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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 objectives of the facility are:
  1. Provide structural and biophysical 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:
  • Protein Expression and Purification
  • Biophysical Characterization of macromolecules and small molecules
  • Crystallization
  • Diffraction quality
  • Data collections
  • Structure determination
 
 
Research reported in this publication includes 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.

Assessment of authorship will be determined by the level of intellectual input, assay design and data analysis provided by Core leadership and staff members for each project individually based on NIH authorship guidelines.

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.  To complement structural data, the core provides state-of-the-art instrumentation to measure the affinities and kinetics of macromolecules with ligands and/or other macromolecules.  Finally, the core has assembled a 1250 member, fragment library to screen for potential lead compounds through differential scanning fluorimetry, SPR, ITC and diffraction methods.

Sample Analysis and Preparation

Consultation and in silico analysis
• Protein expression and purification (bacteria, insect or mammalian cell expression/ affinity or tag-less purification)
• Domain structure, including disorder and secondary structure prediction

Physical Analysis

• Native and SDS PAGE
• Limited Proteolysis and characterization by SDS-PAGE
• Size Exclusion Chromatography (analytical and preparative grade)
• Sedimentation Equilibrium analysis by Analytical Ultracentrifugation (AUC)
• Sedimentation Velocity analysis by AUC
• Surface Plasmon Resonance (SPR)
• Circular Dichroism (CD) Spectroscopy
• Isothermal Titration Calorimetry (ITC)
• Kinetic Exclusion Assay (KinExA)
• Differential Scanning Fluorimetry (DSF)

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 factorials at 3 protein concentrations and 2 temperatures)

2. Optimization – additive screens, factorial overlays, macro and micro seeding

3. Automated imaging available for4 ºC and 20 ºC )

Diffraction quality

1. Test diffraction quality (loop and capillary mounting available)

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 (~18 microL/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.   It is also useful for formulation of biologics.
   
Waters NanoITC
Isothermal Titaction Calorimetry measures the transfers of thermal energy upon ligand binding.    Rom these measurements, one directly observes the enthalpy and measures the association constant of the interaction.  This in turn affords a measure of the entropy involved in an interaction.  These values provide important information about the nature of an interaction and are useful for drug design efforts.
   
Biacore T100 SPR
The GE Healthsciences Biacore provides kinetic and thermodynamic information for macromolecular interactions (protein-protein, DNA-protein, etc.), ligand-protein interactions, and fragments.
   
Jasco 815 Circular Dichroism
The Jasco 825 CD with fluorescence detection provides information vis-à-vis secondary structure.  Combined with a Peltier thermal control, this instrument is useful for determining melting temperatures and how point mutations affect protein stability.  In addition, the fluorescence detection can be used to determine the affinity protein-protein/protein-DNA interactions, how potential therapeutics affect such interactions, etc.
   
Sapidyne Kinexa 3200
The Sapidyne Kinexa determines binding constants and kinetics of an interaction by measuring the free ligand available (as opposed to SPR which measures the complex).  This allows the Kinexa to measure much tighter affinities (down to 10 femtomolar).
 
 
 
 
 
 
 

Abstract for Grants

The X-ray core at Beckman Research Institute of City of Hope is a state-of-the-art crystallization and X-ray facility for basic and translational science.  To facilitate investigators, the core also provides protein expression and purification as well as numerous biophysical methods to characterize stability, oligomerizaiton, and kinetic and thermodynamic properties.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), To follow crystallization trials, two Formulatrix Imaging Robots at different temperatures 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. A Rigaku 007HF generator,  R-axis IV++ imager and an Oxford cryojet (for data collection at 100 K) is used for in-house diffraction studies and access to SSRL and other synchrotrons is available The facility has all the software and computational hardware necessary to determine structures of macromolecular complexes.
 
In addition, our core facility has capabilities to express and purify large quantities of high quality protein (1 to 100 mg +) using a variety of expression systems (bacteria, insect and mammalian cells). Biophysical characterization of macromolecules and small molecules can be carried out using SPR, ITC, AUC, analytical SEC, KinExa and CD and fluorescent spectroscopy.
 

Pricing

Prices and availability vary. Please contact us or visit our site on iLab Solutions for current information.

