Copyright (c)JCPDS-International Centre for Diffraction Data 2002, Advances in X-ray Analysis, Volume 45.

FOCUSING POLYCAPILLARY OPTICS FOR DIFFRACTION H. Huanga, C. A. MacDonalda, W. M. Gibsona,b, J.R. Rublec, J. X. Hoc, D.C. Carterc, J. Chikd, A. Parsegiand, and I. Ponomarevb a. d.

Center for X-Ray Optics, University at Albany State University of New York, Albany, NY 12222 b. X-Ray Optical System, Inc., 30 Corporate Circle, Albany, NY12203 c. New Century Pharmaceuticals Inc., 85 Martin Rd., Huntsville, AL 35824 Laboratory of Physical and Structural Biology, National Institutes of Health Bethesda, MD 20892

ABSTRACT In this paper, we describe two low power systems using polycapillary focusing optics designed to collect Cu Ka radiation from Oxford ultra-bright micro-focus sources to produce a convergent beam for X-ray powder diffraction and protein diffraction measurements. A collimator with two apertures was used to block high-energy X-rays. A still data set for chicken egg-white lysozyme was collected with the low power source focusing optic combination. This data exhibits high quality when processed with a special software program. Several powder samples were also measured and compared with measurements taken with an Enraf-Nonius FR590 sealed-tube source based system.

1. INTRODUCTION Polycapillary optics are made of thousands of bundled bent hollow glass capillaries to focus and control X-rays or neutrons over broad capture angles (up to 30º) and energy ranges (200 eV - 80 keV) with high efficiency (10-50%). These tiny hollow glass tubes typically have diameters ranging between 5 and 50 micrometers1. X-ray beams are deflected by total external reflection from the capillary inner surface at very small angles2. Total external reflection for small angle scattering at the capillary walls occurs because the index of reflection n of glass is slightly less than the index of refraction from air. X-rays incident at angle less than a critical angle θc are totally reflected. Polycapillary optics have been successfully applied for protein diffraction applications recently.3-7 This paper concerns the development of low power systems using a polycapillary focusing optic combined with a low power micro-focus source for convergent beam X-ray powder diffraction and protein diffraction measurements. It is customary to use parallel or weakly focused X-ray beams for protein structure and powder diffraction measurements. However, the possibility of using a convergent X-ray beam for protein crystallography was first suggested by Wycoff and Agard in 19778. In this theoretical paper it was shown that the use of a one-dimensional convergent beam would not seriously impede structure determinations. More recently, it has been shown that convergent beams in two dimensions can also be used9-11. In such measurements the sample is fixed (not oscillated) during each exposure. Use of a convergent X-ray beam for diffraction, results in tangentially elongated diffraction spots, the basic theory for which is well understood10-11. The diffraction patterns from such measurements cannot be analyzed with standard software packages. Therefore, a measurement and analysis procedure and a special software package, CBMPRO has been developed specifically for processing data collected with the Converging Beam Method (CBM)11. A double-aperture collimator was used to block high-energy X-ray penetration and to define the beam intensity and convergence. A still data set for chicken egg-white lysozyme was collected with a low power source-focusing optic combination. These data give high quality results when processed with the CBMPRO program. Several polymer powder samples were also measured with this system and their qualities are compared with measurements of the same samples taken with an Enraf-Nonius FR590 sealed-tube source system. This investigation shows that the application of polycapillary focusing optics in powder diffraction and protein diffraction has a promising future.

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This document was presented at the Denver X-ray Conference (DXC) on Applications of X-ray Analysis. Sponsored by the International Centre for Diffraction Data (ICDD).

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Copyright (c)JCPDS-International Centre for Diffraction Data 2002, Advances in X-ray Analysis, Volume 45.

2. SYSTEM SETUP Figure 1 shows photos for the powder diffraction system and protein diffraction system.

Figure 1. The systems using polycapillary optics for powder diffraction (left) and protein diffraction (right).

The X-ray sources used in the two systems were Oxford ultra-bright micro-focus Cu sources. Following is the data sheet of the powder diffraction system source from Oxford Company: Anode Voltage (max): Measured Spot size at 20kV/20W:

60kV; Anode Current (max): 2 mA 18 µm; Measured Spot size at 60kV/20W: 15 µm

A double-aperture collimator was used to block high-energy X-rays, which pass directly through the optic and produce undesirable background. Figure 2 shows the combination of source, optic and collimator.

