1. Recent Advances in Protein Powder Diffraction R.B. Von Dreele, XSD/IPNS Argonne National Laboratory, USA “Reaching for High Resolution in Protein Powder Diffraction” Thanks – Peter Stephens, Peter Lee, US DOE/OS/BES

2. 2 What is a powder? - polycrystalline mass All orientations of crystallites possible Sample: 1  l powder of 1  m crystallites - ~10 9 particles Single crystal reciprocal lattice - smeared into spherical shells - the overlap problem – lost information Packing efficiency – typically 50% Spaces – air, solvent, etc.

3. 3 d* 1/ Powder diffraction - reciprocal space soso s Ewald sphere 22 Bragg’s Law Spherical reflection shells Smear in 3D Typical 1-D scan

4. 4 Problem – Severe radiation damage of proteins Much worse at APS!! Happens in 10-20min!! 0 3 6 9 12 15 18 21 24 2 , deg Powder patterns: Lysozyme – Multiple scans @ 1.15Å, RT, ~3hr ea.; ~10mg HEWL Radiation damage – initial observations @NSLS X3b1 Xtal/analyzer detector 1 day

5. 5 Faster data collection Beam focused to IP surface & IP offset 6-10cm up Sample (spun, 1x1mm, <1mg) Beam stop ~700mm ~0.6Å ~350mm Image Plate Detector – MAR345 “Guinier geometry”

6. 6 11BMB – 10min scan1BM/MAR345 – 1sec exposure Compare image plate with analyzer/detector

7. 7 Rings – protein pattern (HEWL) – X-rays 30s @ 20kV on MAR345; <1mg HEWL Inner most ring – d~55Å (110) Reflection, lowest order for tetragonal lysozyme 2  ~ 0.67deg Beam stop holder Texture free sample & no graininess – 1  m “perfect” powder Resolution limit – 1.85Å Residual solvent scattering – background (Air, solvent & Kapton background subtracted) ~9000 F hkl for HEWL >2Å

8. 8 Powder diffraction from 2D image plates (MAR345) Best focusing – best resolution FIT2D cake integration to d min = 2Å “resolution” ~0.035 o FWHM 300  m beam/pt. spread fxn. ~4X sample contribution!

9. 9 HEWL comparison – 30s on 1BM Mar345 vs 10min on 11BM 20keV 30keV

10. 10 Background problem – subtract air, liquid & Kapton Before subtraction: Gave too small R wp (<0.5%) from high background No sensitivity to structure After background subtraction: Weights: Compensate for 2D detector integration effect

11. 11 Radiation damage – reflection intensities & positions APS 1BM - 30s exposures + 150s delay, 300K Full sequence wrt NaCl & pH – effects?? Immediate changes seen 2 stages? - 10min exposure Focus here: a up & c down

12. 12 Multiple patterns – different lattice strains Buffer effect: Actually solvent effect? phthalate (<pH5) Phosphate(>pH5) Focus here Shortest exposure

13. 13 Solvent & radiation damage induced lattice strains for HEWL Salt & pH effects Radiation exposure ~8% loss in 4.5m cf. FWHM 0.035 o 2  ~ 0.02% strain @18 o 2  Compare:  [M] = 0.07M;  pH = 0.07 (  [H+]~-15%) in 4.5m

14. 14 Analysis: Induced lattice strain model – obey Laue symmetry From d-spacing expression partial derivatves wrt g ij of Bragg’s law gives And symmetrized for tetragonal a-axis strain for tetragonal is (c-axis similar) Peak shape function #5 in GSAS

15. 15 2.5-2.0Å resolution range – NaCl sequence 0.25M 1.25M Peak shifts! Obsd & calcd powder patterns Also for rad. dam. – just less

16. 16 Profile fit – 1/5 patterns; R wp =1.84% 4x 20x

17. 17 HEWL – superposition of 3 determinations (NaCl,pH5;NaCl,pH4 & RD) & H 2 O independently detn. H 2 O – many common positions (& some not) Variations? RMSD~0.4Å (all protein atoms)

18. 18 Structure quality? Ramachandran plot – 90% most favored Total OMIT map – protein & H 2 O

19. 19 HEWL I pH4 HEWL II pH5 HEWL III Rad dam HEWL I all/backbone, Å Vary NaCl, pH4 ---0.44/0.330.42/0.34 HEWL II all/backbone, Å Vary NaCl, pH5 0.44/0.33---0.55/0.40 HEWL III all/backbone, Å 1.25M NaCl, pH4, rad dam 0.42/0.340.55/0.40--- 6LYT all/backbone, Å Best RT SC structure 1.35/0.751.37/0.771.38/0.77 194L all/backbone, Å Starting model 1.25/0.751.28/0.761.28/0.77 HEWL results – structure comparisons Appropriate for 2Å resolution?

20. 20 Compare – single pattern result (1JA2) & best single xtal 1JA2 - powder PDB 5 NaCl patterns - this work Rad. Dam. Seq. similar Q=57.63% Q=98.33% ERRAT2 – atom neighbor analysis - packing Compare: best HEWL single crystal result (low temp; xtal from shuttle!) Q=94.22% Howzat? PDB 1IEE

21. 21 Overlap factor – effect of lattice strain better “effective” resolution? Overlap factor, R ij =1-  ij /2FWHM =1 if complete overlap, =0 if no overlap F i =  min(R ij ) for multipatterns 2 patterns 5 patterns  ~0  >2FWHM SC = 0 1 pattern

22. 22 Sample size limit? in situ? 1 st experiments – NSLS X3b1; 1 analyzer/detector – 10mg HEWL slurry; 6hr scan Image plate – APS 1BM; MAR345; ~1mg HEWL slurry; 30s exposure CCD – APS 8BM (now defunct); ~15  g HEWL slurry; 10s exposure 2 of 1536 well plate – xtal growth test – not particularly optimized – corners <3Å

23. 23 High throughput screening for crystallization – X-ray 8BM ~12keV 1536 well plate 1 st “real” expt. 4 plates & look for spots/rings? Compare optical pix ADSC 315 – 20Mb each pix = 5-6 DVDs/plate! ROBOT!! Craig Ogata, et al. & Blessing, et al.

24. 24 Detector development – spatial resolution MAR345 – 300  m ADSC – 100  m 35  m – ideal match with sample

25. 25 Tileable area detectors? Cover “best” part of powder pattern Tilted array – avoid “blind” spots in powder pattern Cover curved area to match resolution? Not necessarily spherical!

26. 26 Conclusion – data combinations in proteins (at least HEWL) Protein powder diffraction  Image plates – lower powder resolution, but Better intensity measurement to higher diffr. resolution Induced lattice strain from RD, pH, salt, etc. variation  multiple powder patterns  lattice variation Recover powder resolution Result – higher powder & diffraction resolution  “better” protein structure (including water molecules) Future – smaller samples; better resolution Structure solution – multiple data set extraction of F o