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Study of Biological
Macromolecules with a Laboratory
XRD system
D. Beckers, T. Degen, G. Nenert, J. Bolze, PANalytical B.V., Almelo The Netherlands
S. Logotheti, S. Saslis, I. Margiolaki, University of Patras, Greece
V. Kogan, Dannalab, Enschede, The Netherlands
1 © Copyright (2015), PANalytical B.V.
This document was presented at PPXRD -Pharmaceutical Powder X-ray Diffraction Symposium
Sponsored by The International Centre for Diffraction Data
This presentation is provided by the International Centre for Diffraction Data in cooperation with the authors and presenters of the PPXRD symposia for the express purpose of educating the scientific community.
All copyrights for the presentation are retained by the original authors.
The ICDD has received permission from the authors to post this material on our website and make the material available for viewing. Usage is restricted for the purposes of education and scientific research.
ICDD Website - www.icdd.comPPXRD Website – www.icdd.com/ppxrd
2
Protein analysis
Challenges
Protein poly-crystallography
In situ humidity studies
SAXS on bio-macromolecules
© Copyright (2015), PANalytical B.V.
The challenge
Proteins are challenging samples:
Weak scatter
high intensity required (and low background)
linear detector / area detector (with high resolution)
Large molecules / cells
good low angle performance (peak position and asymmetry/
resolution)
high angular resolution
Often not stable under radiation
X-ray tube
Sample Focussing mirror
Linear / area
detector
Chosen configuration -
high intensity and
resolution © Copyright (2015), PANalytical B.V.
HEWL – Sample in capillary
HEW Lysozyme
Can easily be extracted
Well-known procedure for crystallization
Crystallization after approx. 46 hours
Pipetting into 0.5 mm capillary (manually compacted)
Tetragonal
Acknowledgement: sample supplied by B. Prugovečki , Laboratory of General and Inorganic Chemistry, Faculty of Science, University of Zagreb, Croatia
© Copyright (2015), PANalytical B.V.
Sample stability over the time - Lysozyme
Intensities drop during ~83 hours radiation (5x17 hours)
50
100
150
200Counts
Position [°2Theta]3.60 3.80 4
protein2_sum_C401_C500 protein2_sum_C301_C400 protein2_sum_C201_C300 protein2_SUM_C101_C200 protein2_sum_C1_C100
15% decay
© Copyright (2015), PANalytical B.V.
HEWL Lysozyme – capillary data
5 10 15 20 25 30 352Theta (°)
0
20000
40000
60000
80000
100000
120000
Inte
nsity (
counts
)
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.52Theta (°)
0
20000
40000
60000
80000
100000
120000
Inte
nsity (
counts
)
Background
corrected scan
FWHM
0.050o
© Copyright (2015), PANalytical B.V.
Tetragonal - Orthorhombic
Position [°2Theta]
2 4 6 8
Counts
0
100000
200000
1 1
0
2 1
0
1 0
10
1 1 1
1 1 2 1
1 1 2
1
1 3
0
3 0
13
2 0
3 1
14
0 0
4 1
01
3 1
3 2
1 0 0
2
4 0
1
4 1
1
2 0
2 2 4
0 1 2
2
1 4
14
3 0 3 4
05
1 1
0 3
24
3 1
3 2
25
2 1
6 0
06
1 0
0 5
1
ortho Crystal
system:
Orthorhombic
(Pawley fit)
a (Å) 68.418(7)
b(Å) 59.459(7)
c (Å) 30.745(3)
V (Å3) 125073
FOM 28.7
HighScore Plus
Position [°2θ] (Cu K-α12)
2 4 6
Counts
0
50000
100000
1 1
0
2 0
0
2 1
01
0 1
1 1
1
2 2
02
0 1
2 1
13
1 0
2 2
13
2 0
3 0
1
3 1
1
4 0
0
4 1
0 3 2
13
3 0 1
0 2
1 1
24
2 0
4 0
14
1 1
2 0
23
3 1 2
1 2
4 2
1 4 3
02
2 2
5 1
03
0 2
3 1
2
5 2
04
3 1
5 0
15
1 1
3 2
2 4 4
0
5 2
14
0 2
5 3
04
1 2
3 3
26
0 0
4 4
1 6 1
04
2 2
5 3
1
6 2
01
0 3
6 0
15
4 0
1 1
36
1 1 4 3
25
0 2
2 0
35
1 2
2 1
36
2 1
6 3
05
4 1 5 2
2 2 2
33
0 3
3 1
36
3 1
4 4
25
5 0
7 1
0
Tetragonal 193L Crystal
system:
Tetragonal
(Pawley fit)
a (Å) 79.095(6)
c (Å) 37.957(4)
V (Å3) 237458
FOM 24.8
© Copyright (2015), PANalytical B.V.
