High Brilliance Synchrotrons for SAXS and WAXS Robert Fischetti - - PowerPoint PPT Presentation

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

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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

X-ray Absorption Spectroscopy (XAS)

SSRL 1984 Cyril Applebee Max Perutz Brittan Chance

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Classical interpretation of XAS

Rh L and K absorption edges Metallic Rh K‐edges – closer look X‐ray interacts with atom and ejects a photoelectron Isolated atom “featureless” Atom in vicinity “interference”

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XAS and on-line optical monitoring

Experimental Setup Optical Spectroscopy FT of Data Processed Data Normalized Data Model data Carboxymyoglobin

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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, rij, (Debye Formula):

I(q) = Σ Ii(q) + 2 ΣΣ Fi(q) Fj(q) (sin(qrij)/(qrij))

Wide Angle X-ray Scattering (WAXS)

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Beamline Configuration – Sector 18 BioCAT

Sector 18 – Advanced Photon Source

Typical WAXS Setup

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.

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Scatter Intensity vs. Protein Concentration

Hb in PBS

0 mg/ml 100 mg/ml 1 mg/ml 10 mg/ml

Raw intensity patterns

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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

Protein density depends on molecular weight

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Protein (x10) Protein solution in capillary Empty capillary buffer

At wide‐angles, buffer scatters X‐rays more strongly than the protein displacing it in the protein solution ! Each data set is composed

• f scattering from

(i) Empty capillary (ii) Buffer in capillary (iii) Protein solution in capillary I(protein) = I(Prot. sol. in cap.) – I(cap.) ‐ (1‐vol%)*[I(buffer in cap.) – I(cap.) ]

Components of a WAXS Pattern (150 mg/ml HB)

Buffer in capillary MUST account for excluded volume

1/d (A‐1) = 2*sin(θ)/λ q (A‐1) =2π/d q(nm‐1) = 10*q(A‐1)

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Wide Angle Scattering Setup – Version 2

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.

guard slits flow cell mica exit window

helium

beam stop 12 µm mylar

1.2 mm pin hole helium Mar 165 CCD 29.6 X 29.6 μm 4k X 4k pixels 2.7:1 taper 2x2 binned 360°

He – no windows O‐ring

vacuum 180 mm q = 0.006 – 0.46 1/A

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1/d

0.0 0.1 0.2 0.3 0.4

intensity

2e+4 4e+4 6e+4 8e+4 1e+5

Minimizing radiation damage

Stationary samples show clear signs of degradation

Radiation damage results in weakening of peaks and filling in of troughs 32 sec data collection No protein in the beam for more than 0.1 sec 5 sec data collection Each protein may be exposed in the beam for up to 5 sec Solution scatter from Hb flow cell (red) stationary sample (black)

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

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B A C D

Secondary structures

Alpha helices

• 1.5 Å axial separation of AA
• 5.4 Å pitch
• 10 Å diameter

Beta sheets

• 4.7 Å strand‐to‐strand distance
• 7.0 Å pleat distance

Quaternary structure (shape and size) Tertiary structure (inter‐domain distances) Secondary structure Primary structure

Molecular Dimension in a WAXS Pattern

1/d

0.0 0.1 0.2 0.3 0.4

intensity

10 20 30 40 50 60

Experimental – red Crysol calculated ‐ black cytochrome C

Cytochrome C

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myoglobin

Myoglobin

Experimental – black Crysol calculated ‐ red

1/d

0.0 0.1 0.2 0.3 0.4

intensity

1000 2000 3000 4000

Myoglobin experimental ‐ black Hemoglobin experimental ‐ red

Peak from quaternary structure Peak at 0.1 A‐1 is indicative of alpha helical content Peak at 0.2‐0.26 A‐1 is relatively invariant Peaks from tertiary structure

hemoglobin myoglobin

Comparison of Myoglobin and Hemoglobin

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1/d

0.0 0.1 0.2 0.3 0.4

intensity

1000 2000 3000 4000 5000

Myoglobin: 88% α‐helical (black) Bovine serum albumin: 78% α‐helical (blue) and superoxide dismutase: 17% α‐helical (red) myoglobin serum albumin

