Re: [ccp4bb] Best compounds for heavy atom soaks
Dear Rhys, You may consider Xenon derivative, which could be prepared simply pressurizing the protein crystals in a xenon chamber. It does not require any modification of mother liquor. It just needs cryo-protectant where crystals are stable for at least one to two mins. Higher Pressure (20 to 40 bar) and 1-2 min incubation time are normally sufficient for binding of Xenon to proteins. Xenon binds to pre existing hydrophobic cavities of proteins by dispersion force. Xenon derivatives are highly isomorphous to native crystals. So SIRAS phasing could be efficient. However if you consider collecting data at longer wavelengths you could get anomalous signal from sulphur too. Weaker SAD or SIRAS phases from Xenon derivative could be used to bootstrap the Sulphur phasing. Similarly Kr pressurisation could be tried. MAD experiment can be performed at any tunable beamline but the disadvantage with this derivative is, it desorps quickly during cooling after pressurisation leaving out with lower than 50-60% occupancy. Success of the Xenon/Krypton derivatisation depends on size of the proteins and how stable your crystals are under cryo-protectant. The bigger the protein, higher the chance of Xe/Kr binding. best Santosh Santosh Panjikar, Ph.D. Scientist Australian Synchrotron 800 Blackburn Road Clayton VIC 3168 Australia Ph: +61-4-67770815 (mobile) +61-3-85404276 (office)
Re: [ccp4bb] Problem in running ARP_WARP in CCP4i
try tcsh auto_tracing.sh datafile ../psp.mtz Santosh Panjikar, Ph.D. Scientist Australian Synchrotron 800 Blackburn Road Clayton VIC 3168 Australia Ph: +61-4-67770815 (mobile) +61-3-85404276 (office) From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of S. Karthikeyan [skart...@imtech.res.in] Sent: Friday, March 08, 2013 12:38 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Problem in running ARP_WARP in CCP4i Dear CCP4BB members, I am trying to run arp_warp classic (7.3 version) through ccp4i (CCP4 6.3) in CentOS system. However, it gives the following error even for the example files provided with Arp/Warp. Any suggestions are welcome. --- QUITTING ... ARP/wARP module stopped with an error message: /home/programs/linux/ccp4/arp_warp_7.3/bin/bin-x86_64-Linux/warp_tracing.sh If I try to run through command line it gives the following error: auto_tracing.sh datafile ../psp.mtz Thu Mar 7 19:09:18 IST 2013 Word too long. QUITTING ... ARP/wARP module stopped with an error message: /home/programs/linux/ccp4/arp_warp_7.3/bin/bin-x86_64-Linux/auto_tracing.sh --- Thanking you With regards S. Karthikeyan __ सूक्ष्मजीव प्रौद्योगिकी संस्थान (वैज्ञानिक औद्योगिक अनुसंधान परिषद) Institute of Microbial Technology (A CONSTITUENT ESTABLISHMENT OF CSIR) सैक्टर 39 ए, चण्डीगढ़ / Sector 39-A, Chandigarh पिन कोड/PIN CODE :160036 दूरभाष/EPABX :0172 6665 201-202
Re: [ccp4bb] a challenge
Hi James, The datasets frac.80.mtz to frac.100.mtz are challenging to solve using SAD phasing. However these datasets can be easily solved using other experimental phasing method. Instead of using anomalous signal we could use isomorphous signal only. For example RIP or SIR phasing method, as there is a difference in intensity between the datasets due to scattering of S and Se. Since frac.80.mtz data contains 20% selenium that is sufficient to solve the structure against the frac.100.mtz. It seems the structure can be solved even as less as 10% selenium content (frac.90.mtz vs frac.100.mtz), and substructure can be solved easily. This is not surprising, the pair of the datasets is quite isomorphous, . We phase all reflections (centric and non-centric) where as anomalous phasing we could phase non-centric reflections only. In fact, Single Isomorphous Replacement phasing method is the first phasing technique. This method has been further extended by Ravelli et al with some deviation by introduction of X-ray or UV RIP phasing. I tried RIP (SIR) phasing protocol of Auto-Rickshaw using frac.90.mtz as "before" and frac.100.mtz as "after". Auto-Rickshaw used SHELXC/D/E and ARP/wARP/REFMAC5 to get the partially refined model (Rfree below 30%) . Cheers Santosh Santosh Panjikar, Ph.D. Scientist Australian Synchrotron 800 Blackburn Road Clayton VIC 3168 Australia Ph: +61-4-67770851 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of James Holton [jmhol...