I am wondering whether part of the benefit of fine slicing is really increased multiplicity in disguise. I have seen in papers that empirically things do not improve much (or can even get slightly worse) past 0.5*SigPhi, which is the XDS-defined version of mosaicity; according to MOSFLM’s definition of mosaicity, which is ~2-3 larger than that from XDS, this would correspond to ~0.15-0.25 * mosaic spread (Mueller et al 2011). Accordingly, a crystal with MOSFLM mosaic spread of 0.6 would be best measured with ~0.1 deg oscillations, meaning, I think, that the spot would be measured at least six times as a partial, leading to some averaging of various errors from frame to frame.
JPK From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Edward Snell Sent: Wednesday, November 30, 2016 8:11 PM To: [email protected] Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers There is a very nice paper by Colin Nave on Matching X-ray beam and detector properties to protein crystals of different perfection,J Synchrotron Radiat. 2014 21, 537–546. Due to the spectral and geometric properties of in house sources there is probably no advantage with an oscillation below 0.2 degrees no matter how good the crystal is. A well conditioned beamline can gain an advantage from a very perfect crystal but once you cryocool them all bets are off. Make sure you consider the instrument resolution parameters (spectral and geometric divergence) and the actual crystal quality. As to resolution dependence, it all comes down to the source of the mosaicity, e.g. domain misalignment, imperfections, domain boundary effects and domain size. There is a rich literature around but speaking from first hand experience, it's not trivial to probe this area. Cheers, Eddie. Edward Snell Ph.D. President and CEO Hauptman-Woodward Medical Research Institute Assistant Prof. Department of Structural Biology, University at Buffalo 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: [email protected]<mailto:[email protected]> Heisenberg was probably here! ________________________________ From: CCP4 bulletin board <[email protected]<mailto:[email protected]>> on behalf of Jeffrey, Philip D. <[email protected]<mailto:[email protected]>> Sent: Wednesday, November 30, 2016 7:36 PM To: [email protected]<mailto:[email protected]> Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers Jacob, If you fine slice and everything is then a partial, isn't that *more* sensitive to lack of synchronization between the shutter and rotation axis than the wide-frame method where there's a larger proportion of fulls that don't approach the frame edges (in rotation space) ? Especially if you're 3D profile fitting ? Is fine slicing more or less beneficial at high resolutions relative to lower ones ? Phil Jeffrey Princeton ________________________________ From: CCP4 bulletin board [[email protected]] on behalf of Keller, Jacob [[email protected]] Sent: Wednesday, November 30, 2016 5:44 PM To: [email protected]<mailto:[email protected]> Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers If the mosaicity is, say, 0.5 deg, and one is measuring 1 deg frames, about half the time is spent measuring non-spot background noise under spots in phi, which is all lumped into the intensity measurement. Fine slicing reduces this. But I am conjecturing that there is also fine-slicing-mediated improvement due to averaging out things like shutter jitter, which would also be averaged out through plain ol’ multiplicity. I guess a third equal-count dataset would be useful as well: one sweep with six-fold finer slicing. So it would be: One sweep, 0.6 deg, 60s Six sweeps, 0.6 deg, 10s One sweep, 0.1 deg, 10s Or something roughly similar. Who will arrange the bets? JPK From: Boaz Shaanan [mailto:[email protected]] Sent: Wednesday, November 30, 2016 5:19 PM To: Keller, Jacob <[email protected]<mailto:[email protected]>>; [email protected]<mailto:[email protected]> Subject: RE: Effects of Multiplicity and Fine Phi with Equivalent Count Numbers Hi Jacob, I may have missed completely your point but as far as my memory goes, the main argument in favour of fine slicing has always been reduction of the noise arising from incoherent scattering, which in the old days arose from the capillary, solvent, air, you name it. The noise reduction in fine slicing is achieved by shortening the exposure time per frame. This argument still holds today although the sources of incoherent scattering could be different. Of course, there are other reasons to go for fine slicing such as long axes and others. In any case it's the recommended method these days, and for good reasons, isn't it? Best regards, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: [email protected]<mailto:[email protected]> Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 ________________________________ From: CCP4 bulletin board [[email protected]] on behalf of Keller, Jacob [[email protected]] Sent: Wednesday, November 30, 2016 11:37 PM To: [email protected]<mailto:[email protected]> Subject: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers Dear Crystallographers, I am curious whether the observed effects of fine phi slicing might in part or in toto be due to simply higher “pseudo-multiplicity.” In other words, under normal experimental conditions, does simply increasing the number of measurements increase the signal and improve precision, even with the same number of total counts in the dataset? As such, I am looking for a paper which, like Pflugrath’s 1999 paper, compares two data sets with equivalent total counts but, in this case, different multiplicities. For example, is a single sweep with 0.5 degree 60s exposures empirically, in real practice, equivalent statistically to six passes with 0.5 degree 10s frames? Better? Worse? Our home source has been donated away to Connecticut, so I can’t do this experiment myself anymore. All the best, Jacob Keller ******************************************* Jacob Pearson Keller, PhD Research Scientist HHMI Janelia Research Campus / Looger lab Phone: (571)209-4000 x3159 Email: [email protected]<mailto:[email protected]> *******************************************