Peak at 0.1 A‐1 is indicative

• f alpha helical content

superoxide dismutase

WAXS is sensitive to a–helical content

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1/d

0.0 0.1 0.2 0.3 0.4

intensity

1000 2000 3000 4000 5000

hemoglobin streptavidin

Comparison of Hemoglobin and Streptavidin

Hemoglobin experimental ‐ blue Streptavidin experimental ‐ red a-helix vs. β-sheet

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1/d

0.0 0.1 0.2 0.3 0.4

intensity

1000 2000 3000 4000 5000 1/d vs calmod-solv-cap 1/d vs Mb-solv-cap

Calmodulin 13.3 mg/ml in HEPES/ 30 µM CaCl2

1/d

0.0 0.1 0.2 0.3 0.4

intensity

1000 2000 3000 4000 5000 1/d vs calmod-solv-cap 1/d vs Mb-solv-cap

Comparison of Calmodulin and Myoglobin

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• pen form

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• 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

Use WAXS to study structural fluctuations

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Difference WAXS ‐ ligand‐bound minus apo

Apo Fe‐bound measured curves CRYSOL‐predicted curves

WAXS data apo versus ligand‐bound

N‐terminal lobe of transferrin in the presence and absence

• f 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 63o rotation of the N2 domain relative to the N1 domain in response to binding .

Apo- and Ligand-bound Transferrin

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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

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Alcohol Dehydrogenase and NADH

Difference WAXS ‐ ligand‐bound minus apo WAXS data apo versus ligand‐bound

measured curves CRYSOL‐predicted curves solid: w/ ligand dashed: w/o ligand

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.

Removed NADH from PDB file for w/o ligand

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Detection of Ligand Binding to HEW Lysozyme

lysozyme (black) lysozyme + NAG (red) lysozyme (black) lysozyme + NAG3 (red) lysozyme (black) lysozyme + dextrose (red) Changes in WAXS pattern reflect relative strength of ligand binding. Dextrose: a non‐binder ( too short to occupy the binding pocket) N‐acetylglucosamine (NAG), a relatively poor binder (NAG)3 , a potent enzyme inhibitor

Note error bars are indicated

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Hb concentration = 25 mg/mL 1/d

Denaturing Hb with Guanidine HCl

Guanidine HCl concentration a‐helical content decreases as protein unravels Tetramer disassociated into ab-dimers

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Forms molten globules at EtOH concentrations of 25‐40%

Molten Globules of b-lactoglobulin

b‐lactoglobulin

Hb and Lysozyme Concentration Series Scaled

Hb 50 mg/ml (black) 4 mg/ml (red) Lysozyme 50 mg/ml (black) 4 mg/ml (red) Superposition of 20 scattering patterns from 1‐300 mg/ml At concentrations above 50 mg/ml the patterns are virtually superimposed Hb 1 – 300 mg/ml Lysozyme 1 – 300 mg/ml

Differences are statistically significant at least to 0.2 Å‐1 Differences are not statistically significant

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1/d = 0.0253 0.0400 0.0981 1/d = 0.0523 0.2216 0.0783

Hb and Lysozyme scattering as a function of concentration

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Scattering patterns were scaled by comparison of scattering at low to moderate angles (1/d = 0.0=0.15) Volume excluded by protein was estimated by comparison of the calculated scattering due to protein with a reference curve at wide angles (1/d = 0.15‐0.35) Both calculations indicated that under the conditions used, Hb had a solubility of about 250 mg/ml

Scaling Hb dilution series

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Scaling of WAXS from lysozyme 1‐300 mg/ml

Scattering patterns were scaled by comparison of scattering at low to moderate angles (1/d = 0.0=0.15) Volume excluded by protein was estimated by comparison of the calculated scattering due to protein with a reference curve at wide angles (1/d = 0.15‐0.35) Both calculations indicated that under the conditions used, lysozyme had a solubility of about 175 mg/ml

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Fluctuations were modelled by replacing every vector in the autocorrelation function by a distribution of vector lengths – The distribution was modeled as a Gaussian that varied in width according to the length of the vector being replaced Autocorrelation function at low concentration is then related to that of a reference structure by: al(r) = ar(r)*exp(‐s(r)2/2r2) s(r) α r0 independent motion around equilibrium positions s(r) α r1/2 cumulative widths s(r) α r ~rigid body motions

