Princeton University   |   Department of Molecular Biology

X-ray diffraction image
v-FLIP MC159 X-ray diffraction from data set at
X29 beam line at NSLS-I
X-ray diffraction image
Diffraction image for tetragonal lysozyme using
the RAXIS-IV++ detector and RuH3R home source

Once you've obtained crystals of a suitable size that diffract fairly well, the collection of X-ray data is one of the most straightforward parts of the process. The facility has a Rigaku RuH3R rotating anode generator operating at 5 kW and generating CuKα x-rays (λ = 1.54 Å) which are the best choice for macromolecular samples. Xenocs Fox 25-25P multilayer optics intensify and collimate the x-ray beam and a Rigaku RAXIS-IV++ image plate area detector records the diffraction data. Data collection is viable for strongly-diffracting crystals in house for crystals that have been cryo-cooled to 100 Kelvin using our Oxford Cryostream model 700 to flash-cool the crystal. Protein crystals accumulate significant radiation damage quite quickly at room temperature but this is radically reduced by cooling them to 100 K in a nitrogen gas stream. Data collection on the in-house source takes in the range 1-2 days.

For an example of what data collection on a well-diffracting crystal looks like, here's a video of a 90° data collection (100 sec/0.5°) crammed into 7 seconds from our in-house source. Proteinase K is a strong diffractor and this data extends well past the edge of the detector at 1.6 Å resolution.

For straightforward cases (strong diffractors, available homologous structures) we can determine structures using data collected in-house. For other cases (novel structures or weak diffractors) we must use the ultra-bright tunable X-ray sources at synchrotrons. Our oft-used favorite is beam line X29 at Brookhaven National Lab which offers a bright beam that is capable of accessing the wavelengths most frequently used in structure determination of novel structures. NSLS-I ceases operations in fall 2014 to facilitate the operations of NSLS-II and in the intervening time between viable NSLS-I and NSLS-II beam lines we will be using synchrotrons at Cornell (CHESS) and Argonne (APS), amongst others.

For the in-house source we support the HKL3000 package for data collection and processing.

HKL3000 screen shot
HKL3000. Click image for larger version

Since any individual data collection package has its strengths and weaknesses we also support XDS, MOSFLM, autoProc and Xia2 processing programs for data collected either in-house or at synchrotrons.