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.

DMM Accident

Chemical & Engineering News May 12, 1997


CHEMICAL SAFETY

Handling dimethylmercury

We report a case of severe mercury toxicity resulting from a
single exposure to dimethylmercury.  Review of research
notes interviews, hair analysis, and statements made by the
patient established the circumstances and events described
here.  Testing of the type of gloves worn by the patient
supports the hypothesis that dimethylmercury rapidly
penetrated them, resulting in transdermal exposure.

It appears that there was only one acute exposure to
dimethylmercury.  The patient recounted spilling one or
several drops (estimated to total 0.1 to 0.5 ml) on
disposable latex gloves during a transfer procedure in a
fume hood while preparing a mercury nuclear magnetic
resonance (Hg NMR) standard.  A severely toxic dose of 100
to 200 mg of mercury absorbed requires absorption of less
than 0.1 ml of liquid (density 3 g per ml.).  The
possibility of inhalation exposure (the vapor pressure at
20C is 50 torr is considered highly unlikely given the brief
time the material was handled, the use of the fume hood,
and the high concentration in the patient's body.

                A profile of the mercury content along a
15cm length of the patient s hair revealed what was probably
a single, large exposure to mercury in mid-August 1996, in
accord with a review of research notes and interviews with
colleagues.  Approximately THREE MONTHS later, the patient
experienced episodes of nausea and vomiting spaced weeks
apart.  Approximately five months after exposure, the
patient noted the onset of ataxia (difficulty with balance),
dysarthria (slurred speech), loss of vision, and loss of
hearing.  Medical evaluation at this time revealed a whole
blood mercury concentration of 4,000 ,ug per L80 times the
usual toxic threshold (50 ,ug per L) and markedly above the
normal range (<10 ,ug per L).

The patient's symptoms progressed rapidly over approximately
three weeks to cognitive deficits and coma.  Chelation
increased the rate of elimination of mercury from the body,
but without clinical improvement.  Whole blood and urine
testing of family members and laboratory coworkers revealed
no other abnormal mercury levels.  Air samples from the
patient's laboratory, office, and home revealed detectable
levels of mercury only near the sealed mercury waste can in
the laboratory hood.

      Permeation tests done by an independent testing
laboratory found that dimethylmercury penetrates disposable
latex gloves in 15 seconds or less, and perhaps
instantaneously.  Individuals working with alkyl mercury
compounds should employ cautions similar to those described
in 'prudent Practices in the Laboratory" (National Research
Council, 1995) for highly toxic substances.

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.  Latex or
PVC gloves have an important role in many laboratory
activities, but they are not suitable for significant,
direct contact with aggressive or highly toxic chemicals.
Medical surveillance measuring mercury concentrations in
whole blood or urine should be considered for repeated or
extended use of alkyl mercury compounds.  In all:  cases,
:the potential hazards associated with dimethylmercury and
related alkyl mercury compounds must not be underestimated.

All laboratories working with such compounds are strongly
encouraged to conduct an assessment of existing work
practices and precautions.  We urge the Hg NMR community to
consider a safer standard compound.

Michael B. Blayney
Environmental Health & Safety
Dartmouth College

John S.  Wiinn
Department of Chemistry
Dartmouth College

David W.  Nierenberg
Departments of Medicine and
Pharmacology/Toxicology
Dartmouth Medical School

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.

DMM Safety Sheet

 

University of California  -  MSDS System              PAGE    1
                                                                        05/16/95
Source of MSDS: VWR SCIENTIFIC CORPORATION

                          MATERIAL SAFETY DATA SHEET

                            EASTMAN KODAK COMPANY
                               343 STATE STREET
                          ROCHESTER, NEW YORK 14650

FOR EMERGENCY HEALTH, SAFETY, AND ENVIRONMENTAL INFORMATION, CALL 716-722-51
FOR ALL OTHER PURPOSES, CALL 800-225-5352, IN NEW YORK STATE CALL 716-458-40

DATE OF PREPARATION:  08/22/86                 KODAK ACCESSION NUMBER:  9083

============================================================================
SECTION I.  IDENTIFICATION

   -  PRODUCT NAME: DIMETHYL MERCURY
   -  SYNONYM(S): METHYL MERCURY
   -  FORMULA: C2 H6 HG
   -  CAT NO(S): 119 8050; 119 8068; 119 8076
   -  CHEM. NO(S): 08340
   -  KODAK'S INTERNAL HAZARD RATING CODES: R:  3    S:  3    F:  3    C:  0
============================================================================
SECTION II.  PRODUCT AND COMPONENT HAZARD DATA
                                                        ACGIH
  COMPONENT(S):                         PERCENT         TLV(R)    CAS REG. N

  DIMETHYL MERCURY                      CA. 100       0.01 MG/M3     593-74-
============================================================================
SECTION III.  PHYSICAL DATA

   -  APPEARANCE: COLORLESS LIQUID
   -  BOILING POINT: 93 C (199 F)
   -  VAPOR PRESSURE: NOT AVAILABLE
   -  EVAPORATION RATE (N-BUTYL ACETATE = 1): NOT AVAILABLE
   -  VOLATILE FRACTION BY WEIGHT: CA. 100 %
   -  SPECIFIC GRAVITY (WATER = 1): 3.19
   -  SOLUBILITY IN WATER (BY WEIGHT): NEGLIGIBLE
============================================================================
SECTION IV.  FIRE AND EXPLOSION HAZARD DATA

   -  FLASH POINT: -4 C (24 F) SETAFLASH CLOSED CUP
   -  EXTINGUISHING MEDIA: WATER SPRAY; DRY CHEMICAL; CARBON DIOXIDE; FOAM
   -   SPECIAL FIRE FIGHTING PROCEDURES: WEAR SELF-CONTAINED BREATHING
      APPARATUS AND PROTECTIVE CLOTHING. USE WATER SPRAY TO KEEP FIRE-EXPOSE
      CONTAINERS COOL.
   -  UNUSUAL FIRE AND EXPLOSION HAZARDS: DANGER, EXTREMELY FLAMMABLE, VAPOR
      MAY CAUSE FLASH FIRE. VAPORS ARE HEAVIER THAN AIR AND MAY TRAVEL ALONG
      THE GROUND OR MAY BE MOVED BY VENTILLATION TO AN IGNITION SOURCE AND
      MAY FLASH BACK. FIRE OR EXCESSIVE HEAT MAY PRODUCE HAZARDOUS
      DECOMPOSITION PRODUCTS.
============================================================================
      R-0142.400A                                                  86-6876

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.

 

nature_pr_coverlarvae_x1 nature_pr_slides_final_diffraction_x1 nature_pr_slides2_toxiccycle05

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

forDOEpage2x

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).