[ccp4bb] Processing Laue data
What programs are available for processing Laue data to produce an intensity data set? Are explanatory notes or publications available? Rex Palmer Birkbeck College
Re: [ccp4bb] Processing Laue data
Dear Rex, I can speak for the 'Daresbury Laue Software Suite', which is available with full documentation and tutorials from :- http://www.srs.ac.uk/px/jwc_laue/laue_top.html A wide range of validation tests involving both biological and chemical crystallography have been made of the software and these tests are summarised in:- Y.P. Nieh, J. Raftery, S. Weisgerber, J. Habash, F. Schotte, T. Ursby, M. Wulff, A. Haedener, J.W. Campbell, Q. Hao and J.R. Helliwell Accurate and highly complete synchrotron protein crystal Laue diffraction data using the ESRF CCD and the Daresbury Laue software (1999) J. Synchrotron Rad. *6*, 995–1006. In most recent times different facilities have made adaptations for reading the diffraction images from their preferred detector such as at Diamond and, for neutrons, the Institut Laue Langevin and LANSCE at Los Alamos. The main paper cited, if you wish to do a literature follow up of the wide and diverse output from these X-ray and neutron facilities is J R Helliwell et al 'The recording and analysis of Laue diffraction photographs' (1989) J. Appl. Cryst. *22*, 483-497. Besides time-resolved and neutron Laue crystallography applications a more recent materials sciences application of the software is Laue diffraction using SR microbeams sometimes including multiple crystallites such as in ancient pottery. Greetings, John On Fri, Jan 28, 2011 at 8:43 AM, REX PALMER rex.pal...@btinternet.comwrote: What programs are available for processing Laue data to produce an intensity data set? Are explanatory notes or publications available? Rex Palmer Birkbeck College -- Professor John R Helliwell DSc
Re: [ccp4bb] Processing Laue data
Dear Rex, the most popular one seems the Daresbury Laboratory Laue Software Suite, available via http://www.srs.ac.uk/px/jwc_laue/laue_top.html I have not, though, managed to compile the whole suite. There is also a commercial product from renzresearch.com, as M. Blakeley has pointed out to me. I would be curious to learn about more options or how to properly install the Daresbury Suite. Tim On Fri, Jan 28, 2011 at 08:43:44AM +, REX PALMER wrote: What programs are available for processing Laue data to produce an intensity data set? Are explanatory notes or publications available? Rex Palmer Birkbeck College -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
[ccp4bb] REMINDER - EMBO 2011 Practical Course - ESRF-EMBL, Grenoble, France, 6 - 10 June 2011
Hi all, Just a reminder, as deadline is approacing DEADLINE TO APPLY FEBRUARY 28th COURSE ANNOUCEMENT EMBO 2011 Practical Course - Exploiting Anomalous Scattering in Macromolecular Structure Determination ESRF-EMBL, Grenoble, France, 6 - 10 June 2011 The EMBO 2011 Practical Course on Exploiting Anomalous Scattering in Macromolecular Structure Determination will be hosted by the ESRF in Grenoble, France from 6 to 10, June 2011. The course aims to impart the theoretical and practical basis for the 3-dimensional structure determination of bio-macromolecules using Anomalous Dispersion techniques (SAD MAD) to young scientists who intend to apply these methods in macromolecular crystallography. Through a series of lectures, software demonstrations, practicals on the ESRF beamlines and tutorials, participants will get insights into all aspects of the structure determination process including beamline instrumentation, data collection and processing, heavy atom substructure determination, phasing and model building. There will also be sessions focusing on automated structure solution procedures and newer methods. Additional sessions will include presentations of distinguished structures solved with these techniques. The number of participants is limited to 20 and the deadline for application is February 28th, 2011. Additional information, course programme and instructions to apply to the course can be found in the course webpages: http://cwp.embo.org/pc11-03/ -- ἀρετή --- Daniele de Sanctis, PhD Structural Biology Group ESRF, Grenoble, France Tel 33 (0)4 76 88 2869
Re: [ccp4bb] Processing Laue data
Dear Hao Quan, Yes I forgot about CHESS; apologies. Charles Ballard helped with the recent installation at the Diamond Test Beamline. The modifications to read the Photonic Science detector Laue images on the Diamond Test Beamline have been made by Karen Ashton and Steve Kinder based at the Daresbury Science and Innovation Centre. Greetings, John On Fri, Jan 28, 2011 at 9:46 AM, HAO, Quan q...@hku.hk wrote: Dear Tim, John, The source code can be downloaded from the CCP4: http://www.ccp4.ac.uk/ccp4bin/viewcvs/laue/ Charles Ballard was my contact at the CCP4. Two years ago, Marian Szebenyi (MacCHESS) successfully modified the code to read an ADSC detector image format. Best, Quan On 1/28/2011 5:33 PM, John R Helliwell wrote: Dear Tim, What problems have you had during installation? Prof Hao Quan can advise on technical and computer platform details. I copy him in. Greetings John cc Rex On Fri, Jan 28, 2011 at 9:02 AM, Tim Gruenet...@shelx.uni-ac.gwdg.de wrote: Dear Rex, the most popular one seems the Daresbury Laboratory Laue Software Suite, available via http://www.srs.ac.uk/px/jwc_laue/laue_top.html I have not, though, managed to compile the whole suite. There is also a commercial product from renzresearch.com, as M. Blakeley has pointed out to me. I would be curious to learn about more options or how to properly install the Daresbury Suite. Tim On Fri, Jan 28, 2011 at 08:43:44AM +, REX PALMER wrote: What programs are available for processing Laue data to produce an intensity data set? Are explanatory notes or publications available? Rex Palmer Birkbeck College -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.9 (GNU/Linux) iD8DBQFNQoY5UxlJ7aRr7hoRAlttAJ9NxTPa72enH0T0gYT7mi/q52H0cgCgxHcc kMjcm0bHy3kVYqVbT1wqE10= =ojc4 -END PGP SIGNATURE- -- Professor John R Helliwell DSc
[ccp4bb] Merging data to increase multiplicity
Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
Jose - you're missing the fact that the same dataset processed in different ways are not statistically independent datasets! Increasing the multiplicity for independent data reduces the uncertainty because the calculation of the SU assumes statistical independence. Cheers -- Ian On Fri, Jan 28, 2011 at 11:46 AM, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