Bovine Insulin on HTS stage
2 4 6 8 10 12 14 16 182Theta (°)
0
500
1000
1500
2000
Inte
nsity
(cou
nts)
FWHM= 0.05O
• Crystallized at ph 5.6
• Placed into a HTS-plate
Data of ~20 min measurement on screening plate
Acknowledgement: sample supplied by I. Margiolaki, ESRF, Grenoble, France © Copyright (2015), PANalytical B.V.
Indexing
Hexagonal crystal system
a = 82.491 Å
c = 33.627 Å
V = 198167 Å3
Position [°2Theta] (Copper (Cu))
5 10 15
Counts
0
1000
2000
Simple Sum_1_insulin screen_B_1
Search Unit Cell Result 1
Resolution sufficient
for “automated”
indexing of Insulin
T3
R
3f
© Copyright (2015), PANalytical B.V.
Position [°2θ] (Cu K-α12)10 20 30
Counts
0
100000
200000
300000
Simple Sum_ProteinHQscan_40
Insulin
0
5000
-5000
10000
-10000
10
Protein polycrystallography
Insulin - Pawley fit, 4563 refined parameters, 5457 data points
© Copyright (2015), PANalytical B.V.
11
Protein in situ humidity study
Protein molecules in microcrystalline precipitates are surrounded by
solvent and their packing arrangement is retained by limited
intermolecular contacts.
A change in the crystal environment first affects the bulk solvent that
fills the intermolecular space, with resulting changes in the crystal
structure.
© Copyright (2015), PANalytical B.V.
12
Protein in situ humidity study
Sample preparation:
Crystallization (HEWL with 50
mg/ml, pH 4.4)
Concentration in centrifuge
Pipetting concentrate onto
transmission holders (~100μl
precipitate per sample) – sample
height varies during humidity cycle
Multiple samples to reduce radiation
damage
Humidity variation: 95% 51%
© Copyright (2015), PANalytical B.V.
1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,52Theta (°)
20000
40000
60000
80000
100000
Inte
nsity
(co
un
ts)
13
Protein in situ humidity study
• 3 hours / humidity step
• 30 min stabilization / 2.5 h data collection (3 scans)
• Here: results of 2nd humidity cycle
Decre
asin
g r
el. h
um
idity
© Copyright (2015), PANalytical B.V.
14
In situ humidity study on HEWL
Surface view showing phase transitions
Decre
asin
g r
el. h
um
idity
© Copyright (2015), PANalytical B.V.
15
In situ humidity study on HEWL
Tetragonal HEWL phase at 95% Humidity
Pawley fit
© Copyright (2015), PANalytical B.V.
In situ humidity study on HEWL
Pawley fit of Tetragonal phase from 95% to 79% rH
38,4
38,5
38,6
38,7
38,8
38,9
39
39,1
39,2
78,3 78,4 78,5 78,6 78,7 78,8 78,9
95%
91%
79%
83%
87%
c[Å
]
a[Å]
c versus a axis for 95% to 79% rH
© Copyright (2015), PANalytical B.V.
Error bar corresponds to
0.2 mm sample
height uncertainty
17
In situ humidity study on HEWL
Peaks of an un-indexed HEWL polymorph after Pawley fit of the Tetragonal
HEWL cell at 75% RH
© Copyright (2015), PANalytical B.V.
