Re: [ccp4bb] Heavy atom sites
Yes - mlphare did, but so does Phaser_ep E On 16 December 2013 18:46, Bosch, Juergen jubo...@jhsph.edu wrote: Didn't mlphare use to print those values in the log file ? Jürgen On Dec 15, 2013, at 4:29 PM, David Schuller dj...@cornell.edu wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Heavy atom sites
Hello David, I would use the SAD target function of refmac5 for the anomalous occupancy. As of isomorphous occupancy and phasing power, I don't know. Best, Tim On 12/15/2013 10:29 PM, David Schuller wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] Heavy atom sites
I would find the sites from the PHIC - you need to use CAD to add Fcalc PHIC and FOM to the original data with Fnative Fderiv DANOderiv etc I usually then use SCALEIT to scale native and derivative to Fcalc - then you know you are roughly on an absolute scale Then feed those sites into Phaser_EP and it will refine occupancy etc and give you phasing power Eleanor On 16 December 2013 09:21, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Hello David, I would use the SAD target function of refmac5 for the anomalous occupancy. As of isomorphous occupancy and phasing power, I don't know. Best, Tim On 12/15/2013 10:29 PM, David Schuller wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] Heavy atom sites
Didn't mlphare use to print those values in the log file ? Jürgen On Dec 15, 2013, at 4:29 PM, David Schuller dj...@cornell.edumailto:dj...@cornell.edu wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edumailto:schul...@cornell.edu .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
[ccp4bb] Heavy atom sites
I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Heavy atom sites?
Hi Pu Obviously your SAD SIRAS solutions can't both be right, one must have the inverted handedness: I would guess it's the SIRAS solution that's wrong, since the SAD solution seems to have given you an interpretable map. The reason for getting the wrong hand in the SIRAS case is probably that the anomalous contribution is smaller relatively, so is unable to discriminate the solutions. But what makes you think that the SIRAS solution you (or the program) have chosen is correct, in particular how do the peak heights in the HA-phased map compare for the SIRAS solution inverted through the origin, i.e. the SAD solution? -- Ian On Fri, Aug 20, 2010 at 5:35 AM, Pu Gao ga...@moon.ibp.ac.cn wrote: Hi all, I recently solved a structure using SAD or SIRAS successfully (refinement using native data). But I came across some questions about the heave atom (Hg) sites. SG : P212121. Cell: 61.000 137.700 142.170 90.00 90.00 90.00 (native and derivative are very similar). 1. I used shelxD finding the Hg sites, but got different results from SAD and SIRAS. Here paste the first 3 sites: X Y Z X Y Z (fractional) (orthogonal) SAD: Hg1 0.598389 0.567345 0.098041 36.759 78.146 13.983 Hg2 0.901955 0.570168 -0.098881 55.407 78.535 -14.102 Hg3 0.727585 0.594368 -0.240330 44.696 81.868 -34.276 SIRAS: Hg1 0.598717 0.432159 0.098363 36.522 59.508 13.984 Hg2 0.901527 0.430199 -0.098437 54.993 59.238 -13.995 Hg3 0.727432 0.406845 -0.240253 44.373 56.023 -34.157 It seems there is a symmetry plane m(x,1/2,z) between SAD sites and SIRAS sites. The above HA coordinates were in real space not the partterson space, right? So could someone tell me why there is a m? PS: the Harker section plots (FFT for patterson in CCP4) of difference patterson and ano-difference patterson are very similar. 2. I put the Hg sites caculated by shelxD (say, the SAD sites) to SHARP and got the density map (looks very good). RESOLVE was used to autobuild the model. The Fo-Fc map (Fo from Hg-data, Fc from the model after refinement) at sig5 obviously showed the Hg sites (around Cys). But the heave atom coordinates (from shelxD or sharp) didn't match the Fo-Fc density. I don't know why is that? Dose it bucause the coordinate system mismatch? Any help would be much appreciated. Thanks, Pu
Re: [ccp4bb] Heavy atom sites?
