High Brilliance Synchrotrons for SAXS and WAXS Robert Fischetti Associate Division Director for Structural Biology and GM/CA Group Leader X ‐ ray Science Division Advanced Photon Source October 24, 2012
Outline � Complementary techniques XAS, MX, SAXS and WAXS � What can we learn from XAS? � MX and microcrystallography – Scientific highlights ‐ GPCRs – SONICC – Micro ‐ beams and radiation damage – Micro ‐ focus endstation � What can we learn from WAXS � SAXS/WAXS capabilities around the world 2
X-ray Absorption Spectroscopy (XAS) What can we learn from X ‐ ray Absorption Spectroscopy? Short range, high resolution probe Local structure about an absorbing atom Precise bond lengths and angles Redox state SSRL 1984 Cyril Applebee Max Perutz Brittan Chance 3
Classical interpretation of XAS Metallic Rh K ‐ edges – closer look Rh L and K absorption edges X ‐ ray interacts with atom and ejects a photoelectron Isolated atom Atom in vicinity “featureless” “interference” 4
XAS and on-line optical monitoring Experimental Setup Optical Spectroscopy Carboxymyoglobin Normalized Data Processed Data FT of Data Model data 5
Wide Angle X-ray Scattering (WAXS) What is WAXS? � Diffraction from proteins in solution � Similar to SAXS, but extends to higher scattering angles � Third generation synchrotrons provide sufficient intensity to extend collection of accurate data to near atomic resolution (>3.0 Å) � WAXS data is unlikely to provide atomic resolution structure of a protein because the scatter patterns are spherically averaged What can WAXS tell us? � Determine degree of effect of drug binding on conformation � Measure native structure in solution � Study biological processes in solution not amenable to standard crystal analysis How is WAXS calculated? Solution scattering as a function of scattering vector can be expressed in terms of interatomic vectors, r ij, (Debye Formula): I (q) = Σ I i (q) + 2 ΣΣ F i (q) F j (q) (sin(qr ij )/(qr ij )) 6
Beamline Configuration – Sector 18 BioCAT Typical WAXS Setup Sector 18 – Advanced Photon Source Undulator A 3.3 cm period 72 poles High brilliance: small beams, low divergence, clean background R.F. Fischetti et al, The BioCAT undulator beamline 18ID: a facility for biological non ‐ crystalline diffraction and X ‐ ray absorption spectroscopy at the Advanced Photon Source (2004) J. Synch. Rad. 11:399 – 405. 7
Scatter Intensity vs. Protein Concentration Hb in PBS 0 mg/ml 1 mg/ml 100 mg/ml 10 mg/ml Raw intensity patterns 8
Protein density depends on molecular weight We also noticed in the curves plotted in Figure 2 from the results of Tsai et al. (1999) that the reported average densities of all the studied proteins, determined theoretically, are about 2.4% higher than those determined experimentally (Tsai et al. 1999). This difference can be qualitatively explained considering that the volume determined experimentally includes an ~3 Å thick water layer around the external surface (Svergun et al. 1998), this effect thus leading to an apparent decrease of the actual average density. Fischer, H., Polikarpov, I. and Craievich, A. Average protein density is a molecular ‐ weight ‐ dependent function Protein Sci. 2004 October; 13(10): 2825–2828. PMCID: PMC2286542 9
Components of a WAXS Pattern (150 mg/ml HB) I(protein) = I(Prot. sol. in cap.) – I(cap.) ‐ (1 ‐ vol%)*[I(buffer in cap.) – I(cap.) ] Each data set is composed of scattering from (i) Empty capillary (ii) Buffer in capillary (iii) Protein solution in Protein (x10) capillary Buffer in capillary At wide ‐ angles, buffer scatters X ‐ rays more strongly than the protein displacing it in the protein solution ! buffer Protein solution MUST account for in capillary excluded volume Empty capillary 1/d (A ‐ 1 ) = 2*sin( θ )/ λ q (A ‐ 1 ) =2 π /d q(nm ‐ 1 ) = 10*q(A ‐ 1 ) 10
Wide Angle Scattering Setup – Version 2 12 µm mylar mica exit window guard slits 1.2 mm Mar 165 CCD pin hole 29.6 X 29.6 μ m 4k X 4k helium helium vacuum pixels 2.7:1 taper 2x2 binned O ‐ ring flow cell 360° beam stop q = 0.006 – 0.46 1/A 180 mm He – no windows Fischetti, R. F., Rodi, D. J., Gore, D. B., and Makowski L., Wide angle x ‐ ray solution scattering as a probe of ligand ‐ induced conformational changes in proteins. Chemistry and Biology Chem. & Biol. 11: 1431 ‐ 1443 . 11
