscalculations. Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies 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.Mrabet at univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 28/07/2014 14:20, Jose Manuel Duarte wrote: I believe that ignoring hydrogen atoms is the default behaviour of any software calculating ASA values. Normally the VdW radii values of the heavy atoms already include the hydrogens implicitly. If your input structure has hydrogens and they were included in the calculation it would result in over-estimating the ASA values. I'm not familiar with AREAIMOL but I guess it must behave in a similar way. Jose On 28/07/14 14:02, Harry Mark Greenblatt wrote: BSD Dear All, I understood from the areaimol documentation that hydrogens are not included as one of the default atoms. But one can add atoms, and so I added an ATOM line for hydrogen. The program quite happily accepted my input line, but later on stated explicitly that it was ignoring the hydrogens. Is there no way to include hydrogens? Thanks Harry - Harry M. Greenblatt Associate Staff Scientist Dept of Structural Biology Weizmann Institute of SciencePhone:972-8-934-3625 234 Herzl St.Facsimile: 972-8-934-4159 Rehovot, 76100 Israel harry.greenbl...@weizmann.ac.il mailto:harry.greenbl...@weizmann.ac.il
[ccp4bb] research technician job at Brown University
Dear colleagues, Please see below an ad for a research technician position at Brown University (Rhode Island). Thank you very much. Sincerely yours, Alexandra Deaconescu *Research Technician, Deaconescu Laboratory at Brown University, Providence, Rhode Island* * Job Summary* The Deaconescu Laboratory in the Department of Molecular Biology, Cell Biology and Biochemistry at Brown University (Providence, Rhode Island) is seeking a highly motivated individual to work as a research technician. More information about the lab at: http://brown.edu/research/labs/deaconescu/home. The desired candidate will be responsible for performing routine lab work including media preparation, cloning, large-scale protein expression and purification, preparation of solutions and stocks and protein crystallization. Additional responsibilities include performing general lab administration, which includes organization and maintenance of lab databases and stocks, ordering, data entry and other duties as assigned. The applicant should be highly motivated with thorough, methodological and accurate work habits, and must be able to work both independently and as part of a team. The ideal candidate has a strong ability to multi-task, has good communication skills, and the ability to work as a part of a team, as well as independently, and has prior experience with molecular cloning, protein expression and protein purification. The laboratory has access to a state-of-the-art structural biology facility at Brown equipped with liquid handling robots, and instrumentation suitable for both X-ray diffraction and small-angle X-ray scattering studies as well as other facilities for macromolecular biophysical characterization. * Required Qualification* - Bachelor's degree in a biological science or chemistry is highly desired - Biological laboratory experience is highly desired To apply, please send a CV, undergraduate transcript (if applicable), a letter detailing your interest in the position and contact information for three references to alexandra_deacone...@brown.edu mailto:alexandra_deacone...@brown.edu. Write technician candidate in the subject line.
