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. Chris Neilson Michael Blum Joe Ferrara Meitian Wang

AB AtzD, a cyanuric acid hydrolase Peat, T. S., Balotra, S., Wilding, M., French, N. G., Briggs, L. J., Panjikar, S., Cowieson, N., Newman, J. & Scott, C. (2013) Cyanuric acid hydrolase: evolutionary innovation by structural concatenation, Mol Microbiol. 88,

Biochemistry suggests Mg ++ Inhibitor/TreatmentResidual Activity (%) PMSF8.5 o-Phenanthroline1.2 EDTA100 1 ApoAtzD plus Mg +2 (50 µM)60 Lysine alkylation7.5 ApoAtzD treated by the addition of 50 μM Zn ++ Na + K + Mn ++ Co ++ Cu ++ Ca ++ or Fe ++ failed to recover any catalytic activity.

AB E297 P351 G353 E347 Metal site: is it Mg ++ ? Or Na + ?

AB Position (Å) Electron density (e - / Å 3 )

Metal site: is it Mg ++ ? Or Na + ?

Sources of error for anomalous differences Shutter jitter (rms 0.5 ms) Beam flicker (0.15%/sqrt(Hz)) Attenuation correction (< 2%) Radiation damage (1%/MGy) Detector calibration (3%)

Detector calibration: 7235 eV

Detector calibration: 7247 eV

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

Dan Schuette PhD Thesis (2008) Fig 6.22 page 198, Gruner Lab, Cornell University. Pilatus is not immune! Spatial Heterogeneity in Sharp Spot Sensitivity

Spatial Heterogeneity in Sharp Spot Sensitivity (SHSSS): Q315r vs Pilatus average change in spot intensity (%) distance between spots (mm) Pilatus Q315r anomalous mates typically > 100 mm apart different modules

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

Data collection parameters: 16 crystals 360° each, inverse beam 7235 eV photon energy < 1 MGy per xtal Australian Synchrotron MX1 –35 kGy/s into 100 μm x 100 μm

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 16 σ

140-fold multiplicity 15σ = PO 4 Phased anomalous difference Fourier

140-fold multiplicity ~2σ = Mg? Phased anomalous difference Fourier

140-fold multiplicity 8.2σ = Mg? DELFAN residual anomalous difference

140-fold multiplicity 7.4σ = Mg? DELFAN residual anomalous difference

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

Competitive occupancy refinement start_004.pdb:HETATM11129 MG A MG M Mg+2 start_004.pdb:HETATM11130 NA B NA M Na+1 start_004.pdb:HETATM11131 MG A MG M Mg+2 start_004.pdb:HETATM11132 NA B NA M Na+1 start_005.pdb:HETATM11129 MG A MG M Mg+2 start_005.pdb:HETATM11130 NA B NA M Na+1 start_005.pdb:HETATM11131 MG A MG M Mg+2 start_005.pdb:HETATM11132 NA B NA M Na+1 start_006.pdb:HETATM11129 MG A MG M Mg+2 start_006.pdb:HETATM11130 NA B NA M Na+1 start_006.pdb:HETATM11131 MG A MG M Mg+2 start_006.pdb:HETATM11132 NA B NA M Na+1 start_007.pdb:HETATM11129 MG A MG M Mg+2 start_007.pdb:HETATM11130 NA B NA M Na+1 start_007.pdb:HETATM11131 MG A MG M Mg+2 start_007.pdb:HETATM11132 NA B NA M Na+1 start_008.pdb:HETATM11129 MG A MG M Mg+2 start_008.pdb:HETATM11130 NA B NA M Na+1 start_008.pdb:HETATM11131 MG A MG M Mg+2 start_008.pdb:HETATM11132 NA B NA M Na+1 start_009.pdb:HETATM11129 MG A MG M Mg+2 start_009.pdb:HETATM11130 NA B NA M Na+1 start_009.pdb:HETATM11131 MG A MG M Mg+2 start_009.pdb:HETATM11132 NA B NA M Na+1

Discerning Na + from Mg ++ macro-round of refinement Mg ++ occupancy refmac

Discerning Na + from Mg ++ macro-round of refinement Mg ++ occupancy phenix.refine

140-fold multiplicity 8.2σ = 60% Mg DELFAN residual anomalous difference

140-fold multiplicity 7.4σ = Na DELFAN residual anomalous difference

Conclusion ? Tom thinks it is Mg ++ James thinks it is Na σ is enough for phasing! Multiplicity must be “genuine” Implications

How do I do Na-SAD ? Be Tom & Janet Reproducibility! Careful with humidity All mounts exactly same time Blow on the cryo Low dose!

How do I do Na-SAD ? Be Tom & Janet Reproducibility! Careful with humidity All mounts exactly same time Blow on the cryo Low dose!

R iso vs dose R iso (%) change in dose (MGy) data taken from Banumathi, et al. (2004) Acta Cryst. D 60,

R iso vs dose R iso (%) change in dose (MGy) data taken from Banumathi, et al. (2004) Acta Cryst. D 60, R iso ≈ 0.7 %/MGy

How long will my crystal last?

How do I do Na-SAD ? Be Tom & Janet Reproducibility! Careful with humidity All mounts exactly same time Blow on the cryo Low dose!

plunge cooling foam insulation warm, moist air cold N 2 liquid N 2 ice

cold N 2 plunge cooling foam insulation warm, moist air liquid N 2 ice

Warkentin method foam insulation liquid N 2 Warkentin (2006) J. Appl. Crystallogr. 39,

How do I do Na-SAD ? Be Tom & Janet Reproducibility! Careful with humidity All mounts exactly same time Blow on the cryo Low dose!

First diffraction from protein xtal Bernal, J. & Crowfoot, D. (1934). "X-ray photographs of crystalline pepsin", Nature 133,

RH 84.2% vs 71.9% R iso = 44.5%RMSD = 0.18 Å Non-isomorphism in lysozyme

Dehydration: μm 2 mm = 1.5 μL = 1.0 nL 10 μm = 1.0 pL and 2014?

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 Heterogeneity in Sharp Spot Sensitivity

down Spatial Heterogeneity in Sharp Spot Sensitivity

downup Spatial Heterogeneity in Sharp Spot Sensitivity

downup R separate Spatial Heterogeneity in Sharp Spot Sensitivity

oddeven R mixed Spatial Heterogeneity in Sharp Spot Sensitivity

separate:2.5% Spatial Heterogeneity in Sharp Spot Sensitivity

separate: mixed: 2.5% 0.9% Spatial Heterogeneity in Sharp Spot Sensitivity

separate: mixed: 2.5% 0.9% 2.5% % 2 = 2.3% 2 Spatial Heterogeneity in Sharp Spot Sensitivity

Spatial Heterogeneity in Sharp Spot Sensitivity (SHSSS): Q315r vs Pilatus average change in spot intensity (%) distance between spots (mm) Pilatus Q315r anomalous mates typically > 100 mm apart different modules

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

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

7247 eV

7235 eV

Detector calibration 7223 eV

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