Figure 2. Source, optic and collimator combination.

Table 1 shows the parameters and transmissions for the two systems. Focusing optic 1106 was used for powder diffraction. Focusing optics 1265 and 1291 were used for protein diffraction. Focusing Optic # 1106

F-input (mm) 6.9

F-output (mm) 98

D-input (mm) 1.56

D-output (mm) ~4.0

Capture Angle 13o

Length (mm) 32.6

Transmission. % 19.5

1265

10.0

90

1.88

5.30

11o

48.8

20.0

1291

7.0

88

1.70

4.37

14o

48.8

18.8

Table 1. Optics parameters and transmissions.

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Copyright (c)JCPDS-International Centre for Diffraction Data 2002, Advances in X-ray Analysis, Volume 45.

3. EXPERIMENATAL RESULTS AND ANALYSIS 3.1. Protein diffraction Still diffraction patterns of lysozyme were taken with the source-focusing optic combination. Figure 3 shows one of these images with an enlargement of part of it. These images show that the diffraction spot shapes are tangentially elongated due to the strongly convergent beam. However they can still be analyzed separately by using the special software package.

Figure 3. Still diffraction image taken with source-strongly focusing optic combination.

The source-focusing optic combination system produced a high intensity beam in a small spot. For optic 1291, a whole data set of chicken egg white lyzosyme crystal with 42 still frames were taken, each frame was exposed 30 minutes. The overall R-factor was 9.5%. The beam divergence was about 15 mrad, and the crystal size was less than 0.2 mm.

3.2.

Powder diffraction

Several powder diffraction sets were taken with this low power powder diffraction system for organic powder samples provided by National Institutes of Health. A schematic of the setup for such measurements is shown on Figure 4 and results are shown in Figure 5 through Figure 8.

Setup for Powder Crystallography 12 µm Ni filter

Image Plate

Powder sample

source *

optic

collimator with two apertures

He pig

Figure 4. Setup for powder diffraction measurements.

Beam stop

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Copyright (c)JCPDS-International Centre for Diffraction Data 2002, Advances in X-ray Analysis, Volume 45.

80

328

B of hp01023a

70 60

Intensity

50 40 30 20 10 0 -10 0

5

10

15

20

2 theta (degree)

Figure 6. Radial integration of diffraction pattern with Nonius source.

Figure 5. Diffraction pattern of polymer with 900 W Nonius source, 30 min exposure.

These diffraction patterns have been integrated by a FIT2D program that was developed at the European Synchrotron Research Facility12.

120

B of optic1106 40kv40W7min

100

Intensity

80

60

40

20

0 -2

0

2

4

6

8

10

12

14

16

2 theta (degree)

Figure 8. Diffraction pattern of polymer with focusing optic and 40 W Oxford Ultrabrite source, 7 min. exposure.

Figure 7. Radial integration of diffraction pattern with Nonius source.

Copyright (c)JCPDS-International Centre for Diffraction Data 2002, Advances in X-ray Analysis, Volume 45.

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Table 2. Diffraction pattern analysis results by Gaussian fitting the diffraction peaks for 2 theta 5.5o to 10.5o of the Nonius system and the focusing optic system.

System

Collection Time (min)

Nonius system

30

Y0 16.7

Xc 8.4

W 2.5

A 66

Optic 1106 system

5

21.3

8.4

3.1

169

Optic 1106 system

7

36.3

8.4

3.3

260

In Table 2, Y0 is the Gaussian fitting base height to indicate the background level. Xc was used to determine the width of the diffraction ring 2 theta which was used to determine the unit cell space. The reason that the Xc values were not exactly the same was because the distance between the image plate and the sample and the direct beam position were not precisely measured. W is the Guassian fitting width which for the optic 1106 system was a little larger than that of Nonius system. This is because, compared to the Nonius system, the polycapillary optic beam had more high energy X-rays as well as some white radiation. A is the integration of the diffracted peaks which was used to compare the beam raw intensity. Compared with the Nonius system, 1106 system provided 15 times higher intensity, at 4.5% of the x-ray source power. Measurements were also made with a polycrystalline polymer powder sample. The diffraction pattern for this measurement is shown in Figure 9 and the radial integration is shown in Figure 10. Although this pattern has not been analyzed, it is clear that fine features in the pattern are clearly resolved.