 

 

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 objectives of the facility are:
  1. Provide structural and biophysical 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:
  • Protein Expression and Purification
  • Biophysical Characterization of macromolecules and small molecules
  • Crystallization
  • Diffraction quality
  • Data collections
  • Structure determination
 
 
Research reported in this publication includes 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.

Assessment of authorship will be determined by the level of intellectual input, assay design and data analysis provided by Core leadership and staff members for each project individually based on NIH authorship guidelines.

Services

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.  To complement structural data, the core provides state-of-the-art instrumentation to measure the affinities and kinetics of macromolecules with ligands and/or other macromolecules.  Finally, the core has assembled a 1250 member, fragment library to screen for potential lead compounds through differential scanning fluorimetry, SPR, ITC and diffraction methods.

Sample Analysis and Preparation

Consultation and in silico analysis
• Protein expression and purification (bacteria, insect or mammalian cell expression/ affinity or tag-less purification)
• Domain structure, including disorder and secondary structure prediction

Physical Analysis

• Native and SDS PAGE
• Limited Proteolysis and characterization by SDS-PAGE
• Size Exclusion Chromatography (analytical and preparative grade)
• Sedimentation Equilibrium analysis by Analytical Ultracentrifugation (AUC)
• Sedimentation Velocity analysis by AUC
• Surface Plasmon Resonance (SPR)
• Circular Dichroism (CD) Spectroscopy
• Isothermal Titration Calorimetry (ITC)
• Kinetic Exclusion Assay (KinExA)
• Differential Scanning Fluorimetry (DSF)

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 factorials at 3 protein concentrations and 2 temperatures)

2. Optimization – additive screens, factorial overlays, macro and micro seeding

3. Automated imaging available for4 ºC and 20 ºC )

Diffraction quality

1. Test diffraction quality (loop and capillary mounting available)

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

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 (~18 microL/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.   It is also useful for formulation of biologics.
   
Waters NanoITC
Isothermal Titaction Calorimetry measures the transfers of thermal energy upon ligand binding.    Rom these measurements, one directly observes the enthalpy and measures the association constant of the interaction.  This in turn affords a measure of the entropy involved in an interaction.  These values provide important information about the nature of an interaction and are useful for drug design efforts.
   
Biacore T100 SPR
The GE Healthsciences Biacore provides kinetic and thermodynamic information for macromolecular interactions (protein-protein, DNA-protein, etc.), ligand-protein interactions, and fragments.
   
Jasco 815 Circular Dichroism
The Jasco 825 CD with fluorescence detection provides information vis-à-vis secondary structure.  Combined with a Peltier thermal control, this instrument is useful for determining melting temperatures and how point mutations affect protein stability.  In addition, the fluorescence detection can be used to determine the affinity protein-protein/protein-DNA interactions, how potential therapeutics affect such interactions, etc.
   
Sapidyne Kinexa 3200
The Sapidyne Kinexa determines binding constants and kinetics of an interaction by measuring the free ligand available (as opposed to SPR which measures the complex).  This allows the Kinexa to measure much tighter affinities (down to 10 femtomolar).
 
 
 
 
 
 
 

Abstract for Grants

Abstract for Grants

The X-ray core at Beckman Research Institute of City of Hope is a state-of-the-art crystallization and X-ray facility for basic and translational science.  To facilitate investigators, the core also provides protein expression and purification as well as numerous biophysical methods to characterize stability, oligomerizaiton, and kinetic and thermodynamic properties.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), To follow crystallization trials, two Formulatrix Imaging Robots at different temperatures 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. A Rigaku 007HF generator,  R-axis IV++ imager and an Oxford cryojet (for data collection at 100 K) is used for in-house diffraction studies and access to SSRL and other synchrotrons is available The facility has all the software and computational hardware necessary to determine structures of macromolecular complexes.
 
In addition, our core facility has capabilities to express and purify large quantities of high quality protein (1 to 100 mg +) using a variety of expression systems (bacteria, insect and mammalian cells). Biophysical characterization of macromolecules and small molecules can be carried out using SPR, ITC, AUC, analytical SEC, KinExa and CD and fluorescent spectroscopy.
 

Pricing

Pricing

Prices and availability vary. Please contact us or visit our site on iLab Solutions for current information.

 

 
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.
 
 
 
 
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800-888-5323
dgrignetti@coh.org

 

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213-241-7160
ssprester@coh.org

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213-241-7112
cnassr@coh.org

 
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