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Enhancing Crystallographic Data Collection: Best Practices for Structure Determination

Learn how to optimize data collection for crystallography, understand solvability thresholds, correct density errors, and improve signal-to-noise ratios. Explore the challenges and techniques for successful structure determination.

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Enhancing Crystallographic Data Collection: Best Practices for Structure Determination

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  1. PowerPoint File available: http://bl831.als.lbl.gov/ ~jamesh/powerpoint/ ECM_SvN_2012.ppt

  2. Acknowledgements Chris Neilson Michael Blum Joe Ferrara Meitian Wang ALS 8.3.1 creator: Tom Alber 8.3.1 PRT head: Jamie Cate Center for Structure of Membrane Proteins Membrane Protein Expression Center II Center for HIV Accessory and Regulatory Complexes W. M. Keck Foundation Plexxikon, Inc. M D Anderson CRC University of California Berkeley University of California San Francisco National Science Foundation University of California Campus-Laboratory Collaboration Grant Henry Wheeler The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the US Department of Energy under contract No. DE-AC02-05CH11231 at Lawrence Berkeley National Laboratory.

  3. Getting the Best Data from Your Crystal aka: Getting anything out of Your Crystals!

  4. “Success rate” of structures ~2500 datasets/year ~100 PDBs/year

  5. signalvsnoise

  6. signalvsnoise

  7. signalvsnoise “If you don’t have good data,then you have no data at all.” -Sung-Hou Kim

  8. signalvsnoise easy hard impossible

  9. signalvsnoise easy hard impossible threshold of “solvability”

  10. MR simulation corrupted data Correlation coefficient to correct density Signal to noise ratio

  11. MR simulation corrupted model Correlation coefficient to correct density Rmsd from perfect search model (Å)

  12. threshold of “solvability” SAD phasing simulation mlphare results Correlation coefficient to correct density Anomalous signal to noise ratio

  13. signalvsnoise “If you don’t have good data,then you must learn statistics.” -James Holton

  14. Adding noise

  15. Adding noise 12 + 12 = 1.42

  16. Adding noise 12 + 12 = 1.42 32 + 12 = 3.22 σtotal2 = σ12 + σ22

  17. Adding noise 12 + 12 = 1.42 32 + 12 = 3.22 σtotal2 = σ12 + σ22

  18. Adding noise 12 + 12 = 1.42 32 + 12 = 3.22 σtotal2 = σ12 + σ22

  19. Adding noise 12 + 12 = 1.42 32 + 12 = 3.22 102 + 12 = 10.052

  20. What is holding us back? ( if not rad dam! ) • Weak spots (high-res) background • MAD/SAD (small differences) fractional errors

  21. $100,000.00 $100,000.00 $100,000.00 $100,000.00 $100,000.00 $100,000.00 $100,000.00 $100,000.00 Background scattering real estate is expensive use it!

  22. Background scattering

  23. Σ Vxtal VASU a Diffuse scattering R. W. James (1962) Ids = Ibeam t re2 P A |fa(s)|2(1-exp(-2Ba∙s2)) Ids - scattered photons/steradian Ibeam - incident (photons/s/m2 ) t - exposure time (s) re - classical electron radius (2.818x10-15 m) Vxtal - volume of crystal (in m3) VASU - asymmetric unit (in m3) P - polarization factor A - attenuation factor a - particular atom in the ASU fa(s) - atomic structure amplitude (electrons) s - scattering length (sin(θ)/λ) Ba- atomic B factor

  24. background background Fine Slicing Pflugrath, J. W. (1999)."The finer things in X-ray diffraction data collection", Acta Cryst. D55, 1718-1725.

  25. Dose slicing unacceptable damage crystal’s useful life N photons N photons unacceptable read noise N photons

  26. adjust exposure so this is ~100 Optimal exposure time(faint spots) thr Optimal exposure time for data set (s) tref exposure time of reference image (s) bgref background level near weak spots on reference image (ADU) bg0 ADC offset of detector (ADU) bghr optimal background level (via thr) σ0rms read-out noise (ADU) gain ADU/photon m multiplicity of data set (including partials)

  27. Multi-crystal strategies Kendrew et al. (1960) "Structure of Myoglobin” Nature185, 422-427.

  28. What is holding us back? ( if not rad dam! ) • Weak spots (high-res) background • MAD/SAD (small differences) fractional errors

  29. anomalous signal √ ΔF F # sites MW (Da) ≈1.2 f” World record! ΔF/F = 0.5% Wang, Dauter & Dauter (2006) Acta Cryst. D62, 1475-1483. Crick, F. H. C. & Magdoff, B. S. (1956) Acta Crystallogr.9, 901-908. Hendrickson, W. A. & Teeter, M. M. (1981) Nature290, 107-113.

  30. 0.1% error? Jenkins et al. (2009)."Evidence of correlations between nuclear decay rates and Earth–Sun distance", Astroparticle Physics32, 42-46.

  31. 0.1% error? Jenkins et al. (2009)."Evidence of correlations between nuclear decay rates and Earth–Sun distance", Astroparticle Physics32, 42-46.

  32. 238Pu decay rate in space Cooper, P. S. (2009)."Searching for modifications to the exponential radioactive decay law with the Cassini spacecraft", Astroparticle Physics31, 267-269.