Figure 9. Diffraction pattern of polycrystalline polymer powder sample with focusing optic and 40 W Ultrabrite source.

Figure 10. Radial integration of polymer powder diffraction pattern.

Copyright (c)JCPDS-International Centre for Diffraction Data 2002, Advances in X-ray Analysis, Volume 45.

4. CONCLUSIONS Strongly focusing polycapillary optics can be used for protein diffraction. This will have particular application for screening of small crystals for purity, quality and to obtain preliminary structural information, for example, during development of the crystal growth process. The high X-ray intensity and small spot size could reduce the data collection time as well as the crystal size requirement. These results also provide the basis for considering convergent beam diffraction for X-ray microdiffraction studies of strain and texture distributions with low power sources. In addition they suggest that convergent beam neutron diffraction may be viable for diffraction of proteins and other large molecules and for X-ray and neutron diffraction of small samples at low temperature or high pressure. This kind of optic can also be used for solving simple structure powder samples with lattice space less than ~100 Å (this is constrained by the system beam divergence.

5. REFERENCES 1. 2.

http://www.xrayoptics.com/products.htm “Polycapillary and Multichannel Plate X-Ray Optics”, C.A. MacDonald, chapter 30 in Handbook of Optics, Volume III, M. Bass, ed, (McGraw-Hill, New York, 2000) 30.1-30.1 3. “Polycapillary X-Ray Optics for Macromolecular Crystallography”, S.M. Owens, J.B. Ullrich, I.Yu. Ponomarev, D.C. Carter, R.C. Sisk, J.X. Ho, and W.M. Gibson, in Hard X-Ray/Gamma-Ray and Neutron Optics, SPIE Proc., Vol. 2859, 200-9 (1996). 4. “Low Power Polycapillary Based System for X-Ray Protein Crystallography”, F.A. Hofmann, W.M. Gibson, C.A. MacDonald, D.A. Carter, J.X. Ho, and J.R. Ruble, in Advances in X-Ray Analysis, Vol 43, Proc. of the 48th Annual Denver X-Ray Conf. (1999). 5. “First Results from a Macromolecular Crystallography System with a Polycapillary Collimating Optic and a Microfocus X-ray Generator”, M. Gubarev, E. Ciszak, I. Ponomarev, W. Gibson, and M. Joy, Jour. Appl. Cryst., 33 (3), 882-887 (2000) 6. “A Compact X-ray System for Macromolecular Crystallography”, M. Gubarev, E. Ciszak, I. Ponomarev, W. Gibson, and M. Joy, Rev. Sci. Instr., 71, 3900 - 05 (2000). 7. “Polycapillary Optic-Source Combinations for Protein Crystallography”, F.A. Hofmann, W.M. Gibson, C.A. MacDonald, D.A. Carter, and J.R. Ruble, J. Appl. Cryst., 34, 330-335 (2001) 8. “Pseudo-rotation Diffraction: The Use of a Convergent Beam of X-rays to Obtain Diffraction Patterns from Protein Crystals”, H.W. Wyckoff and D. Agard, Chapter 13 fro The Rotation Method in Crystallography, eds. U.W. Arndt and A.J. Wacott (North Holland, Amsterdam, 1977). 9. “Microdiffraction using Collimating and Convergent Beam Polycapillary Optics”, S.M. Owens, F.A. Hofmann, C.A. MacDonald, and W.M. Gibson, in Advances in X-Ray Analysis, Proc. Of the 46th Ann. Denver X-ray Conf., Vol. 41, 314-318 (1997). 10. “Stationary Crystal Diffraction with a Monochromatic Convergent X-Ray Source and Application for Macromolecular Crystal Data Collection”, J.X. Ho, E.H. Snell, C.R. Sisk, J.R. Ruble, D.C. Carter, S.M. Owens, and W.M. Gibson, Acta Cryst., D54, 200-14 (1998) 11. “Convergent Beam Method in Macromolecular Crystallography”, J.X. Ho, R.R. Ruble, T.R. McInnis, D.C. Carter, H. Huang and W.M. Gibson, Acta Cryst. (2002) (submitted for publication). 12. http://biocat1.iit.edu/fit2d/

6. ACKNOWLEDGEMENTS We would like to acknowledge the support of NASA under contracts NAS8-97247 and NCC8125 and also technical help from Thomas R. McInnis, Brenda S. Wright and R.C. Sisk.

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