R

20 40 60 80 100 120 140

pair correlation function * R**2

100 200 300 400 500 600 R vs 47*R**2 R vs 4*R**2

Modeling fluctuations at low concentration

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Disorder modeling

1/d

0.0 0.1 0.2 0.3 0.4

relative intensity

500 1000 1500 2000 2500 3000 1/d vs hb47 1/d vs 0.09 1/d vs 0.06

1/d

0.0 0.1 0.2 0.3 0.4

relative intensity

500 1000 1500 2000 2500 3000 1/d vs 0.0 1/d vs 0.45 1/d vs 0.36

1/d

0.0 0.1 0.2 0.3 0.4

relative intensity

500 1000 1500 2000 2500 3000 1/d vs hb47 1/d vs 0.9 1/d vs 1.26

Crystal fluctuation model: σ is constant; al(r) = ar(r)*exp(‐σ2/2r2) Cumulative disorder model: σ α r0.5 ; al(r) = ar(r)*exp(‐rσ2/2r2) Rigid body motion model: σ α r1.0 ; al(r) = ar(r)*exp(‐r2σ2/2r2)

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XAS and WAXS

Used XAS to measure Fe K‐edge and monitor redox state

feed tube to pump

Portable WAXS setup with sample Autoloader

APS Beamline 12ID‐B New Endstation

SAXS WAXS

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All are motorized: Sample stages(x,y,θ) SAXS detector SAXS chamber beamstops Dedicated for : Material SAXS/WAXS Bio‐SAXS/WAXS GISAXS

12ID‐B is a new beamline built during 12ID upgrade.

• pinhole camera with variable sample –

detector distance

• evacuated flight path (0.2m – 3.5m)
• semi‐automatic distance change
• large area CCD detector MX225HE
• fast PAD detector Pilatus 300k

SAXS Instrument at SSRL BL4‐2

• beamstop with integrated photodiode

(transmitted intensity)

• variety of sample environment
• customized Blu‐ICE control system for the

whole instrumental hardware (including

• ptics, detectors and sample environments)

SSRL ‐ Automated Solution SAXS

test

ESRF BioSAXS BM29:

ESRF Bio-SAXS Beamline BM29

Highly automated SAXS from macromolecules in solution

ESRF BioSAXS BM29:

ESRF Bio-SAXS Beamline BM29

• In use since September 2010
• Sample capacity up to 3x96 well

plates from 0.2 to 2 mL

• Pipetting and mixing functions

enabling remote sample manipulation

• Scriptable operation for completely

automatic data collection

• Now installed at Beamline
• Online purification immediately

before data collection

• Automatic calculation of Rg, I0,

Dmax and Volume for every frame

• Automatic merging of all like

frames (based on I0 and Rg) Automated SC developed by EMBL-GR, EMBL-HH and ESRF Online HPLC Pilatus 1M

• Noise free and shutter less

readout enabling

• acquisition of timeframes to

monitor radiation damage

• Increased active area giving

greater resolution

• s=0.05 to 6 nm-1 (s=4πSinθ/λ)

Highly automated SAXS from macromolecules in solution

GM/CA dual canted undulator beamlines at the APS

23‐ID‐D 5 – 20 keV 20 x 65 μm2 23‐ID‐B 3.5 – 20 keV 25 x 120 μm2 23‐ID‐B 3.5 – 20 keV 25 x 120 μm2 Bimorph mirrors 5, 10, 20 μm 1 μm 3.0 3.3 Undulator period (cm) Zone plate

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3.0 cm device optimized for Se MAD phasing Rapid energy tunability High intensity and positional stability Large unit cells of biomolecules low convergence optics

Micro-crystallography developments

Goniometer: 1 μm SOC peak‐to‐peak Goniometer head: nano‐positoning Active beamstop: Photoelectric effect On‐axis sample visualization 6x15 um2 Sample environment Quad mini‐beam collimator: 5, 10, 20‐µm beams and 300‐ µm scatter guard

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SONICC

Rapid beam size selection

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Image of beam at sample position on YAG crystal Quad mini‐beam collimator

