Re: [ccp4bb] decrease of background with distance?
John Another point which might resolve some of the confusion regarding detector distances (or add to the confusion) " that moving the detector back was not the setting required but a small collimator (0.2mm) and slitting down the divergence to control the spot size versus the broader halo of acoustic scattering" This is because the detector was x-ray film with a very good spatial resolution. For a detector with poorer resolution, the angle subtended by one resolution element at short distances can be greater then the angular divergence of the x-ray beam (due to incident beam divergence or crystal imperfections). Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of John R Helliwell Sent: 27 November 2009 09:49 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] decrease of background with distance? Dear Richard, A most interesting discussion has ensued! The balance of elastic versus inelastic scattering becomes the core point re benefit of moving back the detector as mentioned by Ian. It should be easier now ie with much more beamtime available to measure this as a function of wavelength. Colin I believe has made a start in this direction. The acoustic scattering discussion needs to recall from:- I.D. Glover, G.W. Harris, J.R. Helliwell and D.S. Moss 'The variety of X-ray diffuse scattering from macromolecular crystals and its respective components' Acta Cryst. (1991) B47, 960-968. and page 966 in particular that moving the detector back was not the setting required but a small collimator (0.2mm) and slitting down the divergence to control the spot size versus the broader halo of acoustic scattering. These days much more readily accomplished with an undulator. These are both important points then for the growing categories of microcrystals, which I know you have been very usefully surveying, and ever larger molecular weight complexes ie both of which are challenged by S/N for the Bragg spots notably at higher resolution. Best wishes, John Professor John R Helliwell DSc beam divergence. On Mon, Nov 23, 2009 at 10:54 PM, Richard Gillilan wrote: It seems to be widely known and observed that diffuse background scattering decreases more rapidly with increasing detector-to-sample distance than Bragg reflections. For example, Jim Pflugrath, in his 1999 paper (Acta Cryst 1999 D55 1718-1725) says "Since the X-ray background falls off as the square of the distance, the expectation is that a larger crystal-to-detector distance is better for reduction of the x-ray background. ..." Does anyone know of a more rigorous discussion of why background scatter fades while Bragg reflections remain collimated with distance? Richard Gillilan MacCHESS -- -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] Bad geometry for alt. conformation refined in Refmac5
I think I've finally squashed this particular bug which had been annoying me for quite some time! It appears that Intel Fortran v11.0 has an optimisation bug in s/r CHK_ALT (i.e. checking alternate atom codes), & possibly also 10.x - I don't have those to test - though v9.1 & possibly earlier seem to be bug-free (at least this bug anyway!). Possibly this is fixed in the current version of ifort (11.1), again I don't have it to test, maybe someone who does can try it. This affects compilations using optimisation levels -O2, -Os and -O3, but -O0 and -O1 are unaffected. So my solution is to recompile with -O1. The CCP4 6.1.2 distribution uses -O2 so you need either to fix the makefile, or download Garib's source code and make sure the makefile you use has the appropriate flag settings. As was pointed out earlier, having to use the gfortran-compiled code is an issue, because at least for the OS I'm using (Centos 4.6), gfortran-compiled code runs ~ 300% slower than the ifort code!! Hope this helps! -- Ian > -Original Message- > From: owner-ccp...@jiscmail.ac.uk > [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Garib Murshudov > Sent: 25 November 2009 16:27 > To: john.pas...