Cs Potter

Polyvalent Display of Heme on Hepatitis B Virus Capsid Protein through Coordination to Hexahistidine Tags

The addition of a hexahistidine tag to the N terminus of the hepatitis B capsid protein gives rise to a self-assembled particle with 80 sites of high local density of histidine side chains. Iron protoporphyrin IX has been found to bind tightly at each of these sites, making a polyvalent system of well-defined spacing between metalloporphyrin complexes. The spectroscopic and redox properties of the resulting particle are consistent with the presence of 80 site-isolated bis(histidine)-bound heme centers, comprising a polyvalent b-type cytochrome mimic.

Applications of Automated Electron Microscopy: Using Leginon to Study the Structure of COPII Protein Complexes

COPII proteins are responsible for forming the vesicles that transport proteins from the endoplasmic reticulum to the Golgi apparatus. The COPII proteins form a coat around the budding vesicle and are responsible for both selecting the protein cargo and drawing the ER membrane up and pinching it into a vesicle. COPII coats consist of three components: Sar1, a GTPase; Sec23/24, a GTPase activating protein (GAP); and Sec13/31, a GAP stimulator which has also been implicated in inducing membrane curvature. While structures of Sar1 and Sec23/24 have been solved, little is known about the structure of Sec13/31 or the structure of the COPII lattice. We are investigating the structure of the COPII lattice using cryo-electron microscopy (cryoEM).

The impact of data reduction and lossy image formats on electron microscope images

Recent advances in instrumentation and software for cryoEM have increased the applicability and utility of this method. Coupled with the adoption of automated pipelines, significant infrastructure support is required to sustain high throughput workflows. In particular, data generation rates may outpace the ability to deploy data storage and archival solutions. We have investigated what effects data compression and conversion to different file formats have on the ability to obtain high resolution cryoEM reconstructions. Standard lossless data compression strategies have a high impact on reducing the size of direct detector electron counting movie stacks, but provide more modest gains for aligned summed images. We show that EM images can be compressed using standard lossy methods to reduce file storage to 5–10% of the size of the original aligned sum or movie stack file and yet still retain enough information such that modern image processing pipelines will provide sub-2Å reconstructions from the compressed data.

Automated TEM Data Acquisition Applications Using Robotic Grid Loading

Data acquisition spanning multiple specimen grids using a TEM is conventionally done by an operator manually loading and imaging each grid. During the previous meeting we reported on using a robotic grid loading system to collect low resolution atlases for sets of 96 grids without the need for human interaction [1,2]. Here we report on new developments of the system for routinely collecting higher resolution images selected from areas of interest in the atlas.