Protocols for some Crystallization Stock Solutions

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.

How to Prepare Heavy Atom Derivatives with Dimethyl Mercury and Tetraethyl Lead

How to Prepare Heavy Atom Derivatives with Dimethyl Mercury and Tetraethyl Lead

 

Duilio Cascio and Dan Anderson

This technique was first used by Ward Smith at UCLA in the early 80s with RuBisCo. These are some of the first heavy atoms we try at UCLA. The success rate has been very high.

DANGER: DIMETHYL MERCURY HAS NOW CAUSED THE DEATH OF A LABORATORY SCIENTIST AT DARTMOUTH, AND ITS USE SHOULD BE DISCONTINUED AS A PROTEIN HEAVY ATOM DERIVATIVE

DMM or Dimethyl Mercury ( (CH3)2 Hg ) and TEL or Tetraethyl Lead ( (C2H5)4 Pb ) are liquids at room temperature and not miscible with water, but their vapors can diffuse into protein crystals and produce useful derivatives. Both compounds are extremely toxic by inhalation and skin contact. They are fat soluble, and therefore absorption is probably permanent. Please use extreme caution in handling these compounds and in disposing of contaminated objects and crystals after the experiment is done. DMM will permeate in few seconds through disposable latex gloves. A highly resistant laminate glove (Silver Shield or 4H) should be worn under a pair of long cuffed, unsupported neoprene, nitrile, or similar heavy-duty gloves. See report on DMM accident.

  1. Here at UCLA, the management has already purchased DMM and TEL from ALDRICH (catalog numbers 32,808-1 ($120.00) and 40,269-9 ($47.50)). Please, labmates, don’t duplicate our efforts; this is nasty stuff to store.
  2. At UCLA, the management does this: In a fume hood break open an ampule of the liquid and transfer the whole contents to several previously labeled 1 ml REACTI-VIAL vials from PIERCE (catalog 13221Z; $69 for 12 vials; the cap liner must have a Teflon face to contain these materials). The transfer is safest with a positive displacement pipet (Gilson Microman M250). DO NOT use an air displacement pipet (Pasteur or Gilson Pipetman). Close each vial with the special cap provided. Wrap the cap with a strip of Parafilm. Each vial is stored inside a 50 ml polypropylene centrifuge tube with a plug seal screw cap. Cover the cap with Parafilm. Store each individual centrifuge tube inside a larger container (glass jar or metal) sealed with parafilm and label it EXTREMELY TOXIC.These compounds should be stored in a freezer to minimize their vapor pressures. No food should be stored in this freezer. In our lab we keep these vials in a freezer in room 219.
  3. To prepare the protein heavy atom:
    • Keep all your tools used for handling DMM and TEL inside a fume hood. Leave the reagent in its vial in its centrifuge tube in the hood to warm it to room temperature (to minimize condensation of water). Preheat the wax melter in the hood and keep it on.
    • Outside the fume hood: Mount the crystal using your usual technique. Seal one end of the capillary with wax. Insert into the open end a small piece of filter paper. Leave the paper filter flush with the end of the capillary. Pay attention to the size of the paper so it doesn’t touch the crystal.
    • The rest of the procedure MUST be done with double gloves and inside the fume hood (preferably dissimilar glove materials, such as Silver Shield over long cuffed nitrile, neoprene or similar heavy duty gloves).
    • Quickly move the mounted crystal to the fume hood, open the vial with DMM or TEL and with a 10 ul Hamilton syringe add just enough liquid to completely wet the paper. Be careful and do not leave a heavy atom liquid plug inside the capillary.
    • Seal the other end of the capillary: If you use wax, be careful not to warm the paper strip because this causes distillation of the compound onto the nearest cooler thing (your crystal). With 5 minute epoxy, there isn’t a heating problem, but the gyrations needed to hold the liquid epoxy on the end of a capillary sometimes causes the paper strip to fall into the epoxy.
    • Wait for 10-15 minutes to be sure the vapor diffuses into the crystal (see the references, below). TEL is less volatile than DMM, so maybe on average it takes longer. Your crystal is now read for data collection.