• match beam and crystal size
• use small beam to probe large crystal

Beam size FWHM (μm) Intensity (Ph./sec) 20 x 65 2.0 x 1013 20 ∅ 1.0 x 1012 10 ∅ 5.2 x 1011 5 ∅ 5.4 x 1010 1 ∅ 3.0 x 109

JBluIce‐EPICS GUI

Benefit: matching beam size to crystal – improved I/σ

70 x 25 μm standard beam

Reflection at d ≈ 2.9 Å

Sanishvili et al. Acta Cryst. D64, 425‐435 (2008)

Reflection at d ≈ 25 Å

8 x 6 μm mini‐beam

Peak 159 Bkgr 90 S/N = 1.7 Peak 80 Bkgr 27 S/N = 2.6 Peak 5180 Bkgr 200 S/N = 20 Peak 3627 Bkgr 45 S/N = 65

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Ratio of S/N: 1.53 Ratio of S/N: 3.25

Benefit: finding a homogeneous region of a large crystal

Same slice of diffraction space from an inhomogeneous crystal

Nukri Sanishvili

Mini‐beam (10 μm) “Standard” beam (120 x 30 μm)

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Additional benefits of the mini-beam

Finding best spots of the crystal for data collection

30 μm 0.175 0.200 0.100 0.080 0.100 0.110 0.190 0.200 0.190 0.210 0.310

10 frames of 0.2o data were collected from each of the 11 spots along the 250 micron long crystal. Mosaicity values are shown as refined by HKL2000

Best region of the crystal Nukri Sanishvili Beware of bent crystals!

Finding/centering invisible crystals or mapping quality

Ranking by “distl” Nick Sauter

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Grid search developed at ID13 Big beam – beam sample implemented at SSRL (J. Syn.

• Rad. (2007) 14, 1891‐195)

GM/CA large‐beam (coarse grid) and mini‐beam (fine grid) implementation (J. R. Soc. (2009) Interface , 6, S587‐S597) Diamond and now many others have implemented rastering Acta Cryst. D, 66, 1032‐1035 (2010)

M.Hilgart, R.Sanishvili, C.Ogata, M.Becker, N.Venugopalan, S.Stepanov, O.Makarov, J.L.Smith, and R.F.Fischetti, Automated sample scanning methods for radiation damage mitigation and diffraction‐ based centering of macromolecular crystals, JSR (2011) 18, 717‐722

AutoFind

• Produces a search area

(polygon) definition

• First performs optical

centering if needed

Takes four images at angles 0, 30, 60, 90 Uses XREC to generate loop

• utline

Sets the sample to face‐on orientation Total time is about 40 seconds

• Adds a critical link from

screening to analysis

• The next step in

automation is to link this to the screening tab

AutoFind automatically generates a search polygon

Dealing with radiation damage – automated collection along a user defined vector

Efficient use of large homogeneous crystals

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Mark Hilgart and Craig Ogata

G(guanine nucleotide) Protein Coupled Receptors

GPCRs are very important trans‐membrane proteins

• they transmit signals across the cellular membrane
• they are the target of many therapeutic drugs such as

beta blockers, and histamine

• they allow us to see and smell
• common structure of 7 trans‐membrane α‐helices
• know to be >800 receptors in the family

Structural Biology 2000 – Bovine Rhodopsin, photoreceptor (2.8Å) [ESRF] 2006 – Kobilka worked on Human β2‐adrenergic receptor Initial experiments at the ESRF’s ID‐13 and ID‐23‐2 Manfred Burghammer and Gebhard Schertler, 2007 – Kobilka brought the project to APS’s GM/CA Ruslan (Nukri) Sanishvili and Robert Fischetti

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Soren G.F. Rasmussen, …, Manfred Burghammer, …, Ruslan Sanishvili, Robert F. Fischetti, Gebhard F.X. Schertler, William I. Weis, Brian K. Kobilka, "Crystal structure of the human adrenergic β2-Adrenergic G-protein-coupled receptor," Nature 450, 383-387 (2007). DOI: 10.1038/nature06325

Crystals diffracted weakly and were radiation sensitive

Brian Kobilka & Bill Weis: Human β2‐adrenergic receptor

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Quad mini‐beam collimator: 5, 10, or 20‐µm beam or 300‐ µm scatter guard

