Re: [ccp4bb] Zinc or Iron binding protein, that is a question!
Hi Xuan, I guess your protein is not an E.coli protein. There are several examples that eukaryotic Zn-proteins expressed in E.coli contain Fe instead of Zn. I am sceptic whether IMAC with different metal ions will give the solution of the problem. If you really want to get information on the metal ion binding properties you will have to do some matallo biochemistry: preparing apo protein, reconstitution with metal ions, UV-Vis spectroscopy, EPR would be great, ... Dear Sir or Madam, The ICP-ES results indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 Academic email: ya...@moon.ibp.ac.cn mailto:ya...@moon.ibp.ac.cn We will either find a way or make one.
Re: [ccp4bb] refmac5 error message
Vesna Serrano wrote: Dear all, I am using Refmac_5.5.0088 in CCP4 6.1.0, linux version. The program runs well normally, but all of a sudden it failed with the following message: Refmac_5.5.0088: Open failed: File: /tmp/vesna/refmac5_temp1.06489.txt. Here is the log file of the run: #CCP4I VERSION CCP4Interface 2.0.4 #CCP4I SCRIPT LOG refmac5 #CCP4I DATE 05 Aug 2009 02:04:38 #CCP4I USER vesna #CCP4I PROJECT wtDHPAB #CCP4I JOB_ID 19 #CCP4I SCRATCH /tmp/vesna #CCP4I HOSTNAME localhost.localdomain #CCP4I PID 6486 html !-- CCP4 HTML LOGFILE -- hr pre ### ### ### ### CCP4 6.1: Refmac_5.5.0088 version 5.5.0088 : 08/12/08## ### User: unknown Run date: 5/ 8/2009 Run time: 02:04:38 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. $TEXT:Reference1: $$ comment $$ Refinement of Macromolecular Structures by the Maximum-Likelihood Method: G.N. Murshudov, A.A.Vagin and E.J.Dodson,(1997) Acta Crystallogr. D53, 240-255 EU Validation contract: BIO2CT-92-0524 $$ $SUMMARY :Reference1: $$ Refmac: $$ :TEXT:Reference1: $$ Open failed: Unit: 7, File: /tmp/vesna/refmac5_temp1.06489.txt (logical: /tmp/vesna/refmac5_temp1.06489.txt) Refmac_5.5.0088: Open failed: File: /tmp/vesna/refmac5_temp1.06489.txt Times: User: 0.0s System:0.0s Elapsed: 0:00 /pre /html *** * Information from CCP4Interface script *** The program run with command: /usr/local/xtal/ccp4-6.1.1/bin/refmac5 XYZIN /home/vesna/wtDHPAB/ABwt4ID50-adj1.pdb XYZOUT /home/vesna/wtDHPAB/wtDHPAB_19_2_pdb_1.tmp HKLIN /home/vesna/wtDHPAB/full_ABwtID4.mtz HKLOUT /home/vesna/wtDHPAB/wtDHPAB_19_3_mtz_1.tmp LIBOUT /home/vesna/wtDHPAB/wtDHPAB_19_lib.cif has failed with error message Last system error message: Permission denied Refmac_5.5.0088: Refmac_5.5.0088 *** #CCP4I TERMINATION STATUS 0 Last system error message: Permission denied Refmac_5.5.0088: Open failed: File: /tmp/vesna/refmac5_temp1.06489.txt #CCP4I TERMINATION TIME 05 Aug 2009 02:04:38 #CCP4I MESSAGE Task failed I would very much appreciate help in solving this problem. Regards, Vesna Serrano Dept. of Chemistry NC State Univ. Raleigh, NC 27695 Can you write to the directory /tmp/vesna? Does it exist? The last error is permission denied... This could happen if something wiped the files in /tmp - including the directory, or if someone else created the directory there and you cannot write to it I would check ls -ld /tmp/vesna to see that the directory exists and the permissions are correct. Another possibility is that someone changed the permissions on the directory /tmp - and made it not world writable... ls -ld /tmp would tell you if this was the problem. Good luck... Ezra
Re: [ccp4bb] Zinc or Iron binding protein, that is a question!