Minimizing radiation damage Solution scatter from Hb flow cell (red) Stationary samples show clear signs of degradation stationary sample (black) 1e+5 8e+4 32 sec data collection 6e+4 No protein in the Radiation intensity beam for more damage results than 0.1 sec in weakening of 4e+4 peaks and filling in of troughs 5 sec data 2e+4 collection Each protein may 0 be exposed in the 0.0 0.1 0.2 0.3 0.4 beam for up to 5 1/d sec Fischetti, R. F., Rodi, D. J., Mirza, A., Irving, T. C., Kondrashkina, E., and Makowski, L. (2003) High ‐ resolution wide ‐ angle x ‐ ray scattering of protein solutions: effect of beam dose on protein integrity. J. Synch. Rad., 10: 398 ‐ 404 12
Molecular Dimension in a WAXS Pattern Quaternary structure (shape and size) Tertiary structure (inter ‐ domain distances) Secondary structures Secondary structure Alpha helices Primary structure • 1.5 Å axial separation of AA • 5.4 Å pitch A B C D • 10 Å diameter Beta sheets • 4.7 Å strand ‐ to ‐ strand distance • 7.0 Å pleat distance 13
Cytochrome C Experimental – red Crysol calculated ‐ black 60 50 cytochrome C 40 intensity 30 20 10 0 0.0 0.1 0.2 0.3 0.4 1/d 14
Myoglobin Experimental – black Crysol calculated ‐ red myoglobin 15
Comparison of Myoglobin and Hemoglobin Myoglobin experimental ‐ black Hemoglobin experimental ‐ red 4000 hemoglobin 3000 Peak from quaternary intensity structure 2000 Peaks from 1000 myoglobin tertiary structure 0 0.0 0.1 0.2 0.3 0.4 1/d Peak at 0.1 A ‐ 1 is Peak at 0.2 ‐ 0.26 A ‐ 1 indicative of alpha is relatively invariant helical content 16
WAXS is sensitive to a –helical content Myoglobin: 88% α ‐ helical (black) Bovine serum albumin: 78% α ‐ helical (blue) and superoxide dismutase: 17% α ‐ helical (red) myoglobin 5000 4000 3000 intensity 2000 serum albumin 1000 0 0.0 0.1 0.2 0.3 0.4 1/d Peak at 0.1 A ‐ 1 is indicative superoxide of alpha helical content dismutase 17
Comparison of Hemoglobin and Streptavidin Hemoglobin experimental ‐ blue Streptavidin experimental ‐ red 5000 a -helix vs. β -sheet hemoglobin 4000 3000 intensity 2000 1000 streptavidin 0 0.0 0.1 0.2 0.3 0.4 1/d 18
Comparison of Calmodulin and Myoglobin 5000 5000 4000 4000 3000 3000 intensity intensity open form 2000 2000 1000 1000 0 0 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 1/d 1/d 1/d vs calmod-solv-cap 1/d vs calmod-solv-cap 1/d vs Mb-solv-cap 1/d vs Mb-solv-cap Calmodulin 13.3 mg/ml in HEPES/ 30 µM CaCl2 19
Use WAXS to study structural fluctuations � Substrate binding – Domain movement – Binding constant � Effect of small molecule effectors of structure – Molten globules – Action of denaturants � Studies over 2 orders of magnitude in concentration – (1 ‐ 250 mg/ml) – Macromolecular crowding 20
Apo- and Ligand-bound Transferrin N ‐ terminal lobe of transferrin in the presence and absence of iron. Conversion from the ‘open’ apo form to the ‘closed’ ligand ‐ bound form occurs by bending around a hinge between the two domains in a ‘Venus flytrap’ ‐ like motion. The N ‐ terminal half undergoes a 63 o rotation of the N2 domain relative to the N1 domain in response to binding . Apo Fe ‐ bound WAXS data apo versus ligand ‐ bound Difference WAXS ‐ ligand ‐ bound minus apo measured curves CRYSOL ‐ predicted curves 21
Maltose Binding Protein and Dextrose The conformational change between the sugar ‐ bound and apo forms of MBP involves a 35° hinge bending movement accompanied by an 8° anti ‐ clockwise rotational twisting of the smaller N ‐ domain relative to the C ‐ domain, similar to the flytrap movement of transferrin. Difference WAXS ‐ ligand ‐ bound minus apo WAXS data apo versus ligand ‐ bound measured curves CRYSOL ‐ predicted curves 22
Alcohol Dehydrogenase and NADH When NAD+ binds the apo ‐ enzyme there is a rotational change of about 7.5° around a hinge axis passing through the contact point of the α‐ helices connecting the two domains. This change, classified as a shear motion according to Chothia and Lesk’s classification of domain motions, results in a change in the shape of the cleft to accommodate the substrate. WAXS data apo versus ligand ‐ bound Difference WAXS ‐ ligand ‐ bound minus apo measured curves CRYSOL ‐ predicted curves solid: w/ ligand dashed: w/o ligand Removed NADH from PDB file for w/o ligand 23
Detection of Ligand Binding to HEW Lysozyme Note error bars are indicated Dextrose: a non ‐ binder ( too lysozyme (black) short to occupy the binding lysozyme + dextrose (red) pocket) lysozyme (black) N ‐ acetylglucosamine (NAG), a lysozyme + NAG (red) relatively poor binder lysozyme (black) lysozyme + NAG3 (red) (NAG)3 , a potent enzyme inhibitor Changes in WAXS pattern reflect relative strength of ligand binding. 24
Denaturing Hb with Guanidine HCl Guanidine HCl concentration 1/d a ‐ helical content decreases as protein unravels Tetramer disassociated into ab- dimers Hb concentration = 25 mg/mL 25
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