Mini‐beam development was driven by science starting in 2007

Kobilka team collected data 7 times at GM/CA in 2007

Rasmussen et.al. & Kobilka, Nature 450, 383‐387 (2007) Cherezov et. al. & Kobilka and Stevens, Science 318, 1258‐1265 (2007)

In‐meso phase crystallization of G‐protein coupled receptors

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Cherezov et. al. & Kobilka and Stevens, Science 318, 1258‐1265 (2007)

10 µ crystals in meso or LCP

Vadim Cherezov (Martin Caffrey’s Lab) Technology to Ray Stevens Lab

GPCR Structures solved by the Kobilka group

β2 adrenergic receptor‐ Gs protein complex

Data from APS and ESRF

Soren G.F. Rasmussen, …, Manfred Burghammer, …, Ruslan Sanishvili, Robert F. Fischetti, Gebhard F.X. Schertler, William I. Weis, Brian K. Kobilka, "Crystal structure of the human adrenergic β2-Adrenergic G-protein-coupled receptor," Nature 450, 383-387 (2007). DOI: 10.1038/nature06325

Data from APS

Vadim Cherezov, , William I. Weis, Brian K. Kobilka, Raymond C. Stevens, "High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor," Science 318, 1258-1265 (2007). DOI: 10.1126/science.1150577 Daniel M. Rosenbaum, …, William I. Weis, Raymond C. Stevens, Brian K. Kobilka, "GPCR Engineering Yields High-Resolution Structural Insights into β2-Adrenergic Receptor Function," Science 318, 1266-1273 (2007). DOI: 10.1126/science.1150609 Michael P. Bokoch, … William I. Weis, …, Brian K. Kobilka, "Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor," Nature 463, 108-112 (2010). DOI: 10.1038/nature08650 Soren G.F. Rasmussen, …, Roger K. Sunahara, …, William I. Weis, Brian K. Kobilka, "Structure of a nanobody-stabilized active state of the β2-Adrenoceptor," Nature 469, 175-180 (2011). DOI: 10.1038/nature09648 Daniel M. Rosenbaum, …, William I. Weis, Martin Caffrey, Peter Gmeiner, Brian K. Kobilka, "Structure and function of an irreversible agonist-β2-Adrenoceptor complex," Nature 469, 236-240 (2011). DOI: 10.1038/nature09665 Søren G.F. Rasmussen,.., M. Caffrey,.., W.I. Weis, R.K. Sunahara, B.K. Kobilka, "Crystal structure of the β2-Adrenergic receptor–Gs protein complex," Nature 477, 549-555 (2011). DOI: 10.1038/nature10361

GPCR Structures solved by the Kobilka group-continued

A pair of u‐opioid receptors

Data form APS

Kazuko Haga, …, Brian K. Kobilka, Tatsuya Haga, Takuya Kobayashi, "Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist," Nature 482, 547-551 (2012). DOI: 10.1038/nature10753 Andrew C. Kruse, …, William I. Weis, Jürgen Wess, Brian K. Kobilka, "Structure and dynamics of the M3 muscarinic acetylcholine receptor," Nature 482, 552-556 (2012). DOI: 10.1038/nature10867 Aashish Manglik, …, William I. Weis, Brian K. Kobilka, Sébastien Granier, "Crystal structure of the µ-opioid receptor bound to a morphinan antagonist," Nature 485, 321-326 (2012). DOI: 10.1038/nature10954 Sébastien Granier, …, William I. Weis, Brian K. Kobilka, "Structure of the d-opioid receptor bound to naltrindole," Nature 485, 400-404 (2012). DOI: 10.1038/nature11111

2012 Nobel Prize in Chemistry

Structural Biology of GPCRs 2007 ‐ Human β2‐adrenergic receptor (3.4Å) 2007 ‐ Human β2‐adrenergic receptor (2.4Å) 2008 ‐ Human A2A Adenosine receptor (2.6Å) 2010 ‐ Human β2‐adrenergic receptor ‐ ACTIVE State 2011 – Human β2‐adrenergic receptor‐active‐complex

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Brian Kobilka Stanford University Robert Lefkowitz Duke University Discovery of GPCR Using radioisotopes

APS, ESRF and SSRL all posted congratulations Brian and Bob

G‐protein‐coupled receptor (blue) turned on by a signaling molecule (yellow spheres, top) and activating a G protein (red, gold and green).