Ah, yes, I was missing that. The statistics will be wrong. But in principle I will get an mtz with better data, because I am integrating more observations which would have been rejected by being missed at low resolution if the mosaicity was set too low or being rejected by overlaps at high resolution if the mosaicity is increased. So the question is - can I use this data for refinement? Or should I stick with the best of the datasets (the one with the highest completeness and multiplicity)? Thanks! Jose On Jan 28, 2011, at 28/1/11 - 11:59, Ian Tickle wrote: Jose - you're missing the fact that the same dataset processed in different ways are not statistically independent datasets! Increasing the multiplicity for independent data reduces the uncertainty because the calculation of the SU assumes statistical independence. Cheers -- Ian On Fri, Jan 28, 2011 at 11:46 AM, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Merging data to increase multiplicity
I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.ptmailto:trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
Re: [ccp4bb] Merging data to increase multiplicity
I have heard this before. I'm wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
Yes. But we have not published this. Mark Robien and I did a systematic study on about 30 data sets while we were at SGPP. The easy cases can be processed with anything the difficult cases worked only with XDS. This was mostly SeMet data or HA data, so de novo phasing no MR stuff. If you compare the signal of the SeMet peaks between the different processing options you got the strongest peaks via XDS. Success was assessed using ShelxD for finding HA sites. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/http://web.me.com/bosch_lab/ On Jan 28, 2011, at 8:37 AM, Van Den Berg, Bert wrote: I have heard this before. I’m wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edux-msg://257/jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.ptx-msg://257/trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
Hi Bert, here is one anecdotal evidence: a couple of years ago, I had one real in-house 3 A data set from a crystal after a quick iodide soak and processed the images with denzo/scalepack, mosflm/scala and xds/xscale. I got lower Rsym, higher I/sig(I) and better anomalous signal with xds. More importantly, I could solve the iodide substructure easily with SHELXC/D at different high resolution limits up to 3.5 A with the xds data set. For the other data sets, I had to cut the higher resolution limit down to 4-5 A, and there were fewer solutions for the substructure. Best regards, Dirk. Am 28.01.11 14:37, schrieb Van Den Berg, Bert: I have heard this before. I'm wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] Merging data to increase multiplicity
Interesting! But when will it be published? :-) On 1/28/11 8:45 AM, Bosch, Juergen jubo...@jhsph.edu wrote: Yes. But we have not published this. Mark Robien and I did a systematic study on about 30 data sets while we were at SGPP. The easy cases can be processed with anything the difficult cases worked only with XDS. This was mostly SeMet data or HA data, so de novo phasing no MR stuff. If you compare the signal of the SeMet peaks between the different processing options you got the strongest peaks via XDS. Success was assessed using ShelxD for finding HA sites. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 8:37 AM, Van Den Berg, Bert wrote: I have heard this before. I'm wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu x-msg://257/jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt x-msg://257/trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
... but, back to the main point, my advice would be to only limit the mosaicity, to get better completeness by avoiding overlaps. Its not ideal, in the sense that you would be over-estimating the partial fraction of most partial reflections, and thus systematically underestimating intensities. (I hope I got my overs and unders right here ...) But these errors would not matter much for refinement purposes, where you would rather have a slightly systematically wrong estimate for all data, rather than not have the 15% of the data at all. Or at least thats what I thought back in '99 refining MutS ... where I did refine a lot with both datasets and liked the 'fixed mosaicity' one better. A. On Jan 28, 2011, at 13:26, José Trincão wrote: Ah, yes, I was missing that. The statistics will be wrong. But in principle I will get an mtz with better data, because I am integrating more observations which would have been rejected by being missed at low resolution if the mosaicity was set too low or being rejected by overlaps at high resolution if the mosaicity is increased. So the question is - can I use this data for refinement? Or should I stick with the best of the datasets (the one with the highest completeness and multiplicity)? Thanks! Jose On Jan 28, 2011, at 28/1/11 - 11:59, Ian Tickle wrote: Jose - you're missing the fact that the same dataset processed in different ways are not statistically independent datasets! Increasing the multiplicity for independent data reduces the uncertainty because the calculation of the SU assumes statistical independence. Cheers -- Ian On Fri, Jan 28, 2011 at 11:46 AM, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
[ccp4bb] AW: [ccp4bb] Merging data to increase multiplicity
James Swindell, UGeorgia had a presentation Evaluating the role of data reduction approach on the success rate on Sulfur-SAS phasing for a moderately diffracting crystal at PittCon, Oct 2009, comparing HKL, d*TREK, XDS, Proteum2 Mosflm MfG, rgds, Eric Phone: +49 (721) 50997-5311 / +31 (15) 2152-501 Mobile: +49 (173) 7000-615 Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Van Den Berg, Bert Gesendet: Freitag, 28. Januar 2011 14:38 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Merging data to increase multiplicity I have heard this before. I'm wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr Bruker AXS GmbH, Karlsruhe HRB 107524 Amtsgericht Mannheim, Umsatzsteuer-Ident.Nr. DE812037551, Geschäftsführer - Dr. Frank Burgäzy, Bernard Kolodziej, Stephan Franz Westermann Der Inhalt dieser E-Mail ist vertraulich und ausschliesslich fuer den bezeichneten Adressaten bestimmt. Wenn Sie nicht der vorgesehene Adressat dieser E-Mail oder dessen Vertreter sein sollten, so beachten Sie bitte, dass jede Form der Kenntnisnahme, Veroeffentlichung, Vervielfaeltigung oder Weitergabe des Inhalts dieser E-Mail unzulaessig ist. Wir bitten Sie, sich in diesem Fall mit dem Absender der E-Mail in Verbindung zu setzen. The information contained in this email is confidential. It is intended solely for the addressee. Access to this email by anyone else is unauthorized. If you are not the intended recipient, any form of disclosure, reproduction, distribution or any action taken or refrained from in reliance on it, is prohibited and may be unlawful. Please notify the sender immediately.