Maybe I need to elaborate on Ian's answer. SHELXD or other heavy atom location programs based on Patterson and direct methods have a 50% chance of getting either heavy atom enantiomorph, and in addition different solutions may be related by allowed shifts of the cell origin (8 in P212121). This means that when you have found the heavy atoms, you must try both the original heavy atom coordinates and the inverted heavy atom coordinates for density modification. This might also involve inverting the symmetry operators (e.g. P41212 becomes P43212, but that is not a problem for P212121. For SAD only one heavy atom enantiomorph will lead to a sensible map. There is a special case in SAD phasing when the heavy atom arrangement is itself centrosymmetric, then both heavy atom enantiomers should lead to the correct map (e.g. one heavy atom in P21). SIR is less convenient. You still have to try both heavy atom enantiomers, but pure density modification will appear to work (or not) for both. However one of the maps will be a mirror image of the true map, e.g. the alpha helices will have the wrong hand. Automatic tracing programs like the SHELXE beta test will only trace the correct map, not the inverted map, so this is a quick way to find out which heavy atom enantiomer is correct. It is also the best check to see whether you have really solved the structure. However if the SIR heavy atom substructure happens to be centrosymmetric, density modification will not be able to break the enantiomer ambiguity. MAD and SIRAS will in general behave like SAD. However if your isomorphous difference is large and the anomalous signal is lost in the noise, they might be dominated by it and so tend to behave more like SIR. 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 Fri, 20 Aug 2010, Ian Tickle wrote: Hi Pu Obviously your SAD SIRAS solutions can't both be right, one must have the inverted handedness: I would guess it's the SIRAS solution that's wrong, since the SAD solution seems to have given you an interpretable map. The reason for getting the wrong hand in the SIRAS case is probably that the anomalous contribution is smaller relatively, so is unable to discriminate the solutions. But what makes you think that the SIRAS solution you (or the program) have chosen is correct, in particular how do the peak heights in the HA-phased map compare for the SIRAS solution inverted through the origin, i.e. the SAD solution? -- Ian On Fri, Aug 20, 2010 at 5:35 AM, Pu Gao ga...@moon.ibp.ac.cn wrote: Hi all, I recently solved a structure using SAD or SIRAS successfully (refinement using native data). But I came across some questions about the heave atom (Hg) sites. SG : P212121. Cell: 61.000 137.700 142.170 90.00 90.00 90.00 (native and derivative are very similar). 1. I used shelxD finding the Hg sites, but got different results from SAD and SIRAS. Here paste the first 3 sites: X Y Z X Y Z (fractional) (orthogonal) SAD: Hg1 0.598389 0.567345 0.098041 36.759 78.146 13.983 Hg2 0.901955 0.570168 -0.098881 55.407 78.535 -14.102 Hg3 0.727585 0.594368 -0.240330 44.696 81.868 -34.276 SIRAS: Hg1 0.598717 0.432159 0.098363 36.522 59.508 13.984 Hg2 0.901527 0.430199 -0.098437 54.993 59.238 -13.995 Hg3 0.727432 0.406845 -0.240253 44.373 56.023 -34.157 It seems there is a symmetry plane m(x,1/2,z) between SAD sites and SIRAS sites. The above HA coordinates were in real space not the partterson space, right? So could someone tell me why there is a m? PS: the Harker section plots (FFT for patterson in CCP4) of difference patterson and ano-difference patterson are very similar. 2. I put the Hg sites caculated by shelxD (say, the SAD sites) to SHARP and got the density map (looks very good). RESOLVE was used to autobuild the model. The Fo-Fc map (Fo from Hg-data, Fc from the model after refinement) at sig5 obviously showed the Hg sites (around Cys). But the heave atom coordinates (from shelxD or sharp) didn't match the Fo-Fc density. I don't know why is that? Dose it bucause the coordinate system mismatch? Any help would be much appreciated. Thanks, Pu
Re: [ccp4bb] Heavy atom sites?
Dear Pro. George, Many thanks for your detailed and clear exposition, which gives me a more clear idea about this question (Although I still don't really understand the basc mathematic things behind it). I rechecked the sharp logs, and found that the original SAD sites were wrong, which had to be turned to its inverted ones for calculating the final density (while the SIRAS sites are correct, the final density was calculated directly from the original sites). And the SIRAS sites matched the Fo-Fc quite well~~~ Thanks, Pu
Re: [ccp4bb] Heavy atom sites?
MAD and SIRAS will in general behave like SAD. However if your isomorphous difference is large and the anomalous signal is lost in the noise, they might be dominated by it and so tend to behave more like SIR. I thought that MAD and SIRAS had no hand ambiguity--not true? Jacob Keller
Re: [ccp4bb] Heavy atom sites?
Not true. For MAD and SIRAS you still have to try both hands of the heavy atom substructure (unless the heavy atom arrangement is itself centrosymmetric, then both hands are correct). Maybe I should also mention for completeness, that for the space groups I41, I4122 and F4122 the heavy atoms have to be inverted at a point that is not at the origin (e.g. x, y, z - 1-x, 0.5-y, 0.25-z for I4122). Fortunately SHELXE and some other programs know this and apply the correct inversion automatically. 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 Fri, 20 Aug 2010, Jacob Keller wrote: MAD and SIRAS will in general behave like SAD. However if your isomorphous difference is large and the anomalous signal is lost in the noise, they might be dominated by it and so tend to behave more like SIR. I thought that MAD and SIRAS had no hand ambiguity--not true? Jacob Keller
Re: [ccp4bb] Heavy atom sites?
