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?

© 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

© 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

© 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

© 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

© 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

© 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

© 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|

© 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

© 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)

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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

© 2008 Accelrys, Inc. 12

Example: 2 – questionable ligand density

• 1nhu (2.0 Å)

Fo-Fc

-(Fo-Fc)

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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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Example: 3 – questionable structure

• 2q76 (B:134 – B:137) ( 2.0 Å)

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Example: 4 – SBDD - Kinases

• 1nu3 (1.75 Å) – bad ligand geometry… 147.49° vs. 104°

2Fo-Fc

Fo-Fc -(Fo-Fc)

© 2008 Accelrys, Inc. 16

Automatic ED Map Generation

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Advanced PDB search functionality in DS

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Interactive Kinome Viewer

• Kinome Viewer: http://accelrys.com/events/webinars/human-kinome/

© 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

© 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.