Recent Advances in Protein Powder Diffraction

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

2. What is a powder? - polycrystalline mass All orientations of crystallites possible Sample: 1ml powder of 1mm crystallites - ~109 particles Packing efficiency – typically 50% Spaces – air, solvent, etc. Single crystal reciprocal lattice - smeared into spherical shells - the overlap problem – lost information

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

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

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

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

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

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

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

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

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 & >10min exposure Focus here: a up & c down

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

13. Solvent & radiation damage induced lattice strains for HEWL Salt & pH effects Radiation exposure ~8% loss in 4.5m Compare: D[M] = 0.07M; DpH = 0.07 (D[H+]~-15%) in 4.5m cf. FWHM 0.035o2Q ~ 0.02% strain @18o2Q

14. Analysis: Induced lattice strain model – obey Laue symmetry From d-spacing expression partial derivatves wrt gij 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. 2.5-2.0Å resolution range – NaCl sequence Obsd & calcd powder patterns 0.25M Peak shifts! 1.25M Also for rad. dam. – just less

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

17. HEWL – superposition of 3 determinations (NaCl,pH5;NaCl,pH4 & RD) & H2O independently detn. H2O – many common positions (& some not) Variations? RMSD~0.4Å (all protein atoms)

18. Structure quality? Ramachandran plot – 90% most favored Total OMIT map – protein & H2O

19. HEWL results – structure comparisons Appropriate for 2Å resolution?

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

21. Overlap factor – effect of lattice strain better “effective” resolution? 1 pattern 5 patterns 2 patterns SC = 0 Overlap factor, Rij=1-Dij/2FWHM =1 if complete overlap, =0 if no overlap Fi=Smin(Rij) for multipatterns D~0 D>2FWHM

22. Sample size limit? in situ? • 1st 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); ~15mg HEWL slurry; 10s exposure 2 of 1536 well plate – xtal growth test – not particularly optimized – corners <3Å

23. High throughput screening for crystallization – X-ray 8BM ~12keV 1536 well plate 1st “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. Detector development – spatial resolution ADSC – 100mm MAR345 – 300mm 35mm – ideal match with sample

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. 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 Fo