Other GPCR Highlights from 2012 GM/CA

Data from APS

Liu, W., …, Cherezov, V., and Stevens R.C. (2012), Science 337, 232-236. Human A2a adenosine receptor Chun, E., …, Cherezov, V., Hanson, M.A., and Stevens, R.C. (2012), Structure 20, 967-976. Methods Wu, H., …, Cherezov, V., and Stevens, R.C. (2012), Nature 485, 327-332. Human κ-opioid receptor in complex with JDTic Thompson, A.A., …, Cherezov, V., and Stevens, R.C. (2012), Nature 485, 395-399. Nociceptin/orphanin FQ receptor in complex with a peptide mimetic Hanson, M.A., …, Kuhn, P., Rosen, H. and Stevens, R.C. (2012), Science 335, 851 – 855. Lipid G Protein-Coupled Receptor Li, D., Lee, J. and Caffrey, M. (2011), Cryst Growth Des 11, 530 – 537 Methods White, J, … Grishmann, R. – (2012) Nature , www.nature.com/doifinder/10.1038/nature11558 Against-bound Neurotensin Receptor

Other Sources

Gebhard Schertler - Paul Scherrer Institut, Villigen PSI, 5232, Switzerland Turkey, β1 Adrenergic Receptor A pair of u‐opioid receptors

Other Membrane Protein Publications in past 12 months

• Liao, J., …, and Jiang, Y. (2012), Science 335, 686‐690.

Sodium/calcium exchanger

• Brohawn, S. G., …, and MacKinnon, R. (2012) , Science 335, 436‐441.

Human K2P TRAAK, a lipid‐ and mechano‐sensitive K+ ion channel

• Whorton, M. R., and MacKinnon, R. (2011) , Cell 147, 199‐208.

Mammalian GIRK2 K+ channel and gating regulation by G proteins, PIP2, and sodium

• Uysal, S., …, Kossiakoff, A. A., and Perozo, E. (2011) , Proc Natl Acad Sci U S A 108, 11896‐11899.

Activation gating in the full‐length KcsA K+ channel

• Shi, N., …, and Jiang, Y. (2011), J Mol Biol 411, 27‐35.

Determinants of K channel conductance and gating.

• Sauer, …, and Jiang, Y. (2011) , Proc Natl Acad Sci U S A 108, 16634‐16639.

Protein interactions central to stabilizing the K+ channel selectivity filter.

• Derebe, M. G., …, and Jiang, Y. (2011) , Proc Natl Acad Sci U S A 108, 598‐602.

Tuning ion selectivity of tetrameric cation channels by changing the number of ion binding sites

• Noinaj, N., ..., and Buchanan, S. K. (2012), Nature 483, 53‐58.

Structural basis for iron piracy by pathogenic Neisseria

• Fairman, J. W., ..., Cherezov, V., and Buchanan, S. K. (2012), Structure 20, 1233‐1243.

Outer Membrane Domain of Intimin and Invasin from Enterohemorrhagic E. coli and Enteropathogenic Y. pseudotuberculosis

• Oldham, M. L., and Chen, J. (2011), P Natl Acad Sci USA 108, 15152‐15156.

Maltose transporter during ATP hydrolysis

• Symersky, J., ..., and Mueller, D. M. (2012), Nat Struct Mol Biol 19, 485‐491

c(10) ring of the yeast mitochondrial ATP synthase in the open conformation

• Tiefenbrunn, T., ..., and Cherezov, V. (2011) , PLoS One 6, e22348.

ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment

Crystal structure of the Na+/Ca2+ exchanger embedded in a membrane bilayer Y east mitochondrial A TP synthase c10-ring at pH 8.3

SONICC on the beamline Garth Simpson’s Group at Perdue U.

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Second Order Nonlinear Imaging in Chiral Crystals Sample SONICC receiver lens on translation stage SONICC modulator Phenylalanine hydroxylase (Judith A. Ronau & Chittaranjan Das, Purdue) SONICC image ~40 sec Diffractive raster image ~15 min (20μm x 20μm raster cells) No laser induced radiation damage (manuscript submitted) Mike Becker & Chris Dettmar Purdue IMCA SBC GM/CA

How long can you collect on one spot?

