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PowerPoint File available:. http://bl831.als.lbl.gov/ ~jamesh/powerpoint/ ACA_SvN_2011.ppt. 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

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  1. PowerPoint File available: http://bl831.als.lbl.gov/ ~jamesh/powerpoint/ ACA_SvN_2011.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. Why do structures fail? Overlaps Radiation Damage Signal to noise

  4. c avoiding overlaps c

  5. avoiding overlaps c http://www.mitegen.com/mic_catalog.php?c=DTMicroMounts

  6. c avoiding overlaps http://www.mitegen.com/mic_catalog.php?c=orientedmounts

  7. Why do structures fail? Overlaps Radiation Damage Signal to noise

  8. Why do structures fail? Overlaps Radiation Damage Signal to noise

  9. Why do structures fail? Overlaps Radiation Damage Signal to noise

  10. signalvsnoise

  11. signalvsnoise

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

  13. signalvsnoise easy hard impossible

  14. signalvsnoise easy hard impossible threshold of “solvability”

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

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

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

  18. Adding noise

  19. Adding noise 12 + 12 = 1.42

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

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

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

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

  24. FH != ΔFano threshold of “solvability” ΔFano << ΔF SAD phasing simulation mlphare results Correlation coefficient to correct density Anomalous signal to noise ratio (ΔFano/ΔF)

  25. exposure time threshold of “solvability” ΔFano << ΔF MAD phasing simulation mlphare results Correlation coefficient to correct density Anomalous signal to noise ratio (ΔFano/ΔF)

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

  27. Background scattering Se edge with detector at 100 mm you want: air gap < 1000x xtal size Photons/s/pixel  7.5 3.8 2.5 1.9 1.5 1.2 1.1 Resolution (Ǻ)

  28. 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

  29. $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!

  30. Background scattering

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

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

  33. 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)

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

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

  36. 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.

  37. High multiplicity is not enough correlation coefficient of ΔFano to calculated observations per hkl index (multiplicity)

  38. 0.1% error? Jenkins et al. (2009)."Evidence of correlations between nuclear decay rates and Earth–Sun distance", Astroparticle Physics32, 42-46.

  39. 0.1% error? Jenkins et al. (2009)."Evidence of correlations between nuclear decay rates and Earth–Sun distance", Astroparticle Physics32, 42-46.

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

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

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

  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. 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)

  46. 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.

  47. 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.

  48. 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.

  49. 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.

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

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