Microdroplet Sample Mounting Techniques in Small Angle X-Ray. Scattering. Andrew P. Huang

Transcription

1 Microdroplet Sample Mounting Techniques in Small Angle X-Ray Scattering Andrew P. Huang Office of Science, Science Undergraduate Laboratory Internship (SULI) Washington University in St. Louis Stanford Linear Accelerator Center Stanford, CA August 21, 2012 Prepared in partial fulfillment of the requirements of the Office of Science, Department of Energy s Science Undergraduate Laboratory Internship under the direction of Thomas Weiss at the Stanford Synchrotron Radiation Lightsource (SSRL), Stanford Linear Accelerator Center. Participant: Signature Research Advisor: Signature

2 TABLE OF CONTENTS Abstract ii Introduction 1 Basic SAXS Methodology 1 Apparatus 2 Results 3 Discussion and Conclusions 3 Acknowledgments 3 References 3 i

3 ABSTRACT Microdroplet Sample Mounting Techniques in Small Angle X-Ray Scattering. ANDREW P. HUANG (Washington University in St. Louis, St. Louis, MO 63105) THOMAS WEISS (Stanford Synchrotron Radiation Lightsource (SSRL), Stanford Linear Accelerator Center, Stanford, CA 94025) As the need for protein analysis methods expands in the coming years, synchrotron beamtime becomes increasingly precious and new forays into increasing the efficiency of measurement equipment is critical. The concept of the new automatic sampling device is centered about delivering a hanging droplet into the path of an X-Ray beamline for Small Angle X-Ray analysis. This paper details the methods utilized in overcoming the obstacles encountered in the design process of such an apparatus such as compensating for radiation damage, droplet consistency, droplet evaporation, and maintaining cleanliness of the droplet dispenser system. ii

4 INTRODUCTION To improve data collection efficiency and sample economy in Small Angle X-Ray Scattering (SAXS) techniques on protein solutions, development of a new sample mounting procedure intended to replace the current autosampler apparatus on SAXS beamline 4-2 at the Stanford Synchrotron Radiation Lightsource (SSRL) was undertaken. Currently a flow-through capillary is used into which small aliquots ( 30ul) of protein (or buffer) solutions are aspirated and held into the X-ray beam for measurement. Because of imperfections in the glass walls of the capillary tubes causing no two capillary tubes to be of the exact same size, changing capillary tubes would cause unacceptable errors in scattering data. Therefore the same capillary is used for the different protein and buffer solutions during an experiment requiring extensive washing of the capillary in between scans, requiring approximately 3.5 minutes. This is a vast inefficiency especially when it is taken into consideration that the actual scan of the sample takes approximately 30 seconds. The basic premise behind this new mounting procedure is abandoning the capillary methodology and freely suspending a small droplet of the liquid sample ( 3uL) while performing the X-ray measurement on the hanging droplet. By eliminating washing cycles the new sample mounting procedure allows for a substantial increase in the speed of operation at the beamline. However in order for the new mounting procedure to be capable of mimicking the reliability of the current autosampler on the beamline, the issues of preventing droplet evaporation rates, radiation damage to the sample, and maintaining droplet consistency needed to be resolved. BASIC SAXS METHODOLOGY Solution based Small Angle X-Ray Scattering (SAXS) is a low resolution methodology for analyzing the basic molecular structure of proteins suspended in solution. Small angle scat- 1

5 tering is observed from the secondary wavelets produced by X-ray waves interacting with individual atoms. [1] Because of the rotational and dynamic nature of the proteins of the solution, the resolution of the output data is limited but SAXS can provide relatively precise information with respect to size and shape of the molecules in solution. [3] From the scattering pattern on the detector, intensity of the scattering observed is integrated radially about the center thereby yielding the SAXS scattering profile. However, in the measuring of the protein first the buffer background is measured and is subtracted from the protein and buffer solution scattering data to get the protein scattering data as (ρ(r) ρ s ) 2. There the scattering signal is proportional to the square of the electron density between the particle and the solvent as I(q) ρ 2. APPARATUS The current autosampler is designed to be a reliable dispensary of protein (see Figure 1) and buffer solutions into an X-ray beam for SAXS but unfortunately has several failings that are resolved by the next generation sample mounting procedures. The first of these is the difficulties caused by the capillary tubes installed on the apparatus. Imperfections in the fabrication process of the capillary tubes cause error in the scanning and buffer subtraction methodology involved in SAXS procedure. Also, the wash process undertaken in attempt to avoid error due to differences between different capillary tubes takes significantly longer to complete than the actual scan of the protein itself. Because beamtime is valuable, these inefficiencies are magnified by the large number of samples that usually need to be scanned on a SAXS beamline per given experiment leading to a significant amount of beamtime spent simply washing the sample stage. Also, the current autosampler requires a large amount of sample volume for operation, which is oftentimes impossible to acquire for certain scarce protein samples. 2

6 My modifications to the spare autosampler which ultimately became the centerpiece of this project was to remove the installed capillary tube so that when the needle is lowered the droplet would have an open area to hang. In the sampling tube a microscope is pointed at the droplet perpendicular to the path of the X-Ray beam as a means of optical imaging of the droplet for droplet shape analysis. RESULTS In the process of making results presentable DISCUSSION AND CONCLUSIONS DISCUSSION HERE ACKNOWLEDGMENTS This work was made possible by funding from the United States Department of Energy through the Summer Undergraduate Laboratory Internship (SULI) Program. I want to thank my mentor, Thomas Weiss, for giving me the opportunity to work at SLAC, for his help which permitted the completion of the project, and for exposing me to the projects being undertaken at the SSRL. REFERENCES [1] Jacques, David A., and Jill Trewhella. Small-angle Scattering for Structural Biology- Expanding the Frontier While Avoiding the Pitfalls. Protein Science 19.4 (2010): Print. 3

7 [2] Lipfert, Jan, and Sebastian Doniach. Small-Angle X-Ray Scattering from RNA, Proteins, and Protein Complexes. Annual Review of Biophysics and Biomolecular Structure 36.1 (2007): Print. [3] Neylon C (2008) Small angle neutron and X-ray scattering in structural biology: recent examples from the literature. Eur Biophys J Biophys Lett 37: [4] Putnam, Christopher D., Michal Hammel, Greg L. Hura, and John A. Tainer. X-ray Solution Scattering (SAXS) Combined with Crystallography and Computation: Defining Accurate Macromolecular Structures, Conformations and Assemblies in Solution. Quarterly Reviews of Biophysics (2007): n. pag. Print. 4

8 FIGURES Figure 1: Diagram depicting SAXS [2] 5

9 Figure 2: The sample syringe (B) is positioned by a triaxial system of motors (A) which extracts samples from a 96-sample well tray (C) and dispenses into the capillary tube holder (D) which is positioned in the path of the X-Ray beam. Solutions such as the sample and wash fluids are removed from the target area by a tube under the sample stage (E). 6

10 Figure 3: This next generation autosampler works under the same fundamental concepts as the autosampler in Figure 1 but has several modifications that permit droplet based sample dispensing. The needle is at a lower position than the original as to permit the formation of a small droplet directly into the beam. Also the sample is returned to an empty capillary position 7