Re: [ccp4bb] Merging data to increase multiplicity
On Jan 28, 2011, at 8:45, Bosch, Juergen jubo...@jhsph.edu wrote: Mark Robien and I did a systematic study on about 30 data sets while we were at SGPP. can you name the detector(s)? -Bryan
Re: [ccp4bb] Merging data to increase multiplicity
My experience (unpublished) is that XDS works very well for high-mosaicity crystals due to the 3-dimensional profile fitting. For low mosaicity crystals, I did not notice much of a difference between different programs. However, since bad crystals tend to have a high to very high mosaicity, I fully agree with Jürgens statement. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Van Den Berg, Bert Sent: Friday, January 28, 2011 2:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity I have heard this before. I'm wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
[ccp4bb] research software developer vacancy at EMBL Hamburg
Dear all, There is a staff member vacancy for a Research Software Developer at the EMBL Unit in Hamburg, Germany. The post holder will have a leading role in technical implementation and scientific development of the ARP/wARP software for crystallographic structure determination and the building of macromolecular models in 3D electron density maps generated from X-ray diffraction and, potentially, from electron microscopy and free-electron laser based data. Application deadline is 13th March, 2011. For details see: http://ig14.i-grasp.com//fe/tpl_embl01.asp?newms=jjid=41327aid=15470 With best regards, Victor
Re: [ccp4bb] Merging data to increase multiplicity
Can people say how high mosaicity is defined. High relative to what? Is it high relative to the rotation range for each image, high relative to the incident beam divergence, high relative to the (angular) detector resolution or something else? Regards Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: 28 January 2011 14:36 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity My experience (unpublished) is that XDS works very well for high-mosaicity crystals due to the 3-dimensional profile fitting. For low mosaicity crystals, I did not notice much of a difference between different programs. However, since bad crystals tend to have a high to very high mosaicity, I fully agree with Jürgens statement. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Van Den Berg, Bert Sent: Friday, January 28, 2011 2:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity I have heard this before. I'm wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhDCQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
For me, it means a reflecting-range (as defined by XDS) of 5-10 or more degrees and spots being visible on at least 5 or more frames (when using 1° frames). Good crystals (in our hands) have reflection-ranges in the order of 0.5-1.0°. Of course we trust that the synchrotron where we measure (ESRF, SLS) has a well-colimated beam with low beam divergence etc. So I guess, my definition would be high relative to the rotation range. I hope this answers your question, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Colin Nave Sent: Friday, January 28, 2011 3:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity Can people say how high mosaicity is defined. High relative to what? Is it high relative to the rotation range for each image, high relative to the incident beam divergence, high relative to the (angular) detector resolution or something else? Regards Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: 28 January 2011 14:36 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity My experience (unpublished) is that XDS works very well for high-mosaicity crystals due to the 3-dimensional profile fitting. For low mosaicity crystals, I did not notice much of a difference between different programs. However, since bad crystals tend to have a high to very high mosaicity, I fully agree with Jürgens statement. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Van Den Berg, Bert Sent: Friday, January 28, 2011 2:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity I have heard this before. I'm wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the
Re: [ccp4bb] Processing Laue data
A version of the Daresbury Laue programs that can process ADSC CCD data (Q-210, Q-315, etc.), and also has some improvements to the indexing routine for the case of sparse diffraction patterns, is available from CHESS, ftp://waterline.chess.cornell.edu/pub Some documentation (as text files) and sample data files are included. Marian Szebenyi MacCHESS REX PALMER wrote: What programs are available for processing Laue data to produce an intensity data set? Are explanatory notes or publications available? Rex Palmer Birkbeck College
Re: [ccp4bb] Merging data to increase multiplicity
On Fri, Jan 28, 2011 at 9:35 AM, herman.schreu...@sanofi-aventis.com wrote: [...] due to the 3-dimensional profile fitting. what is the specific difference between 3-d profile fitting and using a sliding window of more than one image? -Bryan
Re: [ccp4bb] Merging data to increase multiplicity
You should know that your crystal mosaicity is a physical property of your crystals and the diffraction experiment. Generally, it is anisotropic though most programs output a single value. How can that single value describe what is really happening in your experimental data? You can do anything you want to with the processing programs. You can fix mosaicity to any value you want. You can restrict it to a small value to lie to the program that your spots are not overlapped. This should help completeness and redundancy while perhaps degrading accuracy. Will that help you solve the structure? Will that help to find the anomalous substructure? WIll that help to get an initial map for chain tracing? Will you get a better Rfree if you use data that is merged from several crystals? Will you get a better Rfree if you mix and match different mosaicities when processing the diffraction images from different crystals? These are all hypotheses that you can test. I am not sure how to test these hypotheses by querying the internet. _ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis Perrakis Sent: Friday, January 28, 2011 8:11 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity ... but, back to the main point, my advice would be to only limit the mosaicity, to get better completeness by avoiding overlaps. Its not ideal, in the sense that you would be over-estimating the partial fraction of most partial reflections, and thus systematically underestimating intensities. (I hope I got my overs and unders right here ...) But these errors would not matter much for refinement purposes, where you would rather have a slightly systematically wrong estimate for all data, rather than not have the 15% of the data at all. Or at least thats what I thought back in '99 refining MutS ... where I did refine a lot with both datasets and liked the 'fixed mosaicity' one better. A. On Jan 28, 2011, at 13:26, José Trincão wrote: Ah, yes, I was missing that. The statistics will be wrong. But in principle I will get an mtz with better data, because I am integrating more observations which would have been rejected by being missed at low resolution if the mosaicity was set too low or being rejected by overlaps at high resolution if the mosaicity is increased. So the question is - can I use this data for refinement? Or should I stick with the best of the datasets (the one with the highest completeness and multiplicity)? Thanks! Jose On Jan 28, 2011, at 28/1/11 - 11:59, Ian Tickle wrote: Jose - you're missing the fact that the same dataset processed in different ways are not statistically independent datasets! Increasing the multiplicity for independent data reduces the uncertainty because the calculation of the SU assumes statistical independence. Cheers -- Ian On Fri, Jan 28, 2011 at 11:46 AM, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
Re: [ccp4bb] Merging data to increase multiplicity
