Access to experimental X-ray crystallographic data can further structural understanding in the following ways: 1) It provides a huge resource of information that does not require expertise in crystallography to utilize. 2) It helps establish reasonable expectations from X-ray structures by allowing visualization of electron density maps. 3) When combined with software tools, it can serve as a powerful technique for structure-based drug discovery.

1. How can access to experimental X-
ray data further our understanding
of structures used in structure
based studies?
Francisco Hernandez-Guzman, Ph.D.
Product Manager, Life Sciences
ACS 2010 – San Francisco, CA
March 22, 2010

2. Agenda
• Goals
• Background
– X-ray crystallography 101
• Expectations
– Theoretical & Experimental
• Methods for analysis:
– Map theory
– Resolution
– 2Fo-Fc vs. Fo-Fc vs. –(Fo-Fc)
• Examples
• Custom protocols
• Conclusion
• Acknowledgements/References
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3. Goals
Access to experimental structural data can:
1. Be a HUGE resource of information
• DO NOT need to be an expert crystallographer to use this information
2. Help establish reasonable expectations from X-ray structures
3. Right mix technology  powerful tool for structure base discovery
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4. Background:
X-ray crystallography 101 for structure solution:
1
-987
115.986 115.986 44.151 90.000
90.000 120.000 p6
1 0 0 -0.2 0.4
2 -1 0 0.2 0.6
2 -1 2 2471.3 119.7
2 0 1 3166.3 237.6 2602.5 113.7
2 0 2 3257.2 112.9 3532.6 154.7
3 -2 0 895.5 32.6
3 -2 1 3618.7 120.2 5126.5 180.6
3 -2 2 3016.3 106.0 2722.1 122.3
3 -2 3 1124.7 42.6 1003.7 35.4
3 -2 4 6620.5 419.3 7735.3 386.1
3 -1 1 4332.0 425.0
3 -1 2 674.4 17.9 102.5 8.1
3 -1 3 174.2 12.3 52.7 11.9
3 -1 4 15478.4 827.1 12906.3 844.7
3 0 1 10052.2 496.7 10107.3 504.2
3 0 2 15486.2 469.7 15548.0 590.2
3 0 3 4730.2 167.5 4454.7 188.8
Steps: 3 0 4 9840.6 543.6 8136.2 375.4
4 -3 1 23731.2 553.5 20271.4 584.9
• Crystallization (apo or with ligand)
X-ray data model
• Data Collection
• Data Processing
• Phase Determination
• Model Building and Refinement
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5. Points to remember from X-ray steps
• Crystallization:
– Molecules are in the crystal form
• Packing lattice
• pH
• Additives
• Temperature
• Molarity/concentration
• Data collection
– High energy X-rays
• Room temperature vs Cryo x-ray data collection
• Radiation Damage
• Data processing
– Data completion/redundancy
• Space group
– Data resolution
– B-factors
• Phase Determination
– Molecular replacement
– Experimental phasing (isomorphous replacement, anomalous scattering)
• Model Building
– Automated tools and manual tools
• QUANTA, CNX, DS*, PP*, COOT and others. *DS version 2.5.5 and PP version 7.5.4
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6. Other important points to remember
• X-ray structures are models!!!
– Guided by experimental observations
– Subject to interpretation
– Not immune to error
– Hydrogens are usually missing…
• Observations limited to available data
– Reliability depends on questions asked
– Need to clearly define the expectations properly
© 2008 Accelrys, Inc. 6

7. Expectations
THEORETICAL:
• Reasonable representation of the “biologically active” molecule
• Static representations are significant
– Structures  Space and Time averaged
– Protein dynamics can be “simplified”
• End point structures (before and after) can provide useful
information
EXPERIMENTAL:
• Solved structures match experimental observations
• Structures are biologically and chemically meaningful
• Protein-ligand interactions are properly modeled
© 2008 Accelrys, Inc. 7

8. Methods for Analysis: Map theory
• Electron density map equation:
• In the case of standard model based maps:
|Fhkl|e2πiФhkl
Calculated phases from model
Experiment
• So in theory as Fc  Fo:
ρ ≈ 2Fo – Fc ≈ Fo and ρ ≈ Fo – Fc ≈ 0
R = Σ||Fo| – Fc||/ Σ|Fo|
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9. Methods for Analysis: Resolution
• 2Fo-Fc as a function of resolution: (1r2q.pdb)
low high
4.0 Å 3.0 Å 2.0 Å 1.05 Å
In general, level of detail and confidence increases as a function of resolution
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10. Methods for Analysis: 2Fo-Fc, Fo-Fc, & -(Fo-Fc)
• Map analysis of a misplaced residue:
2Fo-Fc
Fo-Fc
-(Fo-Fc)
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11. Example: 1 – questionable chemistry
• 1LEE.pdb (1.9 Å)
RS367 K238
2Fo-Fc Fo-Fc Fo-Fc
? ?
RS370 R238 + SO4
2Fo-Fc Fo-Fc Fo-Fc
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12. Example: 2 – questionable ligand density
• 1nhu (2.0 Å)
Fo-Fc
-(Fo-Fc)
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13. Example: 3 – questionable structure
• 2q76 (B:134 – B:137) ( 2.0 Å)
• Unexplained Fo-Fc density
• Incomplete B:Gln134
• Ramachandran inconclusive
•R= 0.199 (pdb - 0.198)
•R-free= 0.239 (pdb - 0.233)
vs.
•R=0.197
•R-free= 0.230
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14. Example: 3 – questionable structure
• 2q76 (B:134 – B:137) ( 2.0 Å)
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15. Example: 4 – SBDD - Kinases
• 1nu3 (1.75 Å) – bad ligand geometry… 147.49° vs. 104°
2Fo-Fc
Fo-Fc -(Fo-Fc)
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16. Automatic ED Map Generation
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17. Advanced PDB search functionality in DS
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18. Interactive Kinome Viewer
• Kinome Viewer: http://accelrys.com/events/webinars/human-kinome/
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19. Conclusion
1. Crystal structures are time and space averaged models
based on experimental data
• Structures not immune to error
2. Access to X-ray data is critical for assessing
expectations of crystal structures
• Electron Density map visualization is a very useful and powerful tool
3. Software solutions should provide insights into further
discovery
• Discovery Studio / Pipeline Pilot
• Custom protocols
 Kinome Viewer
 RCSB Search
 Automatic ED Map Generation
© 2008 Accelrys, Inc. 19

20. Acknowledgements and References
• Yi-Shiou Chen, Ph.D.  X-ray components and protocols
• Guillaume Paillard, Ph.D. Kinome Viewer
• NOJ Malcom, Ph.D.  Auto ED Map Generation (wrapping)
• Accelrys R&D and Marketing
• H.M. Berman, K. Hendrick, H. Nakamura. (2003) Announcing the worldwide
Protein Data Bank. Nature Structural Biology 10(2):980
• Davids, E. et al. (2008). Drug Discovery Today. 13(19-20), 831-841
• Gert Vried, Radbound Univ. Nijmegen Med. Centre, The Netherlands.
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