Beamline summary 1.Strong PRT and staff 2.Robust optics and endstation 3.Safety: stable, simple operations 4.Funding: Operational funding secure 5.Scientific productivity: high 6.Future: streamlining success

Holton J. M. (2009) J. Synchrotron Rad ALS beamline Diffraction Methods Research

Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom resolution (Å) maximum tolerable dose (MGy)

10 MGy/Å what the is a MGy? damage_rates.pdf Holton J. M. (2009) J. Synchrotron Rad

Radiation Damage Model accumulated dose (MGy) normalized total intensity

Radiation Damage Model accumulated dose (MGy) best-fit B factor Kmetko et. al. (2006): lysozyme: apoferritin: slopes (Å 2 /MGy): lysozyme: apoferritin: 0.016

Simulated diffraction image MLFSOM simulatedreal

Crystal Size crystal size (μm) CC to correct model predicted Glaeser et.al. (2000) 1 μm amyloids Nelson et al Sawaya et al Glaeser et.al. (2000) Sliz et.al. (2003) ~12 μm xylanase Moukhametzianov et al μm cypovirus polyhedra Coulibaly et. al μm (13x) bovine rhodopsin Standfuss et al theoretical

Minimum Crystal Size n xtal - number of crystals needed n 0 - empirical constant (~ 3) d- d-spacing of interest (Å) B- Wilson B factor (Å 2 ) n xtal = n 0 MW V M 2 ℓ x ℓ y ℓ z (d ) exp(-0.5 B/d 2 ) MW- molecular weight (kDa) V M - Matthews number (~2.5 Å 3 /Da) ℓ- crystal size (microns) B ≈ 4 d Holton J. M. (2009) J. Synchrotron Rad

Where:  I  DL - average damage-limited intensity (photons/hkl) at a given resolution converting R from μm to m, r e from m to Å, ρ from g/cm 3 to kg/m 3 and MGy to Gy r e - classical electron radius (2.818 x m/electron) h- Planck’s constant (6.626 x J∙s) c- speed of light ( m/s) f decayed - fractional progress toward completely faded spots at end of data set ρ- density of crystal (~1.2 g/cm 3 ) R- radius of the spherical crystal (μm) λ- X-ray wavelength (Å) f NH - the Nave & Hill (2005) dose capture fraction (1 for large crystals) n ASU - number of proteins in the asymmetric unit M r - molecular weight of the protein (Daltons or g/mol) V M - Matthews’s coefficient (~2.4 Å 3 /Dalton) H- Howells’s criterion (10 MGy/Å) θ- Bragg angle  a 2  - number-averaged squared structure factor per protein atom (electron 2 )  M a  - number-averaged atomic weight of a protein atom (~7.1 Daltons) B- average (Wilson) temperature factor (Å 2 ) μ- attenuation coefficient of sphere material (m -1 ) μ en - mass energy-absorption coefficient of sphere material (m -1 ) Theoretical limit: Holton J. M. and Frankel K. A. (2010) Acta D submitted

Theoretical limit: at ~2.4 Å photon spot μm Holton J. M. and Frankel K. A. (2010) Acta D submitted for lysozyme

Optimum exposure time (faint spots) t hr optimum exposure time for data set (s) t ref exposure time of reference image (s) bg ref background level near weak spots on reference image (ADU) bg 0 ADC offset of detector (ADU) σ 0 rms read-out noise (ADU) gain ADU/photon m multiplicity of data set (including partials) Short answer: bg hr = 90 ADU for ADSC Q315r

Specific Damage

Damage changes absorption spectrum Photon energy (eV) counts 1 0 Holton J. M. (2007) J. Synchrotron Rad

fluorescence probe for damage fluence (10 15 photons/mm 2 ) Fraction unconverted 25mM SeMet in 25% glycerol Exposing at eV Se cross-section at eV Holton J. M. (2007) J. Synchrotron Rad

fluorescence probe for damage Absorbed Dose (MGy) Fraction unconverted Wide range of decay rates seen Half-dose = 41.7 ± 4 MGy “GCN4” in crystal Half-dose = 5.5 ± 0.6 MGy 8 mM SeMet in NaOH Protection factor: 660% ± 94% Holton J. M. (2007) J. Synchrotron Rad

Take-home lesson: radiation damage to metal sites is unpredictable Best strategy: 5 MGy to complete data geometrically increasing exposure Holton J. M. (2007) J. Synchrotron Rad

Minimum required signal (MAD/SAD)

dataset exposure1.0s0.1s1.0s frames x R merge 5.6% 11.2% 4.7% R anom 4.8%4.7% I/sd I/sd (2.0 Ǻ) redundancy PADFPH FOM FOMDM CC(1H87) same total dose with high and low redundancy

Spatial Noise downup R separate

Spatial Noise oddeven R mixed

Spatial Noise separate: mixed: 2.5% 0.9% 2.5% % 2 = 2.3% 2

Spatial Noise mult > ( — ) 2 2.3%

Minimum Crystal Size n xtal - number of crystals needed n for complete data set, 180 for MAD d- d-spacing of interest (Å) B- Wilson B factor (Å 2 ) n xtal = n 0 MW V M 2 ℓ x ℓ y ℓ z (d ) exp(-0.5 B/d 2 ) MW- molecular weight (kDa) V M - Matthews number (~2.5 Å 3 /Da) ℓ- crystal size (microns) B ≈ 4 d Holton J. M. (2009) J. Synchrotron Rad

Take-home lesson: need better crystals for MAD Best strategy: find them

accurate, unattended data colleciton

beamline microscope reference image Re-centering

accurate, unattended screening

sample shadow on detector Cu

sample shadow on detector

X-ray shadow of cryo stream

Plate goniometers