Jim's comment brings us back to the Dauter in us, or better the Dauter which should be in us. Data Data Data ! Before shooting take a moment and think about the experiment. How do you collect the best possible data from a bad crystal. Investing time at the beginning always pays off (unless the beam dumps before you completed your data set). Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/http://web.me.com/bosch_lab/ On Jan 28, 2011, at 11:05 AM, Jim Pflugrath wrote: You should know that your crystal mosaicity is a physical property of your crystals and the diffraction experiment. Generally, it is anisotropic though most programs output a single value. How can that single value describe what is really happening in your experimental data? You can do anything you want to with the processing programs. You can fix mosaicity to any value you want. You can restrict it to a small value to lie to the program that your spots are not overlapped. This should help completeness and redundancy while perhaps degrading accuracy. Will that help you solve the structure? Will that help to find the anomalous substructure? WIll that help to get an initial map for chain tracing? Will you get a better Rfree if you use data that is merged from several crystals? Will you get a better Rfree if you mix and match different mosaicities when processing the diffraction images from different crystals? These are all hypotheses that you can test. I am not sure how to test these hypotheses by querying the internet. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis Perrakis Sent: Friday, January 28, 2011 8:11 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity ... but, back to the main point, my advice would be to only limit the mosaicity, to get better completeness by avoiding overlaps. Its not ideal, in the sense that you would be over-estimating the partial fraction of most partial reflections, and thus systematically underestimating intensities. (I hope I got my overs and unders right here ...) But these errors would not matter much for refinement purposes, where you would rather have a slightly systematically wrong estimate for all data, rather than not have the 15% of the data at all. Or at least thats what I thought back in '99 refining MutS ... where I did refine a lot with both datasets and liked the 'fixed mosaicity' one better. A. On Jan 28, 2011, at 13:26, José Trincão wrote: Ah, yes, I was missing that. The statistics will be wrong. But in principle I will get an mtz with better data, because I am integrating more observations which would have been rejected by being missed at low resolution if the mosaicity was set too low or being rejected by overlaps at high resolution if the mosaicity is increased. So the question is - can I use this data for refinement? Or should I stick with the best of the datasets (the one with the highest completeness and multiplicity)? Thanks! Jose On Jan 28, 2011, at 28/1/11 - 11:59, Ian Tickle wrote: Jose - you're missing the fact that the same dataset processed in different ways are not statistically independent datasets! Increasing the multiplicity for independent data reduces the uncertainty because the calculation of the SU assumes statistical independence. Cheers -- Ian On Fri, Jan 28, 2011 at 11:46 AM, José Trincão trin...@dq.fct.unl.ptmailto:trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr P please don't
Re: [ccp4bb] Merging data to increase multiplicity
A few things that might be worth looking at: 1. How is your beam divergence varying as you fix mosaicity at different levels? Does it look relatively stable at a realistic value for the beamline? If I'm remembering correctly, mosaicity and beam divergence are highly correlated within mosflm. 2. Is the mosaicity stable when not fixed? If not, this may be a sign that you need to reduce the resolution limit for refinement (within integration) - you didn't specify if your different resolution is from a cutoff applied within mosflm or judging by statistics at a later step. 3. Regarding what Ian mentioned, merging the same observations twice will cause issues with your statistics. It might be preferable to combine the results after merging if your concerned about keeping as many reflections as possible (the statistics from merging will still be accurate for the respective processing run, but not reflective of the combined amplitude/intensity set. However, the sigF/sigI values for each reflection would be less skewed). Pete José Trincão wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] Merging data to increase multiplicity
I see two questions here: -Can we assume an unrealistically low mosaicity in order to reduce overlaps. -Is there any benefit in merging data from the same frames integrated with different strategy? As for cheating on the mosaicity, which I euphemistically call peak sampling, I think it can give usable data, at least for refinement if not for heavy atom phasing, from data which is otherwise unusable. Think of the analogy of an HPLC elution profile, where each compund coming through makes a peak on the strip-chart recorder at a particular position. The peaks may or may not be resolved with baseline separation. Ideally, the amount of each compound is determined from the integrated area under the peak, or some approximation like peak height times width at half height. But if the peak has the same shape every time, it could be quantitated by peak height alone, calibrating with a known standard. Since the peak is the point where the signal from the desired compound is greatest relative to that from the overlapping compounds, this may be the best way (short of curve-fitting all the overlapping peaks) to quantitate when peaks are badly overlapped. Now to make it more like diffraction data collection, suppose you don't have a continuous readout but a fraction collector collecting fractions of equal volume, and you read absorbance of each fraction after mixing to get the average absorbance during that volume. If you have baseline separation and the peak comes out in a single fraction (think fully recorded) then absorbance of that fraction gives the answer. If baseline separation but the peak is spread across multiple fractions, you have to add up all these partials to get the answer. If the peaks are badly overlapped, you collect small fractions (fine slicing) so that one near the peak will approximate concentration at the peak, and use that peak absorbance. One problem, since the integrating program doesn't understand that you want to sample the peaks, it will add partials when the peak comes at the border between two frames. Say you have 2* mosaicity, collecting 0.5 degree frames, and telling the program the mosaicity is 0.5 degrees. For most observations two partials will be added, but occasionally the spot will be centered in one frame which will be taken as full. This can lead to up to 2x disagreement between different observations of the same reflection, but after averaging several observations it won't be that bad. Better to collect .25* frames, then either 2 or 3 frames will be averaged for each measurement. Expect Rsym on the order of 12-15% instead of 4-5%, but after refinement Rfree not much higher than 0.1 x resolution; and decent maps. Not good for sulfur SAD phasing, though. Michael Rossmann described some program for resolving overlapped spots (in each single frame?) by 2-D curve fitting. It doesn't seem to have been adopted, maybe wasn't that successful. I hope 3D profile fitting is something like this with the third dimension (frame to frame) included. My fraction collector analogy refers to the third dimension, but most of the spots that are overlapping in 2D (on a single frame) will also be somewhat separated in the third dimension (otherwise cheating on mosaicity wouldn't help) so their effect can be minimized by peak sampling. As for merging multiple strategies, probably won't help completeness, because other datasets would be subsets of the one assuming lowest mosaicity, but may help accuracy for those whose spots could be integrated without resorting to cheating on the mosaicity Ed José Trincão wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr
Re: [ccp4bb] How to align electron density maps?