18
In situ humidity study on HEWL
Peaks could be explained by a second tetragonal phases
Position [°2θ] (Cu K-α12)
2 4 6
Counts
0
10000
20000
30000
40000
Simple Sum_1_protein fine B_pos5_20.9°C_75.0%
Tetragonal 1
Tetragonal 2
0
1000
-1000
2000
-2000
Phase 2
Phase 1
© Copyright (2015), PANalytical B.V.
19
In situ humidity study on HEWL
Pawley fit with two Tetragonal phases below 79% rH
38
38,5
39
39,5
40
40,5
41
41,5
42
42,5
78,2 78,4 78,6 78,8 79 79,2 79,4
79% 75%
71%
67% 63% 59%
79%
75%
71% 67%
63%
59%
c[Å
]
19 a[Å]
c versus a axis for 79% to 59% rH
© Copyright (2015), PANalytical B.V.
BioSAXS
Size
Shape (3D envelope)
Compactness and aggregation
behavior
Folding / unfolding
2nd virial coefficient
(molecule interaction)
The biomacromolecules are studied in their native environment,
i.e. in dilute (approx. 1 wt.%) solution. Crystallization is not
required. Available sample quantities are often very small.
These properties can be studied
as a function of temperature, pH,
buffer composition, etc.
© Copyright (2015), PANalytical B.V.
Glucose isomerase
21
• Enzyme (protein) produced by microorganisms (bacteria)
• Catalyzes the conversion of glucose to fructose
• Massive industrial use for the production of high-fructose syrups
• Popular protein to demonstrate SAXS measurement and analysis capabilities
sample (1.1 wt.%)
buffer
background-corrected
The signal from the protein is
well resolved, even in the
very low intensity region
© Copyright (2015), PANalytical B.V.
9.75
10.00
10.25
10.50
0 0.05 0.10
ln (I)
q2 [nm
-2]
Guinier plot / Pair distance distribution function p(r)
22
Radius of Gyration
Rg= 3.31 nm
Straight line at smallest angles - no
aggregation
0
5000
0 2 4 6 8 r [nm]
p(r)
[a.u.]
p(r) indicates that the protein has an
overall symmetric shape; maximum
dimension is approx. 9.7 nm
Dmax ~ 9.7 nm
Rg from Guinier plot Rg from p(r) Dmax from p(r)
Lab system 3.31 nm 3.32 nm 9.7 nm
Synchrotron (lit) 3.25 nm n.a. 9.7 nm
© Copyright (2015), PANalytical B.V.
Kratky plot
Curve indicates a
compact, folded
structure
0
20000
0 1 2
Iq2
q [nm-1
]
© Copyright (2015), PANalytical B.V.
3D-shape reconstruction
Software:
Dammin (EMBL) 1
De-smeared data
Fit by 3D model
Model fits the
experimental data
very well
1 D. I. Svergun (1999). Restoring low resolution structure of biological macromolecules from solution
scattering using simulated annealing. Biophys J. 2879-2886.
© Copyright (2015), PANalytical B.V.
Comparison with results from single
crystal diffraction
Simulation of SAXS
data from known single
crystal structures (using
CRYSOL1 from the
EMBL):
Experimental SAXS
data is in good
agreement with the
Tetramer structure.
(Rg values from
simulations)
Tetramer Rg = 3.28 nm
Monomer Rg = 2.44 nm
1 0 0
1 0 0 0
1 0 0 0 0
0 1 2
I n t . [ a . u . ]
q [ n m - 1 ]
1Svergun D.I., Barberato C. and Koch M.H.J. (1995) CRYSOL - a Program to Evaluate X-ray Solution
Scattering of Biological Macromolecules from Atomic Coordinates J. Appl. Cryst. , 28, 768-773.
© Copyright (2015), PANalytical B.V.
Conclusion
26
Multi-purpose X-ray laboratory diffraction systems offer a
range of possibilities to study biological macro
molecules:
- Protein polycrystallography (indexing, space group
determination)
- Polymorph identification / screening
- Analysis of structural changes of protein precipitates
upon dehydration and hydration
- Shape analysis, aggregation and folding of diluted
proteins in solution (SAXS).
© Copyright (2015), PANalytical B.V.