Dan Anderson

We have our own water machine to make HPLC grade water. Use this very pure water, and chemicals of ACS grade or better.

Ammonium Sulfate, saturated solution: Weigh about 80 grams of enzyme grade ammonium sulfate. In a 250ml beaker, stir the solid into 100ml of wa ter. Heat the mixture to dissolve the last of the solid. The final volume is about 120ml. Get a 0.2um filter wet with water before use, then filter the solution while it is still hot. The concentration equilibrates several days later, when crystals appear.

Sodium Citrate buffers: Citrate buffers become very hot during titration with NaOH. The pH’s of buffers that get hot are not reproducible. To make a 0.5M citrate buffer without heating, first separately prepare 0.5M citric acid, and 0.5M Na3 citrate. Mix them while watching the pH with a pH meter. No matter how you mix them, it’s still 0.5M total citrate. Calibrate the pH meter around the pH of interest. Our pH meter automatically recognizes pH 1.68 standard buffer, in addition to the more familiar standards.

Lithium Citrate buffers: This one is really a pain because LiOH is not very soluble, and it fizzes as though part of it is LiHCO3. If you need a series of Li citrate buffers, make the highest pH one first, to final concentration, then mix that with the same concentration of citric acid to back-titrate to the low er pH values. The volume of the highest pH buffer has to be enough to make all the lower pH buffers.

Filtration of 2M Lithium Sulfate: The 0.2um filter has to be wet first.

Lithium chloride: LiCl is soluble to at least 8M. Dissolving LiCl is exothermic. Preparation of the concentrated solution generates concentrat ed heat that can melt Parafilm and make a warm mess. Add the LiCl to the water slowly. It takes a long time for 8M LiCl to wet a cellulose acetate filter membrane. 5M LiCl filters much faster.

Polyethylene Glycol: Weigh the desired amount, put the solid into a graduated cylinder of the desired volume. Add water to less than the final volume. Seal with Parafilm. Mix on a Nutator until dissolved, then add water to final volume. For example, for a 30% stock solution, weigh 30g, put it into a 100ml graduat ed cylinder with water up to about the 90ml mark, seal and Nutate. Later on, add water to 100ml.

Polyethylene Glycol 1000: PEG1000 is poured into its container as a melt; it is not flaked or powdered like the other PEG’s. To make a PEG1000 solution, warm the PEG1000 container in a water bath to melt it, then pour out the desired amount. Dissolve it in water before it solidifies, otherwise it will take a lon g time to dissolve.

4M Na/K Phosphate: I don’t know if this results in anybody’s “standard” pH. Decide what final volume you want, then weigh enough of NaH2PO4, Na2HPO4, KH2PO4, K2HPO4 to make that final volume 1M of each one. Add a little at a time of each one to water, with stirring. When all 4 are nearly dissolved, add water to reach the final volume.

Publication in Nature, view here

Abstract

BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals—50 unit cells per edge, on average—has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.

 

We illustrate a method to access the long unrealized potential of
electrons in the crystallographic structural determination of
biological molecules. Electrons, owing to their strong interaction
with matter, produce high resolution diffraction patterns from tiny
three-dimensional biomolecular crystals (MicroED), enticing structural
biologists with a means to gain much needed insight into the detailed
workings of biological systems and disease mechanisms. However,
barriers to successful use of MicroED arise from a phenomenon known as
dynamical scattering and lack of a means of recovering phases from
diffracted electrons. We showed that if the diffraction resolution is
high enough and the crystals are sufficiently small, both barriers are
overcome, unlocking the potential of MicroED for elucidation of novel
biomolecules.

Click here for: PNAS Full Article

 

UCLA Core Technology Centers have created a new database of campus resources at http://www.cores.ucla.edu/

The site contains descriptions and contact information for each core, as well as a new searchable database of services and instrumentation (courtesy of the UCLA-DOE Institute Bioinformatics and Computational Core Technology Center).

Published in the Journal of Biological Chemistry, the work of Dr. Lorena Saelices and coworkers under the supervision of Prof. David Eisenberg establishes a novel therapeutic strategy for transthyretin amyloidosis. The team identified two segments of transthyretin that are responsible for the protein aggregation and determined the structure of their fibrils. The fibril structures were used to design peptide inhibitors that block self-association of the two segments, and hinder transthyretin amyloid formation. The research published in December, 2015 opens a new approach to tackle the life-threatening transthyretin amyloidosis.

Abstract of the manuscript:

The tetrameric thyroxine-transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of the life-threatening transthyretin amyloidosis. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Although aggregation-preventing strategies involving ligands are known, understanding the mechanism of TTR aggregation can lead to additional inhibition approaches. Several models of TTR amyloid fibrils have been proposed, but the segments that drive aggregation of the protein have remained unknown. Here we identify beta-strands F and H as necessary for TTR aggregation. Based on the crystal structures of these segments, we designed two non-natural peptide inhibitors that block aggregation. This work provides the first characterization of peptide inhibitors for TTR aggregation, establishing a novel therapeutic strategy