Dear All, I have the problem as follows, but I can not find the corresponding solution. Can somebody give me some suggestions? Many Thanks, Hui Rong * [PyMOL] aligning electron density maps Eksterowicz, John Wed, 03 Mar 2004 15:03:43 -0800 I have a pdb and xplor file which contain two units of the same protein/inhibitor complex. What I am trying to do is align the two proteins to each other so I can compare the subunits. I can accomplish this by reading in the pdb file twice and aligning the b unit from the second pdb file to the a unit of the first pdb file. I then split everything into objects then delete what i don't want. In the end I have subunits a and b aligned to each other. I would like to be able to do the same thing for the electron denisty map. Is there a way to align maps? If I read in a protein and a map, then align the protein to another protein, the protein moves, but the map remains in it's original location. Can the map coordinates be transformed somehow? Is it possible to create some association with the protein and the map such that when the protein is aligned, the map will follow? Thanks, John * On 1/28/11, RONG hui Rong daiwangs...@gmail.com wrote:
Re: [ccp4bb] How to align electron density maps?
Hi Hui, (sorry for non-ccp4 recipe) IF I understand correctly what you want to do, then I guess you can achieve this using Map Superposition: http://www.phenix-online.org/documentation/superpose_maps.htm and/or Structure Comparison tools in PHENIX: http://www.phenix-online.org/documentation/structure_comparison.htm where you can superpose many models and corresponding maps, and see all them in one display, along with highlighted differences. Nat Echols, the author of both tools, is someone to ask should you have any questions. Pavel. On Fri, Jan 28, 2011 at 10:15 AM, RONG hui Rong daiwangs...@gmail.comwrote: Dear All, I have the problem as follows, but I can not find the corresponding solution. Can somebody give me some suggestions? Many Thanks, Hui Rong * [PyMOL] aligning electron density maps Eksterowicz, John Wed, 03 Mar 2004 15:03:43 -0800 I have a pdb and xplor file which contain two units of the same protein/inhibitor complex. What I am trying to do is align the two proteins to each other so I can compare the subunits. I can accomplish this by reading in the pdb file twice and aligning the b unit from the second pdb file to the a unit of the first pdb file. I then split everything into objects then delete what i don't want. In the end I have subunits a and b aligned to each other. I would like to be able to do the same thing for the electron denisty map. Is there a way to align maps? If I read in a protein and a map, then align the protein to another protein, the protein moves, but the map remains in it's original location. Can the map coordinates be transformed somehow? Is it possible to create some association with the protein and the map such that when the protein is aligned, the map will follow? Thanks, John * On 1/28/11, RONG hui Rong daiwangs...@gmail.com wrote:
[ccp4bb] iMosflm execution error
To the CCP4bb, I am working on installing the latest version of iMosflm (version 1.0.5) on my computer (running Ubuntu 10.10 (Meerkat). I've followed the installation instructions at the following link. I also have CCP4 ver. 6.1 and ActiveTcl ver. 8.4.19.3 installed and working (as far as I know) as per the instructions: http://www.mrc-lmb.cam.ac.uk/harry/imosflm/ver105/installation.html The only problem I encountered is that I had to correct pointers set up by my CCP4 installation to the CCP4-packaged Tcl/Tk and iMosflm components. I believe I have corrected those problems and I am able to get iMosflm loaded with no errors *as reported by the program*. However, after running the iMosflm executable, the following text is produced before the iMosflm GUI is loaded: snip VM% /usr/local/imosflm1.0.5/src/imosflm MOSFLM_EXEC set to /usr/local/imosflm1.0.5/bin/mosflm [: 180: =: unexpected operator testing MOSFLM_WISH (/usr/local/ActiveTcl8.4.19.3/bin/wish8.4) /snip I can't figure out what is generating this 'unexpected operator' error or how to correct it. I haven't recognized anything on the internet or in the documentation to help me identify the source of the problem. Can anyone provide insight on how to trace its origin or what it means? I will also test/confirm the ability to actually process data with the program later tonight, but I'm just puzzled by this error. Thanks for your help and insight, -Andy = Andrew T. Torelli, Ph.D. Department of Chemistry Chemical Biology Baker Laboratory, Cornell University Ithaca, NY 14853-1301 =
Re: [ccp4bb] iMosflm execution error
Hi Andy I haven't seen that one before. I have a Maverick Meerkat at work, so I can check on Monday to see if it's something that is peculiar to that. On my old Jaunty Jackalope at home, I don't see the error when I install the 32-bit Linux version. Can you let me know if iMosflm works in spite of that message? On 28 Jan 2011, at 19:44, Andrew T. Torelli wrote: To the CCP4bb, I am working on installing the latest version of iMosflm (version 1.0.5) on my computer (running Ubuntu 10.10 (Meerkat). I've followed the installation instructions at the following link. I also have CCP4 ver. 6.1 and ActiveTcl ver. 8.4.19.3 installed and working (as far as I know) as per the instructions: http://www.mrc-lmb.cam.ac.uk/harry/imosflm/ver105/installation.html The only problem I encountered is that I had to correct pointers set up by my CCP4 installation to the CCP4-packaged Tcl/Tk and iMosflm components. I believe I have corrected those problems and I am able to get iMosflm loaded with no errors *as reported by the program*. However, after running the iMosflm executable, the following text is produced before the iMosflm GUI is loaded: snip VM% /usr/local/imosflm1.0.5/src/imosflm MOSFLM_EXEC set to /usr/local/imosflm1.0.5/bin/mosflm [: 180: =: unexpected operator testing MOSFLM_WISH (/usr/local/ActiveTcl8.4.19.3/bin/wish8.4) /snip I can't figure out what is generating this 'unexpected operator' error or how to correct it. I haven't recognized anything on the internet or in the documentation to help me identify the source of the problem. Can anyone provide insight on how to trace its origin or what it means? I will also test/confirm the ability to actually process data with the program later tonight, but I'm just puzzled by this error. Thanks for your help and insight, -Andy = Andrew T. Torelli, Ph.D. Department of Chemistry Chemical Biology Baker Laboratory, Cornell University Ithaca, NY 14853-1301 = Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] How to align electron density maps?
