How can access to experimental X-ray data further our understanding of structures used in structure...
-
Upload
biovia -
Category
Technology
-
view
627 -
download
0
description
Transcript of How can access to experimental X-ray data further our understanding of structures used in structure...
![Page 1: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/1.jpg)
Francisco Hernandez-Guzman, Ph.D.
Product Manager, Life Sciences
ACS 2010 – San Francisco, CA
March 22, 2010
How can access to experimental X-ray data further our understanding
of structures used in structure based studies?
![Page 2: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/2.jpg)
© 2008 Accelrys, Inc. 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
![Page 3: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/3.jpg)
© 2008 Accelrys, Inc. 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
![Page 4: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/4.jpg)
© 2008 Accelrys, Inc. 4
Background:
X-ray crystallography 101 for structure solution:
Steps:
• Crystallization (apo or with ligand)
• Data Collection
• Data Processing
• Phase Determination
• Model Building and Refinement
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
3 0 4 9840.6 543.6 8136.2 375.4
4 -3 1 23731.2 553.5 20271.4 584.9
X-ray data model
![Page 5: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/5.jpg)
© 2008 Accelrys, Inc. 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
![Page 6: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/6.jpg)
© 2008 Accelrys, Inc. 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
![Page 7: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/7.jpg)
© 2008 Accelrys, Inc. 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
![Page 8: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/8.jpg)
© 2008 Accelrys, Inc. 8
Methods for Analysis: Map theory
• Electron density map equation:
• In the case of standard model based maps:
• So in theory as Fc Fo:
|Fhkl|e2πiФhkl
ExperimentCalculated phases from model
ρ ≈ 2Fo – Fc ≈ Fo and ρ ≈ Fo – Fc ≈ 0
R = Σ||Fo| – Fc||/ Σ|Fo|
![Page 9: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/9.jpg)
© 2008 Accelrys, Inc. 9
Methods for Analysis: Resolution
• 2Fo-Fc as a function of resolution: (1r2q.pdb)
4.0 Å
low high
3.0 Å 2.0 Å 1.05 Å
In general, level of detail and confidence increases as a function of resolution
![Page 10: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/10.jpg)
© 2008 Accelrys, Inc. 10
Methods for Analysis: 2Fo-Fc, Fo-Fc, & -(Fo-Fc)
• Map analysis of a misplaced residue:
2Fo-Fc
Fo-Fc
-(Fo-Fc)
![Page 11: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/11.jpg)
© 2008 Accelrys, Inc. 11
Example: 1 – questionable chemistry
• 1LEE.pdb (1.9 Å)
??
K238
R238 + SO4
RS367
RS370
2Fo-Fc
2Fo-Fc
Fo-Fc
Fo-Fc
Fo-Fc
Fo-Fc
![Page 12: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/12.jpg)
© 2008 Accelrys, Inc. 12
Example: 2 – questionable ligand density
• 1nhu (2.0 Å)
Fo-Fc
-(Fo-Fc)
![Page 13: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/13.jpg)
© 2008 Accelrys, Inc. 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
![Page 14: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/14.jpg)
© 2008 Accelrys, Inc. 14
Example: 3 – questionable structure
• 2q76 (B:134 – B:137) ( 2.0 Å)
![Page 15: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/15.jpg)
© 2008 Accelrys, Inc. 15
Example: 4 – SBDD - Kinases
• 1nu3 (1.75 Å) – bad ligand geometry… 147.49° vs. 104°
2Fo-Fc
Fo-Fc -(Fo-Fc)
![Page 16: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/16.jpg)
© 2008 Accelrys, Inc. 16
Automatic ED Map Generation
![Page 17: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/17.jpg)
© 2008 Accelrys, Inc. 17
Advanced PDB search functionality in DS
![Page 18: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/18.jpg)
© 2008 Accelrys, Inc. 18
Interactive Kinome Viewer
• Kinome Viewer: http://accelrys.com/events/webinars/human-kinome/
![Page 19: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/19.jpg)
© 2008 Accelrys, Inc. 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
![Page 20: How can access to experimental X-ray data further our understanding of structures used in structure based studies?](https://reader035.fdocuments.us/reader035/viewer/2022081403/5562d7d8d8b42a6c498b5199/html5/thumbnails/20.jpg)
© 2008 Accelrys, Inc. 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.