Dear Pu, sorry for the long(ish) answer ... handedness/enantiomorph, heavy atom sites and consistency (while using SHARP/autoSHARP) are just one of my favourites ;-) On Fri, Aug 20, 2010 at 01:39:58PM +0100, Pu Gao wrote: I rechecked the sharp logs, and found that the original SAD sites were wrong, which had to be turned to its inverted ones for calculating the final density (while the SIRAS sites are correct, the final density was calculated directly from the original sites). If you run autoSHARP including the density modification step (!) it will try and detect which is the correct hand/enantiomorph. It is then important to either take the last SHARP run (inverted hand) or the last but one (original hand) when continuing to work with the current heavy atom model in SHARP (e.g. to then do MAD+native or spherical averaged heavy atom clusters etc). The correct set of heavy atom parameters should be clearly stated in the log file from autoSHARP. If you did run e.g. SHELXD by hand and then used those sites in SHARP you could obviously have the wrong hand/enantiomorph (see George's post). I would generally recommend using the heavy atom sites from SHELXD in an autoSHARP run: unless you do something more complex than SAD/MAD or SIR(AS)/MIR(AS). It will then skip it's own heavy-atom detection step (also using SHELXC/D) and use your supplied sites. Using sites from your own HA-detection attempt is a good idea for tricky cases (where you might have to run a lot of trials), cases where the number of sites is very uncertain and you need to run lots of slightly different trials ... or if you have very low resolution data/signal (5A and lower). In any case: make your life easy and plug them into autoSHARP up to the density modifcation step. Also, if you run several phasing scenaria (SAD, another one SIRAS, different derivatives etc) there is a very useful feature in SHARP that allows input of external (prior) phase information - e.g. in form of Hendrickson-Lattmann coefficients. This way you could use the SHARP phases from your SIRAS job (e.g. nat+Hg) for a SAD run (Se-MET peak - no sites known so far) and detect your Se sites in the log-likelihood gradient maps ... on the same origina (!) as the other sites. Having all sites on the same origina makes your life MUCH easier if you have to deal with lots of derivatives, datasets and slightly different cell dimensions. You could also use your phases from a poor or partial MR solution in the same way to detect your heavy atom sites. This feature (using prior phase information from partial models, MR or other derivatives) has been in SHARP since the very early days and has been useful to us in a lot of cases. See e.g. the 2010 paper by Pietro Roversi (http://scripts.iucr.org/cgi-bin/paper?ba5141) or some earlier references: Kauppi B, Lee K, Carredano E, Parales RE, Gibson DT, Eklund H, Ramaswamy S. (1998). Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase. Structure 6, 571-86. and the manual: http://www.globalphasing.com/sharp/manual/chapter2.html#external http://www.globalphasing.com/sharp/manual/chapter4.html#ExternalPhaseInformation Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Heavy atom sites?
Dear all, My case seems to have two factors: 1. origin choice. and 2. handedness/enantiomorph. And this kind of problem seems to be very common during the experimental phasing process. And I learned a lot informations both theoretical and practical from your replies. Many thanks for all the replies. Pu
Re: [ccp4bb] Heavy atom sites?
Dear Pu, If I may add a few remarks those already made by George and Clemens, I would say that you cannot expect two heavy atom solutions obtained in separate runs of SHELXD (or any other substructure solution program) from two distinct sets of differences to be consistent with each other in terms of origin and enantiomorph - hence the need for checks by cross-difference Fouriers or (much better) log-likelihood gradient maps for each set of differences phased by the other. This could get even worse in the case of polar space groups (e.g. P2(1) ) where a coordinate shift along the polar axis is left undetermined in each substructure solution. This is a very old problem - see for instance the method of Bragg ellipses. One way of avoiding this in your case would have been to treat your isomorphous and anomalous differences jointly by trying to get estimates for FA values, as if you had a 2-wavelength experiment. This was done under the old terminology via the Matthews combined difference Patterson. Then, solving for your substructure from that combined Patterson would have produced an automatically consistent solution for both the isomorphous and the anomalous differences. George: wouldn't XPREP have done that if it had been given the SIRAS data all at once, instead of separately handling these as SAD and SIR data? With best wishes, Gerard. -- On Fri, Aug 20, 2010 at 04:32:37PM +0100, Pu Gao wrote: Dear all, My case seems to have two factors: 1. origin choice. and 2. handedness/enantiomorph. And this kind of problem seems to be very common during the experimental phasing process. And I learned a lot informations both theoretical and practical from your replies. Many thanks for all the replies. Pu -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
Re: [ccp4bb] Heavy atom sites?