It does work well. That is great! Many Thanks! On 1/28/11, Pavel Afonine pafon...@gmail.com wrote: Hi Hui, (sorry for non-ccp4 recipe) IF I understand correctly what you want to do, then I guess you can achieve this using Map Superposition: http://www.phenix-online.org/documentation/superpose_maps.htm and/or Structure Comparison tools in PHENIX: http://www.phenix-online.org/documentation/structure_comparison.htm where you can superpose many models and corresponding maps, and see all them in one display, along with highlighted differences. Nat Echols, the author of both tools, is someone to ask should you have any questions. Pavel. On Fri, Jan 28, 2011 at 10:15 AM, RONG hui Rong daiwangs...@gmail.comwrote: Dear All, I have the problem as follows, but I can not find the corresponding solution. Can somebody give me some suggestions? Many Thanks, Hui Rong * [PyMOL] aligning electron density maps Eksterowicz, John Wed, 03 Mar 2004 15:03:43 -0800 I have a pdb and xplor file which contain two units of the same protein/inhibitor complex. What I am trying to do is align the two proteins to each other so I can compare the subunits. I can accomplish this by reading in the pdb file twice and aligning the b unit from the second pdb file to the a unit of the first pdb file. I then split everything into objects then delete what i don't want. In the end I have subunits a and b aligned to each other. I would like to be able to do the same thing for the electron denisty map. Is there a way to align maps? If I read in a protein and a map, then align the protein to another protein, the protein moves, but the map remains in it's original location. Can the map coordinates be transformed somehow? Is it possible to create some association with the protein and the map such that when the protein is aligned, the map will follow? Thanks, John * On 1/28/11, RONG hui Rong daiwangs...@gmail.com wrote:
[ccp4bb] FW: [ccp4bb] Merging data to increase multiplicity
Herman Thanks. 5-10 degrees is certainly high whatever definition one uses. In the list I gave, I didn't include relative to the angular separation of the diffraction spots in reciprocal space. I would like to say that I didn't include it to see if anyone else came up with this one. In fact, I just forgot it. This definition is (I think) independent of the instrument and depends on things like the resolution of the data and the unit cell size. I guess I am coming to this from the point of view of separating out the intrinsic and instrument dependent features in order to identify the limitations of data collection procedures. Of course, this is no use at all to José who raised the original question so apologies to him and others who have addressed his question more directly. Regards Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: 28 January 2011 15:05 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity For me, it means a reflecting-range (as defined by XDS) of 5-10 or more degrees and spots being visible on at least 5 or more frames (when using 1° frames). Good crystals (in our hands) have reflection-ranges in the order of 0.5-1.0°. Of course we trust that the synchrotron where we measure (ESRF, SLS) has a well-colimated beam with low beam divergence etc. So I guess, my definition would be high relative to the rotation range. I hope this answers your question, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Colin Nave Sent: Friday, January 28, 2011 3:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity Can people say how high mosaicity is defined. High relative to what? Is it high relative to the rotation range for each image, high relative to the incident beam divergence, high relative to the (angular) detector resolution or something else? Regards Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: 28 January 2011 14:36 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity My experience (unpublished) is that XDS works very well for high-mosaicity crystals due to the 3-dimensional profile fitting. For low mosaicity crystals, I did not notice much of a difference between different programs. However, since bad crystals tend to have a high to very high mosaicity, I fully agree with Jürgens statement. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Van Den Berg, Bert Sent: Friday, January 28, 2011 2:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data to increase multiplicity I have heard this before. I'm wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen
Re: [ccp4bb] Noisy difference maps with high solvent content?