  33. Fractional error • no “scale factor” is perfectly known • no source of light is perfectly stable • no shutter is perfectly reproducible • no crystal is perfectly still • no detector is perfectly calibrated

  34. Fractional error • no “scale factor” is perfectly known • no source of light is perfectly stable • no shutter is perfectly reproducible • no crystal is perfectly still • no detector is perfectly calibrated

  35. Intensity of a Bragg spot Ifull≈ |F(hkl)|2

  36. Vxtal λ3 L Vcell ωVcell Darwin’s Formula I(hkl) = Ibeam re2 P A | F(hkl) |2 I(hkl) - photons/spot (fully-recorded) Ibeam - incident (photons/s/m2 ) re - classical electron radius (2.818x10-15 m) Vxtal - volume of crystal (in m3) Vcell - volume of unit cell (in m3) λ - x-ray wavelength (in meters!) ω - rotation speed (radians/s) L - Lorentz factor (speed/speed) P - polarization factor (1+cos2(2θ) -Pfac∙cos(2Φ)sin2(2θ))/2 A - attenuation factor exp(-μxtal∙lpath) F(hkl) - structure amplitude (electrons) C. G. Darwin (1914)

  37. Vxtal λ3 L Vcell ωVcell Darwin’s Formula I(hkl) = Ibeam re2 P A | F(hkl) |2 I(hkl) - photons/spot (fully-recorded) Ibeam - incident (photons/s/m2 ) re - classical electron radius (2.818x10-15 m) Vxtal - volume of crystal (in m3) Vcell - volume of unit cell (in m3) λ - x-ray wavelength (in meters!) ω - rotation speed (radians/s) L - Lorentz factor (speed/speed) P - polarization factor (1+cos2(2θ) -Pfac∙cos(2Φ)sin2(2θ))/2 A - attenuation factor exp(-μxtal∙lpath) F(hkl) - structure amplitude (electrons) C. G. Darwin (1914)

  38. IT Ibeam A = = exp(-μt) t attenuation factor μ Bouguer, P. (1729). Essai d'optique sur la gradation de la lumière. Lambert, J. H. (1760). Photometria: sive De mensura et gradibus luminis, colorum et umbrae. E. Klett. Beer, A. (1852)."Bestimmung der Absorption des rothen Lichts in farbigen Flüssigkeiten", Ann. Phys. Chem86, 78-90.

  39. tout tin IT Ibeam IT Ibeam A = = exp(-μt) t attenuation factor μ A = = exp(-μ(tin+ tout)) Bouguer, P. (1729). Essai d'optique sur la gradation de la lumière. Lambert, J. H. (1760). Photometria: sive De mensura et gradibus luminis, colorum et umbrae. E. Klett. Beer, A. (1852)."Bestimmung der Absorption des rothen Lichts in farbigen Flüssigkeiten", Ann. Phys. Chem86, 78-90.

  40. tout tin tout tin tout IT Ibeam tin attenuation factor μ A = = exp(-μ(tin+ tout)) Bouguer, P. (1729). Essai d'optique sur la gradation de la lumière. Lambert, J. H. (1760). Photometria: sive De mensura et gradibus luminis, colorum et umbrae. E. Klett. Beer, A. (1852)."Bestimmung der Absorption des rothen Lichts in farbigen Flüssigkeiten", Ann. Phys. Chem86, 78-90.

  41. tso txo tsi txi tso txo txi tsi tso txo IT Ibeam txi tsi attenuation factor μsolvent μxtal A = = exp[-μxtal(txi+ txo) -μsolvent(tsi + tso)] Bouguer, P. (1729). Essai d'optique sur la gradation de la lumière. Lambert, J. H. (1760). Photometria: sive De mensura et gradibus luminis, colorum et umbrae. E. Klett. Beer, A. (1852)."Bestimmung der Absorption des rothen Lichts in farbigen Flüssigkeiten", Ann. Phys. Chem86, 78-90.

  42. Transmitted (98%) Where do photons go? Protein 1A x-rays beamstop attenuation correction cannot be > ~2% for 100 μm xtal at 1 Å

  43. Vxtal λ3 L Vcell ωVcell Darwin’s Formula I(hkl) = Ibeam re2 P A | F(hkl) |2 I(hkl) - photons/spot (fully-recorded) Ibeam - incident (photons/s/m2 ) re - classical electron radius (2.818x10-15 m) Vxtal - volume of crystal (in m3) Vcell - volume of unit cell (in m3) λ - x-ray wavelength (in meters!) ω - rotation speed (radians/s) L - Lorentz factor (speed/speed) P - polarization factor (1+cos2(2θ) -Pfac∙cos(2Φ)sin2(2θ))/2 A - attenuation factor exp(-μxtal∙lpath) F(hkl) - structure amplitude (electrons) C. G. Darwin (1914)

  44. Vxtal λ3 L Vcell ωVcell Darwin’s Formula I(hkl) = Ibeam re2 P A | F(hkl) |2 I(hkl) - photons/spot (fully-recorded) Ibeam - incident (photons/s/m2 ) re - classical electron radius (2.818x10-15 m) Vxtal - volume of crystal (in m3) Vcell - volume of unit cell (in m3) λ - x-ray wavelength (in meters!) ω - rotation speed (radians/s) L - Lorentz factor (speed/speed) P - polarization factor (1+cos2(2θ) -Pfac∙cos(2Φ)sin2(2θ))/2 A - attenuation factor exp(-μxtal∙lpath) F(hkl) - structure amplitude (electrons) C. G. Darwin (1914)

  45. Lorentz Factor Ewald sphere (h,k,l)

  46. Lorentz Factor Ewald sphere (h,k,l) d*

  47. Lorentz Factor Ewald sphere (h,k,l) spindle axis d*

  48. Lorentz Factor Ewald sphere Φcircle (h,k,l) spindle axis

  49. Lorentz Factor Ewald sphere Φcircle (h,k,l) spindle axis

  50. Lorentz Factor Ewald sphere Φcircle (h,k,l) spindle axis diffracted ray

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