University of Colorado Cancer Center Structural Biology Shared Core Service

Structure of the Progesterone Receptor DNA Binding domain
bound to DNA.

The Structural Biology Core provides UCCC researchers access to instrumentation and expertise in the use of X-ray Crystallography and Nuclear Magnetic Resonance Spectroscopy. The Core aims to promote structural studies in the molecular basis of cellular processes that regulate the development and progression of cancer. We enable member access to crystallization facilities, X-ray crystallography facilities, and Synchrotron X-ray data collection.

Services Offered

Structure Determination of Macromolecules (Proteins/Nucleic Acids)

Structure determination of macromolecules is performed in collaboration with Core faculty. Faculty and staff consult with investigators in the project design, protein or nucleic acid expression, purification and sample preparation. The core staff performs data collection and data processing and work with investigators to solve and refine structures.

Preliminary Crystallization Screening

The core faculty provides initial screens of crystallization conditions using commercially available and custom designed sparse matrix screens. Core faculty members analyze the results of these initial screens to provide guidance in designing or extending the screens to produce crystals for X-ray diffraction studies.

Preliminary Crystal Screening and Data Collection 

X-ray Crystal Structure of LUSH-cVA complex. 
The VA is completely enveloped within the protein

Core faculty provide initial data collection on crystals to evaluate project viability. These services provide investigators further directions to improve crystal quality with the goal of a complete structure determination. If high-quality crystals are obtained Core staff collect complete data sets that may be used for structure determination.

Solution structural analysis of small molecules

The NMR Core collects data and provides interpretation of that data to users studying small molecules such as potential drugs and other therapeutic agents. Data collected includes simple one-dimensional (1D) ¹H, ¹³C and ³¹P spectra and two-dimensional (2D) COSY, NOESY, ROESY, TOCSY and HSQC correlation spectra.

Molecular Visualization

Core staff provide training and consultation in the use of molecular graphics programs to display and analyze molecular structures. The Structural Biology Core has 3 Silicon Graphics computers equipped with three-dimensional viewing hardware and software for this purpose.

Experimental Design and Training

Faculty provide consultation to Cancer Center members and other investigators on the appropriate design of experimental conditions for structural analysis. For example this includes design of protein constructs, choice of buffer systems, appropriate methods for data collection. Faculty provide training to PIs, post-doctoral and student researchers in the techniques of sample preparation, data collection, data processing and data interpretation.

Equipment

X-ray Instrumentation

• Rigaku/MSC RUH3R x-ray generator equipped with confocal optics
  

  Varian INOVA 500 MHz Spectrometer

• RAXIS IV ++ x-ray area detector
• Rigaku/MSC X-STREAM cryo-cooling apparatus
• RAXIS IV++ area detector
• Confocal optics
• MSC X-STREAM cryo-cooling apparatus

NMR Instrumentation

• Varian INOVA 500 MHz system 
  • 5 mm triple resonance 1H/13C/15N probe with Z-axis gradient with sensitivity of 850:1 (ETB) 
  • 3 mm triple resonance 1H/13C/15N probe with Z-axis gradient with ETB sensitivity of 850:1
• Varian INOVA 600 MHz system
  • 5 mm triple resonance 1H/13C/15N probe with Z-axis gradient 
  • 5mm triple resonance  1H/13C/15N cryogenically cooled probe with Z-axis gradients
• Varian INOVA 800 MHz system 
  • 5 mm triple resonance 1H/13C/15N probe with Z-axis gradient 
• Varian 900 MHz Direct Drive NMR system 
  • 5 mm triple resonance 1H/13C/15N probe with Z-axis gradient