533 J. Appl. Cryst. (1998). 31. 533-543 A Small-Angle X-ray Scattering Apparatus for Studying Biological Macromolecules in Solution ZIMEI Bu, ARTHUR PERLO, GERALD E. JOHNSON, GERALD OLACK, DONALD M. ENGELMAN* AND HAROLD W. WYCKOFF Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney A venue, PO Box 208114, New Haven, CT 06520-8114, USA. E-mail: don@paradigm.csb.yale.edu (Received 30 May 1997; accepted 3 November 1997) Abstract This paper describes the development of a simple laboratory-based small-angle X-ray scattering apparatus for the study of biological macromolecules in solution. The instrument is based on a two-circular-aperture collimation design combined with a conventional rotating-anode Cu Ka X-ray source, a graphite mono- chromator and a multiwire area detector. The geometry of the collimator, the beam-stop-to-detector distance and the thickness of the platinum foil of the defining aperture have been optimized to reduce background scattering. The effective Q range is from 0.01 to 0.33 ~-1, where Q = (4rrsin0)/Jk is the magnitude of the scattering vector, 20 is the scattering angle and ~, is the wavelength of the X-rays. The length of the collimator, the pinhole sizes and the helium-flushed sample-to-detector path can be easily changed depending on the resolution and intensity requirements of an experiment. The diffraction pattern of a poly- crystalline pellet of ammonium sulfate mounted about 2.5 cm in front of the beam stop and 40 cm in front of the detector is used to monitor changes in the incident-beam intensity as well as the differences in absorption of X- rays by the sample solutions and the solvents, to ensure correct background subtractions. Data collection is controlled by a computer through a parallel DMA (direct memory access) I/O module. Data collection and reduction software has been developed. The typical data collection time is about 2 h for a 5 mgml -~ 10 kDa protein dissolved in an aqueous solution. Examples of applications of this small-angle X-ray scattering instru- ment to studying protein size and conformation changes are presented. 1. Introduction Small-angle X-ray scattering (SAXS) is a useful tech- nique for obtaining structural information from macro- molecules. The length-scale range readily probed by SAXS makes it especially suitable for measuring the size, shape and molecular weight of proteins and nucleic acids with radii of gyration (Rg) of 10-50,& and maximum dimensions (Dmax) of 20-300 ,~. Examples of applications include studying conformational changes, ('i 1998 International Union of Crystallography Printed in Great Britain - all rights reserved protein folding/unfolding, ligand-induced oligomeriza- tion and enzyme-substrate interactions (Guinier & Fournet, 1955; Pessen et al., 1973; Glatter & Kratky, 1982; Moore, 1982; Flanagan et al., 1993; Kataoka et al., 1993; Lattman, 1994; Tuzikov et al., 1996; Lemmon et al., 1997). SAXS can also be used for diffraction studies of the dimensions and phase transitions of biological membranes and lipid bilayers (Lewis & Engelman, 1983; Tournois et al., 1987; Batenburg et al., 1988: Hare et al., 1995). The scattered intensity from a solution of biological macromolecules is usually weak because the electron- density contrast between a macromolecule, e.g. a protein, and its aqueous buffer is low. Further, the experiment is conducted in a dilute solution to reduce the effect of intermolecular interactions. The weak scattering requires that the X-ray flux of an SAXS apparatus be maximized and the background scattering be minimized. The minimum accessible angle must be small enough to permit measurement of macro- molecules with Dma x of 10-300 ]k. Maximizing flux consistent with a resolution requirement is especially important when a conventional rotating-anode X-ray source is employed, or measurement times will be unacceptably long. At the same time, the apparatus should be flexible enough to allow easy optical geometry changes to adjust to different resolution requirements. A beam monitor is required to monitor beam-inten- sity changes and the differences in absorption/trans- mission of X-rays by the sample to ensure correct background subtractions. When measuring the mole- cular-weight changes of biological molecules, such as the oligomerization of proteins to form a complex, a beam monitor is usually necessary to normalize the scattering data collected from a series of samples for precise molecular-weight determinations. A well collimated circular beam is also desirable for eliminating the need for desmearing procedures when reducing the SAXS data. Although data acquisition rates are much slower than at a synchrotron source, a laboratory-based SAXS apparatus employing a conventional rotating-anode source is suitable for many of the above purposes. It has the advantages of local availability, source stability and low cost. When an area detector is used, a SAXS Journal of Applied Crystallography ISSN 0021-8898 ( 1998