Since you sent your question to CCP4bb you presumably have crystals! In that case an excellent way to check which metal atoms you have in which sites is to collect datasets at a synchrotron at suitably chosen wavelengths and look at the anomalous maps. For an example see Acta Cryst. D62 (2006) 417-424. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Thu, 6 Aug 2009, Xuan Yang wrote: Dear Sir or Madam, The ICP-ES results indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 Academic email: ya...@moon.ibp.ac.cn We will either find a way or make one.
[ccp4bb] PHASER packing function with DNA
Dear CCP4BBer, I'm using Phaser 2.1.4 to solve a structure containing both protein and DNA. The packing function works find with the protein, but does not recognize the trace of the DNA. Does anyone know precisely what should be the pdb format ? Here is the format that I'am using : ATOM 1 PGd C 1 23.560 11.045 11.025 1.00 53.18 P ATOM 2 OP1 Gd C 1 24.884 10.499 11.417 1.00 54.98 O ATOM 3 OP2 Gd C 1 22.319 10.778 11.791 1.00 44.44 O ATOM 4 O5' Gd C 1 23.716 12.601 10.891 1.00 47.89 O ATOM 5 C5' Gd C 1 24.689 13.001 9.945 1.00 42.99 C ATOM 6 C4' Gd C 1 24.395 14.391 9.437 1.00 39.27 C ATOM 7 O4' Gd C 1 23.172 14.464 8.683 1.00 35.25 O ATOM 8 C3' Gd C 1 24.264 15.347 10.617 1.00 36.72 C ATOM 9 O3' Gd C 1 25.063 16.487 10.335 1.00 34.52 O ATOM 10 C2' Gd C 1 22.807 15.691 10.507 1.00 34.06 C ATOM 11 C1' Gd C 1 22.474 15.641 9.080 1.00 27.89 C ATOM 12 N9 Gd C 1 21.218 15.303 8.921 1.00 20.00 N ATOM 13 C8 Gd C 1 20.436 14.344 9.523 1.00 20.00 C ATOM 14 N7 Gd C 1 19.207 14.292 9.056 1.00 20.00 N ATOM 15 C5 Gd C 1 19.159 15.275 8.076 1.00 20.00 C ATOM 16 C4 Gd C 1 20.381 15.912 7.978 1.00 20.00 C ATOM 17 N1 Gd C 1 18.457 16.733 6.390 1.00 20.00 N ATOM 18 C2 Gd C 1 19.700 17.306 6.353 1.00 20.00 C ATOM 19 N3 Gd C 1 20.705 16.934 7.129 1.00 20.00 N ATOM 20 C6 Gd C 1 18.087 15.691 7.242 1.00 20.00 C ATOM 21 O6 Gd C 1 16.928 15.244 7.199 1.00 20.00 O ATOM 22 N2 Gd C 1 19.883 18.288 5.476 1.00 20.00 N Thanks, Marie-Ln Laboratoire de Biologie Structurale et Radiobiologie CEA/DSV/IBiTec-S/SB2SM bat144, pièce 25 91191 Gif-Sur-Yvette Tel : 33 (0)1 69 08 71 35 E-mail : marie-helene.l...@cea.fr --
Re: [ccp4bb] PHASER packing function with DNA
Hi, Thanks a lot for the replies. It seems that Randy¹s solution works fine. Marie-Ln Le 6/08/09 13:54, « Randy Read » rj...@cam.ac.uk a écrit : Hi, That's actually the last question answered on our FAQ page! http://www-structmed.cimr.cam.ac.uk/phaser/faq.html Basically, you'll want to change lines like this: ATOM 1 PGd C 1 23.560 11.045 11.025 1.00 53.18 P to lines like this: ATOM 1 P G C 1 23.560 11.045 11.025 1.00 53.18 P because Phaser 2.1.4 expects the residue name in one-letter code in column 20. We didn't notice, before releasing version 2.1.4, that one effect of the PDB remediation was to change the nomenclature for nucleic acid residues, so the packing check won't work using files recently obtained from the PDB (where deoxy-G is represented as DG in columns 19-20). This is fixed for version 2.2.x (available as part of Phenix nightly builds, and hopefully from an upcoming CCP4 release); in the meantime, you'll have to edit recent PDB files before running Phaser. Best wishes, Randy Read On 6 Aug 2009, at 11:25, LEDU Marie-Helene 16 