Hi, This sort of problem can occur if you are missing your lowest resolution data and/or your model for the bulk solvent is inappropriate. You might want to double check these issues. With 80% solvent you have to be careful when choosing your contour level. If you are a fan of normalized maps and contouring based on sigma (and it's not a sigma by the way) you should be aware that the normalization factor is calculated over the protein and all that empty space and will be smaller than one calculated for equivalent protein density in a low solvent crystal. Plus/minus 3 contours will be lower and the significance of features will be inflated. One way to calibrate a contour level would be to leave out a known good bit of model and calculate your difference map. Then select a contour level that shows the understood omission well. Other peaks that show up at that level are errors as significant as the one you created. Dale Tronrud On 01/28/11 12:29, Todd Geders wrote: Greetings CCP4bb, *Short version: Very noisy difference maps from a crystal with extremely high solvent content, seeking advice on how best to handle such high solvent content to eliminate noise in difference maps. * *http://strayxray.com/images/coot.jpg* Long version: I'm having trouble with a 3.0Å dataset from a crystal with 80% solvent content. The space group is P4132 and I'm quite confident the high solvent content is real (there is a species-specific set of helices extending into the solvent channels that appears to prevent tighter packing). I was able to get a MR solution using a structurally related enzyme, but the difference maps are terribly noisy (see link). There are lots of negative density in empty spaces between well-defined 2Fo-Fc electron density. http://strayxray.com/images/coot.jpg The 2Fo-Fc density actually looks fairly good. The initial MR maps had clear density correlating to the sequence differences between the MR model and the crystallized protein. After fixing the model as best I could, the refinement statistics are R/Rfree of 27.5/30.3 with a data/parameters ratio of 1.7. The mosaicity ranges from 0.15-0.3, data were collected with 0.5° oscillations and 180° of data were collected. http://strayxray.com/images/diffraction.jpg Since the crystals appeared to suffer from radiation decay (based on scaling statistics), I only use the first 40° of data (which still gives around 8-fold redundancy). Using more minimal wedges of data or more data does not noticeably make better or noisier maps. Any advice on improving the maps? Could the noisy maps be due to the extraordinarily high solvent content? I'd appreciate any advice or comments. ~Todd Geders
Re: [ccp4bb] Noisy difference maps with high solvent content?
Are you sure that the space group is right? P4132 is cubic, so the spots should have equal spacing on the detector, i.e., squares. Don't you have rectangles? Even the least distorted spots towards the beamstop (bottom right?) seem to be non-square rectangles. JPK On Fri, Jan 28, 2011 at 3:07 PM, Dale Tronrud det...@uoxray.uoregon.edu wrote: Hi, This sort of problem can occur if you are missing your lowest resolution data and/or your model for the bulk solvent is inappropriate. You might want to double check these issues. With 80% solvent you have to be careful when choosing your contour level. If you are a fan of normalized maps and contouring based on sigma (and it's not a sigma by the way) you should be aware that the normalization factor is calculated over the protein and all that empty space and will be smaller than one calculated for equivalent protein density in a low solvent crystal. Plus/minus 3 contours will be lower and the significance of features will be inflated. One way to calibrate a contour level would be to leave out a known good bit of model and calculate your difference map. Then select a contour level that shows the understood omission well. Other peaks that show up at that level are errors as significant as the one you created. Dale Tronrud On 01/28/11 12:29, Todd Geders wrote: Greetings CCP4bb, *Short version: Very noisy difference maps from a crystal with extremely high solvent content, seeking advice on how best to handle such high solvent content to eliminate noise in difference maps. * *http://strayxray.com/images/coot.jpg* Long version: I'm having trouble with a 3.0Å dataset from a crystal with 80% solvent content. The space group is P4132 and I'm quite confident the high solvent content is real (there is a species-specific set of helices extending into the solvent channels that appears to prevent tighter packing). I was able to get a MR solution using a structurally related enzyme, but the difference maps are terribly noisy (see link). There are lots of negative density in empty spaces between well-defined 2Fo-Fc electron density. http://strayxray.com/images/coot.jpg The 2Fo-Fc density actually looks fairly good. The initial MR maps had clear density correlating to the sequence differences between the MR model and the crystallized protein. After fixing the model as best I could, the refinement statistics are R/Rfree of 27.5/30.3 with a data/parameters ratio of 1.7. The mosaicity ranges from 0.15-0.3, data were collected with 0.5° oscillations and 180° of data were collected. http://strayxray.com/images/diffraction.jpg Since the crystals appeared to suffer from radiation decay (based on scaling statistics), I only use the first 40° of data (which still gives around 8-fold redundancy). Using more minimal wedges of data or more data does not noticeably make better or noisier maps. Any advice on improving the maps? Could the noisy maps be due to the extraordinarily high solvent content? I'd appreciate any advice or comments. ~Todd Geders -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Merging data to increase multiplicity
Hi Dirk, This example compares integration software in combination with the scaling program, which is what usually happens. Obviously, the scaling program does more than just scaling, it also handles rejections. It is possibly this procedure that makes most of the difference. For example, the default settings for rejections in scala are quite lenient (i.e. very few observations are rejected). We have some indication that changing these defaults makes some difference to the success or otherwise of phasing, but we have not looked at this in enough detail to say there is 'evidence'. Maybe one day... Cheers, Johan Dr. Johan P. Turkenburg X-ray facilities manager York Structural Biology Laboratory University of York Phone (+) 44 1904 328251 York YO10 5DD UK Fax (+) 44 1904 328266 On 28 January 2011 13:49, Dirk Kostrewa kostr...@genzentrum.lmu.de wrote: Hi Bert, here is one anecdotal evidence: a couple of years ago, I had one real in-house 3 A data set from a crystal after a quick iodide soak and processed the images with denzo/scalepack, mosflm/scala and xds/xscale. I got lower Rsym, higher I/sig(I) and better anomalous signal with xds. More importantly, I could solve the iodide substructure easily with SHELXC/D at different high resolution limits up to 3.5 A with the xds data set. For the other data sets, I had to cut the higher resolution limit down to 4-5 A, and there were fewer solutions for the substructure. Best regards, Dirk. Am 28.01.11 14:37, schrieb Van Den Berg, Bert: I have heard this before. I’m wondering though, does anybody know of a systematic study where different data processing programs are compared with real-life, non-lysozyme data? Bert On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote: I was a bit reductive with my statement (iPhone) The equation below is suppose to read: If you have bad data, then you need to process with XDS in order to get the maximum out of your data. Thanks Tim, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote: Dear Jürgen, is this an assignment operator or an equal sign? For if it's the latter it could read that the result of processing data with XDS are bad data, which is rather rude and probably not what you meant. Tim On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote: Bad data = processing with XDS Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote: Hello all, I have been trying to squeeze the most out of a bad data set (P1, anisotropic, crystals not reproducible). I had very incomplete data due to high mosaicity and lots of overlaps. The completeness was about 80% overall to ~3A. Yesterday I noticed that I could process the data much better fixing the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with a multiplicity of 1.7. I tried to integrate the same data fixing the mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in completeness, Rmerge and multiplicity. Now, is there any reason why I should not just merge all these together and feed them to scala in order to increase multiplicity? Am I missing something? Thanks for any comments! Jose José Trincão, PhD CQFB@FCT-UNL 2829-516 Caparica, Portugal It's very hard to make predictions... especially about the future - Niels Bohr -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone:+49-89-2180-76845 Fax: +49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW: www.genzentrum.lmu.de ***
[ccp4bb] Buffers for crystallization
Hi Chandan, The answer to your question about buffers is to use any buffer that does not bind to metal ions like zinc for example. I have used TRIS and MES myself successfully for crystallization of zinc finger proteins. What kind of zinc fingers do you have? If there is histidine then you cannot go below pH 5.0 or you will lose the zinc. High pH is not a problem up to pH 10.0 as long as you keep the reducing agent concentration low at say 1 mM. Obviously do not use EDTA or any other metal ion chelators in the prep, or in the crystallization mix. Have you checked the protein for activity or binding or proteolysis? Cheers. Ray Ray Brown PhD 3DBioScience LLC Url: www.3dbioscience.com
Re: [ccp4bb] Noisy difference maps with high solvent content?