@lbl.gov] Sent: Monday, January 14, 2013 8:12 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] a challenge I am absolutely delighted at the response I have gotten to my little "John Henry Challenge"! Three people already have managed to do the "impossible". Congratulations to George Sheldrick, Pavol Skubak and Raj Pannu for finding ways to improve the phases over the ones I originally obtained (using the default settings of mlphare and dm) and build their way out of it. This is quite useful information! At least it is to me. Nevertheless, I do think Frances Reyes has a point. This was meant to be a map interpretation challenge, and not a SAD-phasing challenge. I appreciate that the two are linked, but the reason I did not initially provide the anomalous data is because I thought it would be too much to ask people to re-do all the phasing, etc. Yes, there do appear to be ways to improve the maps beyond the particular way I phased them, but no matter how good your phasing program is, there will always be a level of anomalous signal that will lead to phases that are "off" enough to make building the model "impossible". Basically, once the map gets bad enough that just as many "wrong" atoms get built in as "right" atoms, then there is no escape. However, I think human beings should still have an advantage when it comes to pattern recognition, and I remain curious to see if an insightful crystallographer can tip that balance in the right direction. I am also still curious to see if tweaking some setting on some automated building program will do that too. So, my original question remains: are automated building programs better than humans? Any human? I therefore declare the John Henry Challenge still open. But yes, improving the phases can tip the balance too, and the accuracy of the anomalous differences will ultimately affect the accuracy of the phases, and so on. This is a much broader challenge. And I think the best way to frame it is with the question: "How low can the anomalous signal be before any conceivable approach fails?" and perhaps: "What is the best procedure to use for weak anomalous signal?" For those who are interested in joining George, Pavol, Raj and others in this new challenge, the full spectrum of "difficulty" from trivial (100% Se incorporation) to a complete waste of time (0% Se, 100% S) is here: http://bl831.als.lbl.gov/~jamesh/challenge/occ_scan/ The "impossible.mtz" for the John Henry Map Interpretation Challenge was derived from "frac0.79.mtz" and "possible.mtz" from "frac0.78.mtz". These simulated 31% and 32% Se incorporation into Met side chains (respectively). It has now been shown that both of these can be solved automatically if you do the phasing right. But what about frac0.80.mtz? Or frac0.90.mtz ? At least on this one "coordinate" of Se incorporation, the prowess of a particular approach can be given a "score". For example, a "score" of 0.78 means that the indicated procedure could solve the frac0.78.mtz dataset, but not the frac0.79.mtz dataset. Based on the reports I have gotten back so far, the "difficulty score" lineup is: score method 0.86 xds, xscale, right sites, crank2 (Pavol Skubak) 0.78 xds, xscale, right sites, mlphare, d
Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase
Dear Jiamu, Try out MRSAD protocol of Auto-Rickshaw (http://www.embl-hamburg.de/Auto-Rickshaw). You can start this just by providing your selenomet data, starting model, sequence information and space group on the Auto-Rickshaw server. It would do substructure determination based on your model phase, experimental phasing, phase combination, density modification, model building and refinement of the built model. Iteratively it improves phases and complete your model as much as possible. Best Santosh Quoting "Jiamu Du" : Dear All, I am now working on a low resolution phase determination (around 3.3 A with Se anomalous signal around 3.8 A). I can find the Se site and get the phase, but the density map is not so good. Some part of the protein (about 1/3) has a homologue model which is also can be found using Phaser. The homologue region has a good map while other region only show a poor map. I think the combination of experimental phase and MR phase might improve the map. Is there anybody can help find which program can work on this? Thanks. -- Jiamu Du, Ph.D. Postdoctoral Research Fellow Laboratory of Structural Biology Memorial Sloan-Kettering Cancer Center RRL 269, 430 E 67th Street New York, NY, 10021 E-mail: d...