CLEANING

Leave the Hamilton syringe in the fume hood for few days. Label it so it is only used for Hg or Pb Heavy Atom experiments.

The gloves, wax and any other disposable tools that touched the compound should be disposed in a appropriate container (We have a container labeled HEAVY SOFT). After data collection the crystal and capillary should be disposed in the sharp Heavy atom waste container. (labeled HEAVY SHARPS)/.

DMM MSDS safety sheet

De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure

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.

 

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UCLA-DOE PI’s visit the Pacific Northwest National Laboratory

UCLA-DOE PI’s visit the Pacific Northwest National Laboratory to team up on new research projects involving state-of-the-art scientific instrumentation.

forDoepage1x

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Eisenberg lab produces Ab Initio protein structure from prion nanocrystals at atomic resolution by MicroED

sawaya_science_pnas_2x

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

 

Todd Yeates work highlighted in Science


Todd Yeates lab published in Science and highlighted on the cover:

Accurate design of megadalton-scale two-component icosahedral protein complexes.

Included is a Video from Science

Abstract
Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.

UCLA CORES

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).

Uncovering the Mechanism of Aggregation of Human Transthyretin

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

 

Professor David Eisenberg was awarded the Legacy Award at the 2nd Annual UCLA Molecular Biology Institute (MBI) Awards Dinner on November 12th.

MBI Awards Dinner small
The Legacy Award honors outstanding MBI faculty mentors who have worked to inspire, encourage and guide the next generation of scientists and thought leaders.Professor Eisenberg is a Distinguished Professor of Chemistry and Biochemistry and of Biological Chemistry, an HHMI investigator, and the inaugural Chair Holder of the Paul D. Boyer Professorship in Molecular Biology Biochemistry. He served as director of the UCLA-DOE Institute for Genomics and Proteomics from 1993 to 2014.

Nobel Laureate and founding MBI Director Paul D. Boyer and Lyda Boyer attended the event which was held at the UCLA Hershey Hall Grand Salon.

Professor David Eisenberg was presented the Legacy Award by MBI Director Professor Luisa Iruela-Arispe.

Professor David Eisenberg was presented the Legacy Award by MBI Director Professor Luisa Iruela-Arispe.

Left to right: Lyda Boyer, Professor Paul Boyer, and Lucy Eisenberg.

Left to right: Lyda Boyer, Professor Paul Boyer, and Lucy Eisenberg.

Members of Prof. Eisenberg's group pose for a photo at the event (left to right): Dr. Nicole Wheatley, Dr. Alice Soragni, Dr. Lorena Saelices, Jeannette Bowler, Dr. Rebecca Nelson, Pascal Krotee, Prof. David Eisenberg, Elizabeth Guenther, Sarah Griner, Shannon Essewein, and assistant Cindy Chau.

Members of Prof. Eisenberg’s group pose for a photo at the event (left to right): Dr. Nicole Wheatley, Dr. Alice Soragni, Dr. Lorena Saelices, Jeannette Bowler, Dr. Rebecca Nelson, Pascal Krotee, Prof. David Eisenberg, Elizabeth Guenther, Sarah Griner, Shannon Essewein, and assistant Cindy Chau.

Left to right: Professor Sabeeha Merchant (Chemistry & Biochemistry), Professor Edward De Robertis and Ana De Robertis (Biological Chemistry).

Left to right: Professor Sabeeha Merchant (Chemistry & Biochemistry), Professor Edward De Robertis and Ana De Robertis (Biological Chemistry).

2015 Postdoctoral Awardees

Three Postdocs from MBI Labs were recipients of the 2015 Postdoc Awards.

  • Crysten Blaby from the Merchant lab
  • Luz Orozco from the Pellegrini lab
  • Jose Rodriguez from the Eisenberg lab

The events was held on Oct. 9th at the California NanoSystems Institute (CNSI).

PostDoc Awards 151009

PostDoc Awards 2015

More information can be found here

 

Jose Rodriguez and Luz Orozco

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Crysten Blaby

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