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X-ray Crystallography Core Facility

X-ray Crystallography Core Facility
The X-ray Crystallography Core Facility is one of the Shared Resources facilities at Beckman Research Institute of City of Hope.
 
The objective of the facility is to:
 
  1. Provide structural information to understand mechanisms of biological systems at the atomic level;
  2. Validate binding sites of lead compounds of therapeutic interest;
  3. Facilitate lead discovery through co-crystallization of therapeutic targets and small molecule libraries.
     
Services provided include:
 
  • Sample analysis
  • Crystallization
  • Diffraction quality
  • Data collections
  • Structure determination
 
Contact Us:
 
John C. Williams, Ph.D., Director
Phone:  626-256-HOPE (4673), ext. 60227
City of Hope
1500 East Duarte Road
Duarte, CA  91010-3000
Flower Building, Room 148
 
Research reported in this publication included work performed in the X-ray Crystallography Core supported by the National Cancer Institute of the National Institutes of Health under award number P30CA33572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Services

The X-ray crystallography Core provides a state-of-the-art facility for the generation of crystals and structure determination of macromolecules including proteins, DNA, RNA, and complexes between macromolecules and their ligands.
 
Sample Analysis:
 
Consultation andin silicoanalysis
  • Protein expression and purification
  • Domain structure, including disorder and secondary structure prediction
 
Physical Analysis
 
  • Native PAGE using GE HealthSciences PhastGel
  • Limited Proteolysis and characterization by SDS-PAGE
  • Size Exclusion Chromatography
  • Sedimentation Equilibrium analysis by AUC
  • Sedimentation Velocity analysis by AUC.
 
Crystallization:
 
  1. Set up crystallization trials at 4 ºC and/or 20 ºC.
    a.Initial trials (e.g., appropriate concentrations)
    b.Full scale trials (4 different 96 well factorialsat 3 protein concentrations and 2 temperatures)
     
  2. Optimization – additive screens and factorial overlays
     
  3. Automated Screening at 2 temperatures (scan daily forfirst week, weekly thereafter, finish experiment at 3 months)
 
Diffraction quality:
 
  1. Test diffraction using capillary mounted crystal
     
  2. Test/screen cryo-conditions
 
Data collection:
 
  1. Collect, reduce and merge data. Generate table of statistics
  2. MAD/SAD phasing – help design, collect, reduce and merge MAD data
  3. Generate table of statistics including anomalous dispersion differences
 
Structure Determination:
 
  1. Solve structure by Molecular Replacement
  2. Solve structure by MAD/SAD phasing
  3. Refine structure
  4. Produce relevant statistics (e.g., R and Rfree, RMS deviations)
  5. Structural analysis (superpositions, electrostatics, etc)
  6. Deposit Structure at PDB
 

Equipment

The facility houses a Mosquito Crystallization Robot, a Formulatrix imaging robot (with Automated Crystal Screening at 4 ºC and 20 ºC), a Rigaku Micromax 007 with an R-AxisIV++, and an Oxford cryojet and relevant software and computational hardware for structure determination.
 
The facility also houses a Beckman XLI analytical ultracentrifuge and a GE Health Phast Gel system for the characterization of macromolecular properties.
 
Mosquito Crystallization Robot
The mosquito crystallization robot permits hanging drop, sitting drop, and batch methods in a 96 well format using minimal amounts of protein (~16mL/trail). A number of commercial crystallization screens are available (Qiagen, Hampton Research, Jena).
   
Formulatrix Automated Visualization of Crystallization Trials
The core houses two Formulatrix imaging robots for visualization at 4 ºC and 20 ºC.
Users can quickly browse images, score potential hits and follow crystal growth in time.
   
Rigaku X-ray Diffractometer
Micromax 007 HF, R-axis V++, and Oxford cryojets allow full structure determination.
   
Beckman XL-I Proteomelab
The Beckman Analytical ultracentrifuge affords accurate measurement of hydrodynamic properties, including the association constant of macromolecular complexes, as well as small molecule-macromolecular interactions
 
 

Abstract for Grants

The X-ray core atBeckman Research Institute of City of Hope is a state-of-the-art crystallization and X-ray facility. This facility houses a Mosquito Crystallization robot that uses 50 to 100 nL volumes and permits three different crystallization formats; hanging drop, sitting drops or batch methods under oil. The facility has multiple commercial crystallization factorials (e.g., Hampton, Jena, Nextal), that are frequently used, such that they are always fresh. It has also developed protocols for rapid optimization of individual conditions.
 
To follow crystallization trials, two Formulatrix Imaging Robots are used that automatically image each drop, according to a preset schedule (e.g., nightly). This allows follow-up of crystallization trends at two temperatures and produces a visual record for analysis.
 
For diffraction studies, a Rigaku 007HF generator with an R-axis IV++ imager has been purchased. An Oxford cryojet permits data collection at 100 K to reduce radiation damage.
 
The facility has all the software and computational hardware necessary to determine structures of macromolecular complexes.
 

Pricing

Prices and availability vary. Please contact us for current information.
 
If you are a City of Hope employee, please visit the new intranet site for pricing.

 

 
Research Shared Services

City of Hope embodies the spirit of scientific collaboration by sharing services and core facilities with colleagues here and around the world.
 

Recognized nationwide for its innovative biomedical research, City of Hope's Beckman Research Institute is home to some of the most tenacious and creative minds in science.
City of Hope is one of only 41 Comprehensive Cancer Centers in the country, the highest designation awarded by the National Cancer Institute to institutions that lead the way in cancer research, treatment, prevention and professional education.
Learn more about City of Hope's institutional distinctions, breakthrough innovations and collaborations.
Support Our Research
By giving to City of Hope, you support breakthrough discoveries in laboratory research that translate into lifesaving treatments for patients with cancer and other serious diseases.
 
 
 
 
Media Inquiries/Social Media

For media inquiries contact:

Dominique Grignetti
800-888-5323
dgrignetti@coh.org

 

For sponsorships inquiries please contact:

Stefanie Sprester
213-241-7160
ssprester@coh.org

Christine Nassr
213-241-7112
cnassr@coh.org

 
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