Garman limit1 ~ 3.0 x 107 Gray (35% intensity loss) Deposited energy in sample – not incident energy!

Beam Size at sample, FWHM (μm) Intensity (Photons/sec) Dose Rate 2 (Grays/sec) Time to Garman Limit (sec) Full 20 x 65 2.0 x 1013 6.1 x 106 4.9 10-μm 10.6 x 11.6 1.3 x 1011 4.2 x 105 71.4 5-μm 4.8 x 6.2 2.7 x 1010 3.6 x 105 83.3 1-μm 1.1 x 1.2 3.0 x 109 5.1 x 105 58.8

1 Owen, R.L., Rudino‐Pinera, E. & Garman, E.F. Proc Natl Acad Sci U S A 103, 4912‐7 (2006) 2 RADDOSE http://biop.ox.ac.uk/www/garman/lab_tools.html

50 μm thick Lysozyme crystal; E= 12.66 keV

58 ACA 2010, Fischetti, et. al.

Cryo‐cooled crystals

Intensity loss as a function of beam size and dose

Damage decreases 3‐fold with beam size

Nukri Sanishvili and Derek Yoder

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Incident X‐ray beam Polarization Vector

θ

Distribution of damage is wider than beam

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HWHM (μm) Ratio Beam profile 0.42 1.0 Horizontal distribution 2.02 4.8 Vertical distribution 1.19 2.8

X‐ray beam Crystal face

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18.5 Cowan, J.A. & Nave, C., JSR 15, 458‐62 (2008).

Inelastic/Elastic Scattering

Comparison of Monte Carlo simulations and our data

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Microfocus Upgrade Motivation

Provide more intensity for challenging projects Membrane proteins in meso‐phase Small (5‐10 µm) and weakly scattering crystals Provide routine access to microfocus beam ‐ ~1 µm Exploit APS high energy source properties Provide high energy and/or small beams Study radiation damage at higher energies µ‐opioid GPCR Brian Kobilka’s lab Radiation damage k‐opioid GPCR Ray Steven’s lab

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Conclusions

WAXS data:

• Can be collected to high q without measurable protein degradation
• Contains information pertinent to the secondary and tertiary structure of proteins
• Contains significant fold information
• Is sensitive to small conformational changes induced by ligand binding
• Sensitive to structural changes due to denaturants
• Can provide information on disorder in the protein

MX is powerful technique

• Require crystals – still the bottle neck, but improving
• Microcrystallography is having a large impact

Acknowledgements

GM/CA CAT is funded by the National Cancer Institute and the National Institute of General Medical Science. The Advanced Photon Source is supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science. GPCR work Stanford University Brian Kobilka Bill Weiss Dan Rosenbaum Hee‐Jung Choi Scripps Research Inst. Ray Stevens Peter Kuhn Vadium Cherezov

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Radiation Damage work Gerd Rosenbaum – U. of Georgia Stefan Vogt – Argonne Nat. Lab. APS Staff Glenn Decker Louis Emery Karen Schroeder WAXS

• Argonne National Laboratory

– Lee Makowski – Diane J. Rodi – David Minh – Satish Devarapalli – Suneeta Mandava

• BioCAT Scientists and Staff

– Tom Irving – David Gore – Elena Kondrashkina – Ahmed Mirza

ESRF Structural Biology Group Thomas Weis at SSRL APS Xiaobing Zuo

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Acknowledgements

• Argonne National Laboratory

– Lee Makowski – Diane J. Rodi – David Minh – Satish Devarapalli – Suneeta Mandava

• BioCAT Scientists and Staff

– Tom Irving – David Gore – Elena Kondrashkina – Ahmed Mirza

GM/CA@APS Staff

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www.gmca.aps.anl.gov

From left to right: Mark Hilgart Craig Ogata Robert Fischetti Sergey Stepanov Dale Ferguson Janet Smith Oleg Makarov Shenglan Xu Michael Becker and Sudhir Babu Pothineni Insets left to right: Sheila Trznadel Ruslan (Nukri) Sanishvili Naga Venugopalan and Stephen Corcoran

Thank you for your attention