wrote: Dear CCP4BBer, I'm using Phaser 2.1.4 to solve a structure containing both protein and DNA. The packing function works find with the protein, but does not recognize the trace of the DNA. Does anyone know precisely what should be the pdb format ? Here is the format that I'am using : ATOM 1 PGd C 1 23.560 11.045 11.025 1.00 53.18 P ATOM 2 OP1 Gd C 1 24.884 10.499 11.417 1.00 54.98 O ATOM 3 OP2 Gd C 1 22.319 10.778 11.791 1.00 44.44 O ATOM 4 O5' Gd C 1 23.716 12.601 10.891 1.00 47.89 O ATOM 5 C5' Gd C 1 24.689 13.001 9.945 1.00 42.99 C ATOM 6 C4' Gd C 1 24.395 14.391 9.437 1.00 39.27 C ATOM 7 O4' Gd C 1 23.172 14.464 8.683 1.00 35.25 O ATOM 8 C3' Gd C 1 24.264 15.347 10.617 1.00 36.72 C ATOM 9 O3' Gd C 1 25.063 16.487 10.335 1.00 34.52 O ATOM 10 C2' Gd C 1 22.807 15.691 10.507 1.00 34.06 C ATOM 11 C1' Gd C 1 22.474 15.641 9.080 1.00 27.89 C ATOM 12 N9 Gd C 1 21.218 15.303 8.921 1.00 20.00 N ATOM 13 C8 Gd C 1 20.436 14.344 9.523 1.00 20.00 C ATOM 14 N7 Gd C 1 19.207 14.292 9.056 1.00 20.00 N ATOM 15 C5 Gd C 1 19.159 15.275 8.076 1.00 20.00 C ATOM 16 C4 Gd C 1 20.381 15.912 7.978 1.00 20.00 C ATOM 17 N1 Gd C 1 18.457 16.733 6.390 1.00 20.00 N ATOM 18 C2 Gd C 1 19.700 17.306 6.353 1.00 20.00 C ATOM 19 N3 Gd C 1 20.705 16.934 7.129 1.00 20.00 N ATOM 20 C6 Gd C 1 18.087 15.691 7.242 1.00 20.00 C ATOM 21 O6 Gd C 1 16.928 15.244 7.199 1.00 20.00 O ATOM 22 N2 Gd C 1 19.883 18.288 5.476 1.00 20.00 N Thanks, Marie-Ln Laboratoire de Biologie Structurale et Radiobiologie CEA/DSV/IBiTec-S/SB2SM bat144, pièce 25 91191 Gif-Sur-Yvette Tel : 33 (0)1 69 08 71 35 E-mail : marie-helene.l...@cea.fr -- -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] Zinc or Iron binding protein, that is a question!
Dear Mr. Fritz, Yes, the protein is not an E.coli protein! Instead, it was cloned from a virus. And since it was a nonstructural viral protein, I thought it might be appropiate to treat it as eukaryotic proteins. E.coli system was quite different from eukaryotic ones, hence I was quite cautious about the ICP-ES result and trying to confirm it via alternative method. Thanks very much for mentioning the examples which suggested that Fe might be contaminants. Indeed, when I cut the protein in two parts (still with MBP) and test them via ICP-ES again, Fe became negligible in both and Zn stoichiometry increaed to 1:1 in the C-terminal part. The result lead me to focus on Zn instead of Fe. But I still want to confirm the idea. Matallo biochemistry was exactly what I dreamed to do. Sincerely, Xuan Yang 2007/8/6 Guenter Fritz guenter.fr...@uni-konstanz.de Hi Xuan, I guess your protein is not an E.coli protein. There are several examples that eukaryotic Zn-proteins expressed in E.coli contain Fe instead of Zn. I am sceptic whether IMAC with different metal ions will give the solution of the problem. If you really want to get information on the metal ion binding properties you will have to do some matallo biochemistry: preparing apo protein, reconstitution with metal ions, UV-Vis spectroscopy, EPR would be great, ... Dear Sir or Madam, The ICP-ES results indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 Academic email: ya...@moon.ibp.ac.cn mailto:ya...@moon.ibp.ac.cn We will either find a way or make one.
Re: [ccp4bb] Zinc or Iron binding protein, that is a question!
