Transcription with a laser: Radiation-damage-free diffraction of RNA Polymerase II crystals.

ABSTRACT

Well-diffracting crystals are essential to obtain relevant structural data that will lead to understanding of RNA Polymerase II (Pol II) transcriptional processes at a molecular level. Here we present a strategy to study Pol II crystals using negative stain transmission electron microscopy (TEM) and a methodology to optimize radiation damage free data collection using free electron laser (FEL) at the Linac Coherent Light Source (LCLS). The use of negative stain TEM allowed visualization and optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nano crystals bearing perfect lattices were seeded and used to grow larger crystals for FEL data collection. Moreover, the use of in house designed crystal loops together with ultra-violet (UV) microscopy for crystal detection facilitated data collection. Such strategy permitted collection of multiple crystals of radiation-free-damage data, resulting in the highest resolution of wild type (WT) Pol II crystals ever observed.