These include: 1) crystal lattice defects 2) anisotropic diffraction and 3) crystal “polluting” by heavy protein aggregates and micro-crystal nuclei. Moreover, TEM has been shown to have an important role to identify crystal pathologies that contribute to poor X-ray diffraction data. In general, the detection by negative stain TEM methods of well-ordered lattices with third order Bragg spots was a good predictor of X-ray diffraction, while samples with disorganized lattices yielded no diffraction. Our experiments have shown that for all protein crystals tested, TEM images reveal details of the crystal lattices that prove to be accurate qualitative indicators of the potential diffraction of the crystal. Here, we will describe reproducible protocols, using TEM to visualize lattices from fragmented crystals and to study details of the crystallization process. Transmission electron microscopy (TEM) is one of the best techniques to do so. However, less effort has been applied towards the discovery, evaluation and optimization of crystals and nano-crystals (NCs). Recent efforts towards improving sample “crystallizability” using techniques such as alanine scanning mutagenesis or the use of chimeric proteins to promote/improve crystal packing has led to structures of important targets. TEM guided optimization of Pol II crystalsĬrystallization of protein targets remains the most significant challenge in the process of structure determination by macromolecular crystallography. Thus, in order to study the molecular mechanism of nucleotide addition in greater detail, i.e., at near atomic resolution, Poll II crystals are suitable candidates for 1) visualization and optimization of crystal lattice quality using TEM and 2) FEL experiments at the LCLS to improve resolution (since diffraction of Pol II crystals is dose dependent), and to obtain radiation-damage-free structures that could allow observation of metals and solvent molecules in Pol II’s active site.ġ. Moreover, electron density for the two Mg 2+ ions in the active site was not clearly discernable, which could be possible attributed to the effect of radiation damage during data collection. This structure illustrated for the first time the trigger loop in the “on” or closed conformation, and the matched nucleotide in the substrate binding pocket however, these studies were hampered by the low resolution of the data. Similarly, structural studies of Pol II bound to a matched nucleotide have revealed the architecture (at 4.0 Å resolution) of Pol II during synthesis of the nascent transcript. A molecular picture of the role of the individual factors during transcription initiation and elongation is beginning to emerge and has generated tremendous progress towards our understanding of gene processing and regulation. Moreover, the remarkable single particle cryo-electron microscopy structures of Pol II in complex with the general transcription factors and mediator (preinitiation complex) and with the elongation factors have provided snapshots of the complexities of the initiation and elongation steps. Structural studies of Pol II have been very successful and have allowed observation of Pol II in its apo form, in the process of initiation and elongation, and during backtracking or paused by DNA lesions. DNA-directed RNA Polymerase II (Pol II) is a highly-conserved protein among eukaryotic organisms and plays a fundamental role in cellular life, specifically the transcription of genes into messenger RNA (mRNA).
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