Long time ago I had a superoxide dismutase that was active with iron as well as manganese. No matter what functional metal (Fe or Mn) was bound a substantial fraction up to 1/3 of the molecules had the (non-functional) Zinc in their metal binding site (found by AAS and EXAFS). Zinc is everywhere, even in plastic bottles for your distilled water, it is extremely hard to get rid of. And it seems to fit to many iron binding sites. When I recall correctly a fairly stable source of Fe(II) is Mohr's salt. However, Fe binds also unspecifically to the protein, so it is very hard to quantify iron binding to their specific site because eg. EPR spectra change with each washing step. Best Marius Hi Xuan, I guess your protein is not an E.coli protein. There are several examples that eukaryotic Zn-proteins expressed in E.coli contain Fe instead of Zn. I am sceptic whether IMAC with different metal ions will give the solution of the problem. If you really want to get information on the metal ion binding properties you will have to do some matallo biochemistry: preparing apo protein, reconstitution with metal ions, UV-Vis spectroscopy, EPR would be great, ... Dear Sir or Madam, The ICP-ES results indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 Academic email: ya...@moon.ibp.ac.cn mailto:ya...@moon.ibp.ac.cn We will either find a way or make one. Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: m-schm...@uwm.edu http://users.physik.tu-muenchen.de/marius/
[ccp4bb] Protein Production Senior Scientist Vacancy
Protein Production Senior Scientist Job Description: Based at Abingdon, Oxfordshire, Evotec offer integrated services which cover the entire drug discovery and development process. Our skills and expertise make us the clear partner of choice for the world's major pharmaceutical and biotechnology companies. We generate value for our customers by accelerating the discovery and development process, managing risk and reducing the time and cost of bringing new drugs to the market. Position Details: A full time position is available for a senior scientist working within a multidisciplinary project team, your main task will be the expression and purification of proteins for structural studies. Experience Requirements: Ideally you will have a B.Sc. or PhD in a life science or related discipline, with proven relevant industrial experience being an advantage. You will have a strong background with hands-on lab experience in the following areas: * Molecular biology * Expression of proteins in E. coli * Purification of proteins by affinity, ion exchange and size exclusion chromatography * Use of AKTA purification system Expertise in high throughput expression and purification for structural studies would be an advantage. You will enjoy working as part of a team, be enthusiastic, hardworking and able to work on your own initiative. Computer literacy, analytical problem solving skills, and excellent verbal and written communication skills are required. Applications must be submitted via the careers page on the Evotec web-site (http://www.evotec.com/en/careers/united_kingdom.aspx) The Closing Date for applications is August 31st. Isabel Moraes, PhD, MRSC Senior Scientist X-Ray crystallography/Drug Discovery Evotec / 114 Milton Park Abingdon, Oxon OX14 4SA UK +44(0) 123583 8842 (Direct Line) Evotec (UK) Ltd is a limited company registered in England and Wales. Registration number:2674265. Registered office: 114 Milton Park, Abingdon, Oxfordshire, OX14 4SA, United Kingdom.
Re: [ccp4bb] Zinc or Iron binding protein, that is a question!
As others have implied, quantifying metals in metalloproteins is very challenging. In my experience, the principal problems are (1) adventitious metal contamination, (2) accurate measurement of protein concentration, and (3) weak metal binding. Zinc and iron are ubiquitous microcontaminants, and crop up in some pretty unusual places. Zinc will get into everything, and plastic bottles and microcentrifuge tubes seem to be a pretty rich source of iron contamination. In addition, we have found that some plasticware will also bind low concentrations of zinc and other metal ions from solution, making standardization of instruments very frustrating. Having said all this, ICP-OES or ICP-MS are absolutely the best methods of quantifying metals in protein solutions. ICP methods are linear over many orders of magnitude, and sample prep can be minimal. We simply dilute protein solution in high-quality deionized water to approximately 0.1-1.0 ppm in glass (acid washed and dried if possible), NOT plastic, and aspirate into an ICP-OES. The protein must be diluted enough to reduce viscosity issues in nebulization. Standards can be prepared in deionized water. We dilute our protein solutions with the same dI water we use to prepare the blank, and there is always some detectable Zn background. (Fe is usually very low in glass containers). The key to accurate measurements is Zn concentrations 0.1 ppm. This has worked very well for us for quantifying native and metal-substituted zinc-metalloproteins. If you are really up against contamination, you can extract solutions with Chelex resin, but