@mskcc.org Tel: (217) - 417 - 9897 Santosh Panjikar, Ph.D. Staff Scientist EMBL-Hamburg Outstation C/o DESY Notkestrasse 85 22603 Hamburg (Germany) panjikar at embl-hamburg.de http://www.embl-hamburg.de/~panjikar
Re: [ccp4bb] Help needed in solving a MAD dataset
Dear All, Not only for beginners, but also for experts who want to solve structures in hurry at the beamline and who want to complete model starting from X-ray data at resolution better than 3.0 A resolution. Santosh Quoting "Ulrich Zander" : Hi Qing Lu, try Autorickshaw: http://www.embl-hamburg.de/Auto-Rickshaw/ It can perform the complete structure solution procedure if the quality of your data is sufficient and I consider it very user-friendly, especially for beginners . Regards, Uli Hi All, I am new to protein crystallography. I would like to know the steps involved in solving a MAD dataset by using the program in CCP4 where you determine the phases and then obtain the trace. The dataset is collected at 3 different wavelengths (peak, inflection and remote) using Se-Met as the scatterer. The crystals diffracted to resolution of 2 Angstrsoms and has a good anomalous signal. Thanks, Qing Lu Santosh Panjikar, Ph.D. Staff Scientist EMBL-Hamburg Outstation C/o DESY Notkestrasse 85 22603 Hamburg (Germany) panjikar at embl-hamburg.de http://www.embl-hamburg.de/~panjikar
[ccp4bb] Auto-Rickshaw: the EMBL-HH automated crystal structure determination platform
Dear Colleagues, Have you ever heard of Auto-Rickshaw? Auto-Rickshaw is the EMBL Hamburg automated structure determination platform, which is now available as a web service. Try it out and solve your structure in minutes. The EMBL-Hamburg beamline user have been successfully using this system for a little over three years. Now, for the new release with a lot of added functionalities, we are happy to announce that Auto-Rickshaw can be accessed by anybody in the Structural Biology community. The Auto-Rickshaw phasing protocols include SAD, SIRAS, MAD, MR and MRSAD. There are two versions: a "beamline version" and an "advanced version". The beamline version is geared towards speed so as to deliver the results in the shortest amount of time whereas the advanced version can be used for more complete model building if the resolution of the data permits. For more detailed information visit http://www.embl-hamburg.de/Auto-Rickshaw The Auto-Rickshaw server is freely accessible to users who have the necessary software licences. Academic usage requires free of charge on-online registration at the same web site. Further inquiries can be made to Santosh Panjikar at [EMAIL PROTECTED] We would like to take this opportunity to thank all the developers of the various crystallographic programs for their kind permission to invoke their software within Auto-Rickshaw. We also gratefully acknowledge the feedback from the EMBL-Hamburg beamline users and everybody who provided test data. Santosh Panjikar on behalf of Auto-Rickshaw team Auto-Rickshaw team (Santosh Panjikar, Venkataraman Parthasarathy, Victor S. Lamzin, Manfred S. Weiss and Paul A. Tucker) Santosh Panjikar, Ph.D. [EMAIL PROTECTED] Staff Scientist EMBL Hamburg outstation http://www.embl-hamburg.de/~panjikar/ DESY, Notkestrasse 85Tel.:+49-40-89902-141 22603, Hamburg, Germany Fax: +49-40-89902-149
[ccp4bb] DANO from PDB
Hi all, Does anybody have a program which can calculate anomalous differences or F+ and F- from a refined structure at given wavelength and resolution ? Thanks Santosh Santosh Panjikar, Ph.D. [EMAIL PROTECTED] Staff Scientist EMBL Hamburg outstation http://www.embl-hamburg.de/~panjikar/ DESY, Notkestrasse 85Tel.:+49-40-89902-141 22603, Hamburg, Germany Fax: +49-40-89902-149
