Acknowledgements Christine Gee Janet Newman Tom Peat 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.

What is holding us back? Weak spots (high-res) background solution: use as few pixels as possible MAD/SAD (small differences) fractional errors solution: use as many pixels as possible ( if not rad dam! )

Required multiplicity mult > ( — ) 2 ~3%

140-fold multiplicity SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr % 3.9% 4.8% % 100.0* 91* % 5.2% 5.5% % 100.0* 86* % 7.2% 7.0% % 100.0* 76* % 7.2% 6.6% % 100.0* 67* % 7.7% 6.7% % 100.0* 59* % 9.4% 8.3% % 100.0* 49* % 11.2% 10.1% % 100.0* 39* % 14.1% 13.9% % 100.0* 30* % 19.5% 20.2% % 100.0* 23* % 29.0% 31.7% % 99.9* 17* % 40.5% 44.8% % 99.8* 11* % 52.8% 58.8% % 99.8* 10* % 67.4% 76.0% % 99.6* % 88.9% 101.2% % 99.2* % 109.3% 125.5% % 98.1* % 138.2% 161.4% % 96.1* % 197.1% 231.7% % 83.5* % 227.3% 268.7% % 46.9* % 154.4% 169.4% % 47.0* % 170.1% 187.0% % 25.7* total % 15.7% 16.4% % 100.0* 12* crystals, 360° each, inverse beam, 7235 eV AS MX1

RESOLUTION COMPLETENESS R-FACTOR I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OF DATA observed Corr % 3.9% % 100.0* 91* % 5.2% % 100.0* 86* % 7.2% % 100.0* 76* % 7.2% % 100.0* 67* % 7.7% % 100.0* 59* % 9.4% % 100.0* 49* % 11.2% % 100.0* 39* % 14.1% % 100.0* 30* % 19.5% % 100.0* 23* % 29.0% % 99.9* 17* % 40.5% % 99.8* 11* % 52.8% % 99.8* 10* % 67.4% % 99.6* % 88.9% % 99.2* % 109.3% % 98.1* % 138.2% % 96.1* % 197.1% % 83.5* % 227.3% % 46.9* % 154.4% % 47.0* % 170.1% % 25.7* total 93.3% 15.7% % 100.0* 12* fold multiplicity: 16 crystals, 360° each, inverse beam, 7235 eV

140-fold multiplicity 18 σ Phased anomalous difference Fourier data Courtesy of Tom & Janet 16 σ

140-fold multiplicity 15σ = PO 4 Phased anomalous difference Fourier data Courtesy of Tom & Janet

140-fold multiplicity ~2σ = Mg? Phased anomalous difference Fourier data Courtesy of Tom & Janet

140-fold multiplicity 8.2σ = Mg? DELFAN residual anomalous difference data Courtesy of Tom & Janet

Discerning Na + from Mg ++ f’’ (electrons) DELFAN peak height (σ) Mg Ne Na F O N

140-fold multiplicity 7.4σ = Na DELFAN residual anomalous difference data Courtesy of Tom & Janet

140-fold multiplicity 7.3σ = S DELFAN residual anomalous difference data Courtesy of Tom & Janet

140-fold multiplicity 12σ = S Phased anomalous difference Fourier data Courtesy of Tom & Janet

140-fold multiplicity Courtesy of Tom & Janet 8σ = ? DELFAN residual anomalous difference data Courtesy of Tom & Janet

23-fold multiplicity in P1 Courtesy of Tom & Janet DELFAN residual anomalous difference data Courtesy of Tom & Janet

Detector calibration: 7247 eV target: oil distance: 900 mm 2θ: 12°

Detector calibration: 7235 eV target: oil distance: 900 mm 2θ: 12°

Detector calibration: ALS % -10%

Detector calibration errors: detector 2

Detector calibration errors: detector 3

Detector calibration calibration error (%) megapixels

Spatial Noise

down

Spatial Noise downup

Spatial Noise downup R separate

Spatial Noise oddeven R mixed

Spatial Noise separate:2.5%

Spatial Noise separate: mixed: 2.5% 0.9%

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

Spatial Noise: Q315r vs Pilatus Holton, Frankel, Gonzalez, Waterman and Wang (2010) in preparation average change in spot intensity (%) distance between spots (mm) Pilatus Q315r anomalous mates typically > 100 mm apart

Detector calibration errors Dan Schuette PhD Thesis (2008) Fig 6.22 page 198, Gruner Lab, Cornell University. Pilatus is not immune!

7247 eV

7235 eV

Detector calibration 7223 eV

Gadox calibration vs energy photon energy (keV) Relative absorption depth same = good! bad!