it is possible that this treatment could also remove loosely bound metals from proteins. If your putative zinc protein has one or more sulfur ligands, this is less likely to be a problem. Measuring protein concentrations accurately is also challenging. Every protein quantification method is subject to idiosyncrasies. The least idiosyncratic chemical methods are the microbiuret method and a variety of UV methods (I like one based on A230 in 0.1% Tween-100 or another detergent.) Measuring protein concentration will be the major source of error in metalloprotein stoichiometry measurements. Protein has to be really homogeneous for any method to be accurate. Also keep in mind that His-tagged proteins (and some proteins with vicinial His residues or other ligands on the surface) may non-specifically bind metal ions, clouding metal stoichiometry. Finally, if your metal is not in a tight binding site, it will be difficult to prepare solutions with the saturated stoichiometry. In these cases, it is possible that microcalorimetry might be useful. Xuan Yang wrote: Dear Mr. Fritz, Yes, the protein is not an E.coli protein! Instead, it was cloned from a virus. And since it was a nonstructural viral protein, I thought it might be appropiate to treat it as eukaryotic proteins. E.coli system was quite different from eukaryotic ones, hence I was quite cautious about the ICP-ES result and trying to confirm it via alternative method. Thanks very much for mentioning the examples which suggested that Fe might be contaminants. Indeed, when I cut the protein in two parts (still with MBP) and test them via ICP-ES again, Fe became negligible in both and Zn stoichiometry increaed to 1:1 in the C-terminal part. The result lead me to focus on Zn instead of Fe. But I still want to confirm the idea. Matallo biochemistry was exactly what I dreamed to do. Sincerely, Xuan Yang 2007/8/6 Guenter Fritz guenter.fr...@uni-konstanz.de Hi Xuan, I guess your protein is not an E.coli protein. There are several examples that eukaryotic Zn-proteins expressed in E.coli contain Fe instead of Zn. I am sceptic whether IMAC with different metal ions will give the solution of the problem. If you really want to get information on the metal ion binding properties you will have to do some matallo biochemistry: preparing apo protein, reconstitution with metal ions, UV-Vis spectroscopy, EPR would be great, ... Dear Sir or Madam, The ICP-ES results indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329
Re: [ccp4bb] Mosaicity beam divergence
Sorry, I meant to say does divergence add to the reported mosaicity value. If so, do actual mosaicity and divergence add in quadrature to give the reported value? On Aug 6, 2009, at 6:14 PM, Richard Gillilan wrote: Does anyone know if beam divergence gets included in the mosaicity values reported by HKL2000? (i.e. does it add to the measured divergence (in quadrature)?) Richard Gillilan MacCHESS
Re: [ccp4bb] Mosaicity beam divergence
Richard Gillilan wrote: Sorry, I meant to say does divergence add to the reported mosaicity value. If so, do actual mosaicity and divergence add in quadrature to give the reported value? Not sure what denzo does, but in general if you convolute two things that have a Gaussian distribution, then the result is itself a Gaussian with width equal to the root-mean-square of the two starting widths (they add in quadrature). However, if you convolve two things that have a square or tophat distribution, then the result is a trapezoid with base width equal to the arithmetic sum of the two starting widths (they just add). On the other hand, the full-width at half-max (FWHM) of the trapezoid is the greater of the two starting widths (not really adding in any normal way). Nevertheless, when you are integrating spots you're not interested in the FWHM, but rather the full width at baseline (whatever that means). Gaussians are easy because the FWHM, the width at 1% max, or whatever width you like will always add in quadrature when you convolute them, but Gaussians are the only shape that does this. If I were to guess, I would say that denzo probably uses the Greenhough-Helliwell formula (international tables C 2.2.7.3 p. 40) for the rocking width (which assumes everything is a tophat function and that the beam divergence is an ellipse, not a rectangle) and then just refines the mosaic spread (greek letter eta) in that formula until the integrated intensity levels off based on some criterion. Again, just a guess. -James Holton MAD Scientist On Aug 6, 2009, at 6:14 PM, Richard Gillilan wrote: Does anyone know if beam divergence gets included in the mosaicity values reported by HKL2000? (i.e. does it add to the measured divergence (in quadrature)?) Richard Gillilan MacCHESS
Re: [ccp4bb] Mosaicity beam divergence
Richard Gillilan wrote: Sorry, I meant to say does divergence add to the reported mosaicity value. If so, do actual mosaicity and divergence add in quadrature to give the reported value? Yes, they do add in quadrature, the total is reported by scalepack and HKL2000 (if postrefinement is used), denzo overestimates it somewhat. -- Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu
