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

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What is a powder? - polycrystalline mass

All orientations of crystallites possible

Sample: 1l powder of 1m crystallites - ~109 particles

Single crystal reciprocal lattice - smeared into spherical shells- the overlap problem – lost information

Packing efficiency – typically 50%Spaces – air, solvent, etc.

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d*

1/

Powder diffraction - reciprocal space

so

s

Ewald sphere

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Bragg’s Law sin* 2d

Spherical reflection shells

Smear in 3D

Typical 1-D scan

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Problem – Severe radiation damage of proteins

Much worse at APS!! Happens in 10-20min!!

0 3 6 9 12 15 18 21 242, deg

Powder patterns: Lysozyme – Multiple scans @ 1.15Å, RT, ~3hr ea.; ~10mg HEWL

Radiation damage – initial observations @NSLS X3b1Xtal/analyzer detector

1 day

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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”

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11BMB – 10min scan 1BM/MAR345 – 1sec exposure

Compare image plate with analyzer/detector

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Rings – protein pattern (HEWL) – X-rays 30s @ 20kV on MAR345; <1mg HEWL

Inner most ring – d~55Å(110) Reflection, lowest order for tetragonal lysozyme2 ~ 0.67deg

Beam stop holder

Texture free sample & no graininess –1m “perfect” powderResolution limit – 1.85ÅResidual solvent scattering –

background

(Air, solvent & Kapton background subtracted) ~9000 Fhkl for HEWL >2Å

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Powder diffraction from 2D image plates (MAR345)

Best focusing – best resolutionFIT2D cake integrationto dmin = 2Å “resolution”

~0.035o FWHM300m beam/pt. spread fxn.~4X sample contribution!

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HEWL comparison – 30s on 1BM Mar345 vs 10min on 11BM

20keV

30keV

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Background problem – subtract air, liquid & Kapton

Before subtraction:Gave too small Rwp (<0.5%)from high backgroundNo sensitivity to structure

After background subtraction:Weights:

Compensate for 2D detectorintegration effect

Iw 01.0

2tan2

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Radiation damage – reflection intensities & positions

APS 1BM - 30s exposures + 150s delay, 300KFull sequence wrt NaCl & pH – effects??Immediate changes seen2 stages? - <10min & >10min exposure

Focus here:a up & c down

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Multiple patterns – different lattice strains

Buffer effect:

Actually solvent effect?

phthalate (<pH5)Phosphate(>pH5)

Focus hereShortest exposure

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Solvent & radiation damage induced lattice strains for HEWL

Salt & pH effects Radiation exposure ~8% loss in 4.5m

cf. FWHM 0.035o2 ~ 0.02% strain @18o2

Compare: [M] = 0.07M; pH = 0.07 ([H+]~-15%) in 4.5m

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tan)()2( 2231312

233

222

211 dklhlhklkh

tan])([)2( 2233

2211 dlkh

%100**)(90 211

aa

a

Analysis: Induced lattice strain model – obey Laue symmetry

231312332

222

1122* 222 klghkghkgglgkghd T ghh

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

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2.5-2.0Å resolution range – NaCl sequence

0.25M

1.25M

Peak shifts!

Obsd & calcd powder patterns

Also for rad. dam.– just less

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Profile fit – 1/5 patterns; Rwp=1.84%

4x20x

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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)

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Structure quality?

Ramachandran plot – 90% most favored

Total OMIT map – protein & H2O

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HEWL IpH4

HEWL IIpH5

HEWL IIIRad dam

HEWL I all/backbone, ÅVary NaCl, pH4

--- 0.44/0.33 0.42/0.34

HEWL II all/backbone, ÅVary NaCl, pH5

0.44/0.33 --- 0.55/0.40

HEWL III all/backbone, Å1.25M NaCl, pH4, rad dam

0.42/0.34 0.55/0.40 ---

6LYT all/backbone, ÅBest RT SC structure

1.35/0.75 1.37/0.77 1.38/0.77

194L all/backbone, ÅStarting model

1.25/0.75 1.28/0.76 1.28/0.77

HEWL results – structure comparisons

Appropriate for 2Å resolution?

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Compare – single pattern result (1JA2) & best single xtal

1JA2 - powder PDB5 NaCl patterns - this workRad. 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

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Overlap factor – effect of lattice strainbetter “effective” resolution?

Overlap factor, Rij=1-ij/2FWHM=1 if complete overlap,=0 if no overlapFi=min(Rij) for multipatterns

2 patterns5 patterns

~0 >2FWHM

SC = 0

1 pattern

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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); ~15g HEWL slurry; 10s exposure

2 of 1536 well plate – xtal growth test – not particularly optimized – corners <3Å

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High throughput screening for crystallization – X-ray

8BM ~12keV1536 well plate1st “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.

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Detector development – spatial resolution

MAR345 – 300m ADSC – 100m

35m – ideal match with sample

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Tileable area detectors?

Cover “best” part of powder patternTilted array – avoid “blind” spots in powder pattern

Cover curved area to match resolution?Not necessarily spherical!

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Conclusion – data combinations in proteins (at least HEWL)

Protein powder diffraction Image plates – lower powder resolution, butBetter intensity measurement to higher diffr. resolutionInduced 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 resolutionStructure solution – multiple data set extraction of Fo