Hi Todd, in addition to all other suggestions, here is some basic sanity check that you can do in no time: - how the R-factor vs Resolution plot looks like? - did the bulk-solvent correction was done right (what are k_sol and B_sol values)? - plot the scatterer graph Fobs vs Fmodel and see if there are severe outliers? Did removing them changes the map? - how the Data completeness vs Resolution plot looks like? - how POLYGON plot looks like? The answers will give some food for thought, and probably will invite some more more specific suggestions. All the best! Pavel. On Fri, Jan 28, 2011 at 12:29 PM, Todd Geders ged...@strayxray.com wrote: Greetings CCP4bb, *Short version: Very noisy difference maps from a crystal with extremely high solvent content, seeking advice on how best to handle such high solvent content to eliminate noise in difference maps. * *http://strayxray.com/images/coot.jpg* Long version: I'm having trouble with a 3.0Å dataset from a crystal with 80% solvent content. The space group is P4132 and I'm quite confident the high solvent content is real (there is a species-specific set of helices extending into the solvent channels that appears to prevent tighter packing). I was able to get a MR solution using a structurally related enzyme, but the difference maps are terribly noisy (see link). There are lots of negative density in empty spaces between well-defined 2Fo-Fc electron density. http://strayxray.com/images/coot.jpg The 2Fo-Fc density actually looks fairly good. The initial MR maps had clear density correlating to the sequence differences between the MR model and the crystallized protein. After fixing the model as best I could, the refinement statistics are R/Rfree of 27.5/30.3 with a data/parameters ratio of 1.7. The mosaicity ranges from 0.15-0.3, data were collected with 0.5° oscillations and 180° of data were collected. http://strayxray.com/images/diffraction.jpg Since the crystals appeared to suffer from radiation decay (based on scaling statistics), I only use the first 40° of data (which still gives around 8-fold redundancy). Using more minimal wedges of data or more data does not noticeably make better or noisier maps. Any advice on improving the maps? Could the noisy maps be due to the extraordinarily high solvent content? I'd appreciate any advice or comments. ~Todd Geders
Re: [ccp4bb] iMosflm execution error
Dear Andy, We have seen this error since forever on our Ubuntu boxes (I believe starting from ubuntu ver. 8.04). This is what the latest version of imosflm outputs on startup: lbbspc2 ~ imosflm MOSFLM_EXEC set to /usr/local/lbbs/ccp4/current/bin/ipmosflm [: 180: =: unexpected operator testing MOSFLM_WISH (/usr/local/lbbs/ccp4/tcltk++/bin/wish) but (!) this error never manifested itself in any program malfunction. imosflm seems to run just fine. Sincerely, Petr On Jan 28, 2011, at 8:44 PM, Andrew T. Torelli wrote: To the CCP4bb, I am working on installing the latest version of iMosflm (version 1.0.5) on my computer (running Ubuntu 10.10 (Meerkat). I've followed the installation instructions at the following link. I also have CCP4 ver. 6.1 and ActiveTcl ver. 8.4.19.3 installed and working (as far as I know) as per the instructions: http://www.mrc-lmb.cam.ac.uk/harry/imosflm/ver105/installation.html The only problem I encountered is that I had to correct pointers set up by my CCP4 installation to the CCP4-packaged Tcl/Tk and iMosflm components. I believe I have corrected those problems and I am able to get iMosflm loaded with no errors *as reported by the program*. However, after running the iMosflm executable, the following text is produced before the iMosflm GUI is loaded: snip VM% /usr/local/imosflm1.0.5/src/imosflm MOSFLM_EXEC set to /usr/local/imosflm1.0.5/bin/mosflm [: 180: =: unexpected operator testing MOSFLM_WISH (/usr/local/ActiveTcl8.4.19.3/bin/wish8.4) /snip I can't figure out what is generating this 'unexpected operator' error or how to correct it. I haven't recognized anything on the internet or in the documentation to help me identify the source of the problem. Can anyone provide insight on how to trace its origin or what it means? I will also test/confirm the ability to actually process data with the program later tonight, but I'm just puzzled by this error. Thanks for your help and insight, -Andy = Andrew T. Torelli, Ph.D. Department of Chemistry Chemical Biology Baker Laboratory, Cornell University Ithaca, NY 14853-1301 =
[ccp4bb] Singapore Research, facts and comments?
Dear All, sorry for this off-topic inquiry. I am interested in knowing about the current scientific research in Singapore but have had hard time finding the most up-to-date articles/comments on this. All I could find are the articles a few years back in Science and Nature. Would appreciate a pointer. Happy Chinese New Year! Thanks, Jacob Wong
