Application Note AN12Mar15
Sulfur-SAD phasing of lysozyme at room temperature with a PILATUS3 R 200K-A detector on a marYX2G micro-beam generator
|Distance crystal-detector [mm]||55||55||55|
|Total PHI range [deg.]||360||360||90|
|Number of images||3600||720||900|
|Exposure time/image [sec]||1||10||4|
|Total exposure time [min]||60||120||60|
|Max. resolution [Ang.]||2.4||1.6||1.6||1.6|
|# unique reflections||8631||30318||19076||30398|
|# measured reflections||48124||172492||43151||324230|
|Completeness1 [%]||97.7 (87.3)||99.7 (99.1)||62.6 (43.9)||99.8 (99.1)|
|R1sym [%]||3.7 (4.8)||5.4 (43.8)||4.5 (37.0)||4.6 (44.6)|
|R1meas [%]||4.1 (6.0)||5.9 (49.8)||5.5 (46.6)||4.8 (49.2)|
|I/σ1||35.7 (16.0)||19.2 (3.3)||13.7 (2.7)||28.0 (3.5)|
|SIG1ano||1.16 (0.83)||0.91 (0.79)||0.97 (0.80)||1.07 (0.78)|
1 Last shell in brackets: 2.56-2.4 Ang for set 1, 1.71-1.60 Ang. for sets 2 and 3, respectively.
Structure solution and refinement
Program SHELXD was instructed to locate 17 anomalous scatterers: 10 sulfur atoms and 7 Cl ions at 1.8 Ang. resolution. The best solution was used for phase calculation and further improvement by density modifications with program SHELXE. The following results were obtained from SHELXD: PATFOM: 1.38 CC all/weak: 37.9 / 16.6 The following results were obtained from SHELXE: Contrast / enantiomorph: 0.48 / 0.32 Pseudo Free CC / enantiomorph: 70.8 / 52.4 The resulting experimental phases from SHELXE were used for chain-tracing from scratch using program arp/warp. The program automatically built all 129 residues and yielded a refined model at 1.6 Ang. resolution that matches the published data.
Residues 50 to 56: initial SHELXE map
Residues 50 to 56: final 2Fo-Fc map
ConclusionPA total of 810 degrees of data collected from a single lysozyme crystal within just 4 hours on an in-house micro-focus generator yielded good enough data for ab initio structure solution. This was done despite of using a non-frozen crystal at room temperature and with the smallest of all DECTRIS pixel detectors, the PILATUS3 R 200K. To achieve higher resolu- tion, we made use of the built-in 2-theta offset of the mardtb goniostat. This study hence nicely demonstrates the amazing capabilities of the entire setup consisting of a very fast and efficient detector, an easy-to-use goniostat and a small but very powerful home source.
In summary, the PILATUS3 R series of detectors are ideally suited for fast crystal screening experiments as well as for complete data collections with the aim of solving and refining protein structures in a home lab.