Perhaps the new concept of one “frame” for a crystal should be a complete 3D sampling of reciprocal space, and then averaging can be done after the fact to improve statistics.
JPK From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Graeme Winter Sent: Thursday, May 01, 2014 3:26 AM To: [email protected] Subject: Re: [ccp4bb] Pilatus and Strategy wrt Radiation Damage Hi All, A major opportunity with Pilatus detectors is the chance to redistribute the dose in reciprocal space i.e. measure a lot more data, with less dose / frame, then decide in hindsight where you probably should have cut off the data set. It is certainly true that "strategies" such as 0.2 s/0.2 degree (I would call this a tactic myself ;oD) seem to work well, and that it often seems that you need a reasonable dose to be able to process the data properly (see below). I would however agree strongly that unless you are not vulnerable to radiation damage the use of a strategy program such as EDNA is critical as continuous readout of a fast detector can let you kill your sample really quickly... and it would be a shame to measure the wrong part of reciprocal space. Also the 0.2s / 0.2 degree rate is very beamline dependent. Here at Diamond it is certainly routine to measure data with 0.05 s / 0.1 degree exposure times with Pilatus2 and end up with very good data, and the latest Pilatus3 machines can run with 0.01s exposure times. As Nukri said earlier, once you start running at these very high rates you become much more sensitive to beamline and source characteristics, so your mileage may vary and so on. It's certainly worth spending some time exploring the capability and what works well for *your* samples. I would however strongly agree with the recommendations for fine slicing, and avoid e.g. 1 degree images. In terms of "a reasonable dose to process the data properly" there are some major challenges when dealing with exceedingly weak data in measuring the reflections at high resolution well: the statistics start to become poorly behaved with current analysis software. One tactic I have been playing with is to record the same wedge of data (for example from an EDNA strategy) with exceedingly low dose perhaps 20 times, then to process this and look for signs of radiation damage. After arbitrarily deciding which "pass" radiation damage kicked in at then *sum* the *raw images* from each pass up to this point e.g. pass_1_0001.cbf + pass_2_0001.cbf + .... pass_N_0001.cbf => sum_0001.cbf Then process these summed images as if this was the original data. Funnily enough you may get better data than processing pass_1 to pass_N separately and then scaling and merging all of the measurements, which leads me to pointing the pointy finger of blame at the behaviour of the statistics, and that statistics and things like background subtraction become hard when you have very sparse data. This summing process may seem like manipulating your raw data (naughty!!) but in essence it is really just performing the same process as when you recorded multiple exposures / passes on a single CCD image. It also has the happy side effect of averaging out any random / high frequency effects induced from source / beamline effects, but will also average in any radiation damage effects as well! This by the way is what I was getting at with redistributing your dose in reciprocal space... Cheerio, Graeme On 30 April 2014 17:41, Harry Powell <[email protected]<mailto:[email protected]>> wrote: Hi Marcus Mueller (from Dectris, who develop and manufacture the Pilatus) did some work on this a couple of years ago and determined that an oscillation angle ~ 0.5x the mosaicity of the crystal (using the XDS value of mosaicity, which is not the same as Mosflm's); the abstract says - The results show that fine ’-slicing can substantially improve scaling statistics and anomalous signal provided that the rotation angle is comparable to half the crystal mosaicity. Acta Cryst. (2012). D68, 42-56 [ doi:10.1107/S0907444911049833 ] Optimal fine -slicing for single-photon-counting pixel detectors M. Mueller, M. Wang and C. Schulze-Briese My reading of this is that there is still a place for strategy calculations. On 30 Apr 2014, at Wed30 Apr 15:06, Sanishvili, Ruslan wrote: Hi Jacob, I'll take a first crack as I am sure many will follow. It is true that with CCD detectors one has to be careful how small an oscillation range to use for a frame before read noise starts to eat into the data quality. Pilatus offers two major new features - is fast and is photon counting as opposed to integrating detector. The speed allows to collect data without a shutter and it is very important as it can dramatically improve data quality. Now there are fast CCD detectors as well on the market. Being a photon counter, Pilatus has no "read" noise which, as you have pointed out, allows you to collect as thin a frame as you want. However, it is if you consider the detector only. In reality, if you go very thin and very fast, you may not have enough flux to record the data. Also, even once we get rid of the shutter, there are still other sources of instabilities and they do affect the fast data collection adversely. One could try going (very) thin sliced and somewhat slow but there is another gotcha there. Most rotation stages used for rotating the sample crystal, do not like going extremely slow which would be the case with thin frames and long exposure times. In this case the speed may not remain as constant as we would like during data collection. I think there was a publication from Diamond Synchrotron discussing strategies of data collection with Pilatus. We've done a little bit of systematic studies as well and while things may well be sample- and facility-dependent, ~0.2 degree frames with ~0.2 sec exposure time seemed to make good compromise between above-mentioned issues. Here I would like to emphasize again - there certainly will be samples which will benefit from somewhat different parameters. Hope it helps, Cheers, N. Ruslan Sanishvili (Nukri) Macromolecular Crystallographer GM/CA@APS X-ray Science Division, ANL 9700 S. Cass Ave. Lemont, IL 60439 Tel: (630)252-0665<tel:%28630%29252-0665> Fax: (630)252-0667<tel:%28630%29252-0667> [email protected]<mailto:[email protected]> ________________________________________ From: CCP4 bulletin board [[email protected]<mailto:[email protected]>] on behalf of Keller, Jacob [[email protected]<mailto:[email protected]>] Sent: Wednesday, April 30, 2014 7:49 AM To: [email protected]<mailto:[email protected]> Subject: [ccp4bb] Pilatus and Strategy wrt Radiation Damage Dear Pilatus/Radiation Damage Cognoscenti, I read a few years ago, before the advent of Pilatus detectors, that the best strategy was a sort of compromise between number of images and detector readout noise "overhead." I have heard that Pilatus detectors, however, have essentially no readout noise, so I am wondering whether strategies have changed in light of this, i.e., is the best practice now to collect as many images as possible at lowest exposure possible? JPK ******************************************* Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: [email protected]<mailto:[email protected]> ******************************************* Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