@jefferson.edu > Cc: CCP4BB@jiscmail.ac.uk > Subject: Re: [ccp4bb] Bad geometry for alt. conformation > refined in Refmac5 > > Could you please try the version from York: > > www.ysbl.york.ac.uk/refmac/latest_refmac.html > > I think probkem you mention is related with compilation or > something. > At least I cannot repat it on my computer > > regards > Garib > > On 25 Nov 2009, at 16:18, John Pascal wrote: > > > Hello All, > > > > We are trying to refine ARG residues with two conformations in > > Refmac5, and the > > refined atom positions in the output PDB file are all over the > > place, as if the > > geometry restraints are not well defined. > > > > We've tried several different formats for the input file, based on > > previous > > postings to the bulletin board and the PDB standard (two examples > > below), but > > the result is always the same. > > > > We are using Refmac5 in CCP4 Suite 6.1.2, GUI 2.0.5 on Mac OSX. > > > > We'd appreciate any suggestions. Thanks. -John > > > > Examples of ARG format: > > > > 1) > > ATOM 1472 N ARG A 1 -5.737 26.887 38.372 1.00 > > 29.53 CN > > ATOM 1473 CA ARG A 1 -5.445 25.560 37.882 1.00 > > 30.24 CC > > ATOM 1474 CB ARG A 1 -5.314 24.548 39.036 1.00 > > 30.63 CC > > ATOM 1475 CG ARG A 1 -5.426 23.052 38.627 1.00 > > 34.81 CC > > ATOM 1476 CD AARG A 1 -4.827 22.075 39.644 0.50 > > 37.09 CC > > ATOM 1477 CD BARG A 1 -4.301 22.419 39.279 0.50 > > 37.09 CC > > ATOM 1478 NE AARG A 1 -3.430 21.777 39.304 0.50 > > 42.71 CN > > ATOM 1479 NE BARG A 1 -4.482 21.902 40.627 0.50 > > 42.71 CN > > ATOM 1480 CZ AARG A 1 -2.998 20.868 38.402 0.50 > > 44.91 CC > > ATOM 1481 CZ BARG A 1 -3.648 22.142 41.638 0.50 > > 44.91 CC > > ATOM 1482 NH1AARG A 1 -3.841 20.117 37.678 0.50 > > 45.20 CN > > ATOM 1483 NH1BARG A 1 -2.584 22.912 41.464 0.50 > > 45.20 CN > > ATOM 1484 NH2AARG A 1 -1.688 20.715 38.210 0.50 > > 44.99 CN > > ATOM 1485 NH2BARG A 1 -3.878 21.619 42.831 0.50 > > 44.99 CN > > ATOM 1486 C ARG A 1 -6.518 25.176 36.830 1.00 > > 29.97 CC > > ATOM 1487 O ARG A 1 -7.675 25.501 36.971 1.00 > > 31.02 CO > > > > 2) > > ATOM 44 N AARG A 1 26.671 62.112 46.990 0.50 > > 30.13 AN > > ATOM 45 CA AARG A 1 26.970 63.346 47.667 0.50 > > 30.65 AC > > ATOM 46 CB AARG A 1 27.172 64.495 46.676 0.50 > > 31.07 AC > > ATOM 47 CG AARG A 1 27.152 65.897 47.322 0.50 > > 34.20 AC > > ATOM 48 CD AARG A 1 27.993 66.976 46.599 0.50 > > 37.16 AC > > ATOM 49 NE AARG A 1 27.726 67.425 45.342 0.50 > > 42.06 AN > > ATOM 50 CZ AARG A 1 28.315 67.639 44.168 0.50 > > 44.78 AC > > ATOM 51 NH1AARG A 1 29.525 67.160 43.918 0.50 > > 45.37 AN > > ATOM 52 NH2AARG A 1 27.690 68.340 43.240 0.50 > > 45.67 AN > > ATOM 53 C AARG A 1 25.839 63.640 48.622 0.50 > > 30.37 AC > > ATOM 54 O AARG A 1 24.690 63.377 48.340 0.50 > > 31.24 AO > > ATOM 55 N BARG A 1 26.667 62.080 47.010 0.50 > > 30.13 AN > > ATOM 56 CA BARG A 1 26.921 63.329 47.640 0.50 > > 30.65 AC > > ATOM 57 CB BARG A 1 27.108 64.390 46.581 0.50 > > 31.07 AC > > ATOM 58 CG BARG A 1 27.138 65.756 47.103
Re: [ccp4bb] AW: [ccp4bb] off-topic: crystal optimization without buffer
Hi Katja, If your protein preparation did have salt, you could try lowering that salt (if your protein tolerates) and grid search with varying (increasing) PEG concentrations. Seeding and Additives should really help. the effect/outcome is difficult to predict but these two techniques are helpful in different cases/extent. you could give it a try. good luck also temperature is a under-utilized variable in crystallization, you could see if that has a drastic/favorable effect Regards, Karthik On Thu, Nov 19, 2009 at 8:39 AM, Jan Schoepe wrote: > Hi Katja, > > it makes perfect sense to add a buffer to your assay. Of course for the > beginning something which buffers well around pH 7 like HEPES, BTP, etc. > would be appropriate. If your purification protocol is optimized I would not > change the conditions of the purification buffer. But this might change > maybe if your crystallization experiments give you some more hints about the > solubility of the protein. > > Good luck, > Jan > > > --- Katja Schleider ** schrieb am *Do, > 19.11.2009: > * > > * > Von: Katja Schleider > Betreff: [ccp4bb] off-topic: crystal optimization without buffer > An: CCP4BB@JISCMAIL.AC.UK > Datum: Donnerstag, 19. November 2009, 10:33 > > * > Hi everybody, > > sorry for my off-topic question. I got small initial crystals in 200mM > NaSulfat and 20% PEG 3350. > There is no buffer in this condition. How can I optimize these crystals? > Just vary the PEG concentration? Or should I add a buffer; or vary the pH > of the buffer the proteinsolution was in? > > Thank you and best regards, > > Katja > * > * > > >
