study of biological macromolecules with a laboratory xrd ...pharmaceutical powder x -ray diffraction...

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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.

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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


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


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


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


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


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


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.


12


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%


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


• 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


14

In situ humidity study on HEWL

Surface view showing phase transitions

Decre

asin

g r

el. h

um

idity


15


Tetragonal HEWL phase at 95% Humidity

Pawley fit



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


Error bar corresponds to

0.2 mm sample

height uncertainty

17


Peaks of an un-indexed HEWL polymorph after Pawley fit of the Tetragonal

HEWL cell at 75% RH


18


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


19


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


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.


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


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


Kratky plot

Curve indicates a

compact, folded

structure

0

20000

0 1 2

Iq2

q [nm-1

]


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.


http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

http://dx.doi.org/10.1016/S0006-3495(99)77443-6

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.


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).


study of biological macromolecules with a laboratory xrd ...pharmaceutical powder x -ray diffraction...

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