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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. What is a powder? - polycrystalline mass. - PowerPoint PPT Presentation
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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
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.
3
• • • • • • • • •
• • • • • • • • •
• • • • • • • • •
• • • • • • • • •
• • • • • • • • •
d*
1/
Powder diffraction - reciprocal space
so
s
Ewald sphere
2
Bragg’s Law sin* 2d
Spherical reflection shells
Smear in 3D
Typical 1-D scan
4
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
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
11BMB – 10min scan 1BM/MAR345 – 1sec exposure
Compare image plate with analyzer/detector
7
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
10
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
11
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
12
Multiple patterns – different lattice strains
Buffer effect:
Actually solvent effect?
phthalate (<pH5)Phosphate(>pH5)
Focus hereShortest exposure
13
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
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
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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)
18
Structure quality?
Ramachandran plot – 90% most favored
Total OMIT map – protein & H2O
19
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?
20
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
21
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
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); ~15g 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 ~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.
24
Detector development – spatial resolution
MAR345 – 300m ADSC – 100m
35m – ideal match with sample
25
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!
26
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