Facilities/Equipment

X-ray diffraction

The crystallization facility, housed in the basement of the building, and led by Dr. Dave McKay, has a Phenix (Art Robbins) crystallization robot and two temperature controlled (20 and 4 ºC) Formulatrix RockImagers for automated crystallization image evaluation .

The Department  recently obtained a Rigako XtaLAB MM003 home source, equipped with a Dectris PILATUS 200K 2D hybrid pixel array area detector.

We have access to the HHMI Beamline at the ALS Berkeley, and at the SAXS beamline at the APS, through collaboration with a former Luger Lab postdoc, Srinivas Chakravarthy.

Analytical Ultracentrifugation

Analytical ultracentrifugation has become the method of choice to analyze structural transitions in chromatin. The method is based on first principles and allows a wide variation of solute conditions. Information on molecular weight, shape, and sample homogeneity can be obtained, using the Ultrascan software packages

Our Beckmann XLA ultracentrifuge is equipped with an Aviv Fluorescence detection system, allowing us to detect labeled compounds at low nanomolar concentration in a complex mixture.

Atomic Force Microscopy

The Atomic Force Microscope (AFM) is a device used to image materials and biological samples at the single-molecule level. It uses an extremely fine tip positioned at the end of a cantilevered bar.

As the tip is moved over the substrate, it periodically taps the surface and bends as it is repelled or attracted to the structure. A laser picks up the deflections, resulting in a direct three-dimensional image of single molecules. Although the resolution of these images does not reach that of structures obtained by x-ray crystallography, a major advantage of this approach lies in the ability to visualize single macromolecules under a variety of conditions.

Our Asylum MFP-3D™ Atomic Force Microscope  is highly suitable to image and manipulate single biological macromolecules in aqueous environments. Single molecules are ‘pulled’ and the forces that are required for defined structural transitions are measured precisely, yielding information on the stability of intermolecular and intramolecular interactions.

ITC

We use isothermal calorimetry (MicroCal VP-ITC) to determine binding constants of protein protein and protein – ligand interactions.

Fluorescence Spectroscopy

Fluorescence spectroscopy is used to investigate the thermodynamics of nucleosomes and chromatin (Stopped flow: Applied Photophysics SX20; Fluorometer: HoribaJobinYvon Fluorolog 3).

We have access to two brandnew Typhoon phosphorimagers; these are used for plate assays as well as for scanning of gels with fluorescent components.

Light Scattering

Wyatt Technologies Dawn Heleos II light scattering device connected to an AKTA HPLC system allows us to analyze the molecular weight of components from complex mixtures, as they elute from a gel filtration column (SEC-MALS). This allows us to rapidly determine the stoichiometry of macromolecular complexes.

We use a Wyatt Calypso II for rapid, quantitative characterization of protein-protein and other macromolecular interactions with composition-gradient multi-angle light scattering.

Hydrogen-Deuterium Exchange Coupled to Mass Spectrometry (HDX-MS)

We use HDX-MS to map protein-protein and protein-DNA interfaces, and to monitor structural changes in chromatin-associated proteins. These experiments are performed in collaboration with Dr. Natalie Ahn and the CU Boulder Mass Spectrometry Facility.