Dear Eleanor at al, On Fri, Aug 20, 2010 at 05:20:05PM +0100, Eleanor Dodson wrote: There is quite a lot of background to these Qs in a variwty of text books, and something on this website. http://www.ccp4.ac.uk/dist/html/pxmaths/index.html Nice page :-) When changing hand you need to consider whether this also involves a change of spacgroup; eg P32 instead of P31 It always does involve a change to the enantiomorph. One only ever needs to check two solutions: * if you started with your dataset in P31 you will have to check (X,Y,Z) P31 and (-X,-Y,-Z) P32 and never: (-X,-Y,-Z) P31 * if you started with P32 just check (X,Y,Z) P32 and (-X,-Y,-Z) P31 and never: (-X,-Y,-Z) P32 It helps to process data always in the same enantiomorph spacegroup (since the extinction rules are identical): e.g. sticking with P61 and P62 at the beginning and never go into P65 and P64 for a start (only after phasing or MR points to that spacegroup). Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Heavy atom sites?- Only them?
The present generation of high throughput structural biologists stays on the intellectual shoulders of the giants of crystallography from past days (modifying GOOGLE). In the military jargon, in the constant wars with the structures, situation described in this exchanges is called encounter. To know all this - is as to know how to calculate without computers. Or to have Mentates to navigate in space without computer technology which is presently prohibited after attempts of computers to take over (citing Dune). Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Aug 20, 2010, at 19:42 , Clemens Vonrhein wrote: Dear Eleanor at al, On Fri, Aug 20, 2010 at 05:20:05PM +0100, Eleanor Dodson wrote: There is quite a lot of background to these Qs in a variwty of text books, and something on this website. http://www.ccp4.ac.uk/dist/html/pxmaths/index.html Nice page :-) When changing hand you need to consider whether this also involves a change of spacgroup; eg P32 instead of P31 It always does involve a change to the enantiomorph. One only ever needs to check two solutions: * if you started with your dataset in P31 you will have to check (X,Y,Z) P31 and (-X,-Y,-Z) P32 and never: (-X,-Y,-Z) P31 * if you started with P32 just check (X,Y,Z) P32 and (-X,-Y,-Z) P31 and never: (-X,-Y,-Z) P32 It helps to process data always in the same enantiomorph spacegroup (since the extinction rules are identical): e.g. sticking with P61 and P62 at the beginning and never go into P65 and P64 for a start (only after phasing or MR points to that spacegroup). Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
[ccp4bb] Heavy atom sites?
Hi all, I recently solved a structure using SAD or SIRAS successfully (refinement using native data). But I came across some questions about the heave atom (Hg) sites. SG : P212121. Cell: 61.000 137.700 142.170 90.00 90.00 90.00 (native and derivative are very similar). 1. I used shelxD finding the Hg sites, but got different results from SAD and SIRAS. Here paste the first 3 sites: X Y Z X Y Z (fractional) (orthogonal) SAD: Hg10.598389 0.567345 0.09804136.759 78.146 13.983 Hg20.901955 0.570168 -0.09888155.407 78.535 -14.102 Hg30.727585 0.594368 -0.24033044.696 81.868 -34.276 SIRAS: Hg10.598717 0.432159 0.09836336.522 59.508 13.984 Hg20.901527 0.430199 -0.09843754.993 59.238 -13.995 Hg30.727432 0.406845 -0.24025344.373 56.023 -34.157 It seems there is a symmetry plane m(x,1/2,z) between SAD sites and SIRAS sites. The above HA coordinates were in real space not the partterson space, right? So could someone tell me why there is a m? PS: the Harker section plots (FFT for patterson in CCP4) of difference patterson and ano-difference patterson are very similar. 2. I put the Hg sites caculated by shelxD (say, the SAD sites) to SHARP and got the density map (looks very good). RESOLVE was used to autobuild the model. The Fo-Fc map (Fo from Hg-data, Fc from the model after refinement) at sig5 obviously showed the Hg sites (around Cys). But the heave atom coordinates (from shelxD or sharp) didn't match the Fo-Fc density. I don't know why is that? Dose it bucause the coordinate system mismatch? Any help would be much appreciated. Thanks, Pu
