research papers J. Appl. Cryst. (2012). 45, 1219–1227 doi:10.1107/S0021889812039945 1219 Journal of Applied Crystallography ISSN 0021-8898 Received 14 June 2012 Accepted 20 September 2012 # 2012 International Union of Crystallography Printed in Singapore – all rights reserved Time-of-flight Bragg scattering from aligned stacks of lipid bilayers using the Liquids Reflectometer at the Spallation Neutron Source Jianjun Pan, a * Frederick A. Heberle, a Justin R. Carmichael, a John F. Ankner a and John Katsaras a,b,c a Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA, b Joint Institute for Neutron Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA, and c Canadian Neutron Beam Centre, National Research Council, Chalk River, Ontario, Canada K0J 1J0. Correspondence e-mail: panj@ornl.gov Time-of-flight (TOF) neutron diffraction experiments on aligned stacks of lipid bilayers using the horizontal Liquids Reflectometer at the Spallation Neutron Source are reported. Specific details are given regarding the instrumental setup, data collection and reduction, phase determination of the structure factors, and reconstruction of the one-dimensional neutron scattering length density (NSLD) profile. The validity of using TOF measurements to determine the one-dimensional NSLD profile is demonstrated by reproducing the results of two well known lipid bilayer structures. The method is then applied to show how an antimicrobial peptide affects membranes with and without cholesterol. 1. Introduction Nondestructive neutron diffraction from positionally corre- lated multibilayer samples has traditionally been used to study the distribution of matter within bilayers. This technique is particularly relevant for studies of biological membranes, which are inherently enriched in hydrogen and whose heavier isotope (i.e. deuterium) possesses a coherent neutron scat- tering length that is remarkably different (both in sign and in magnitude). Thus by exchanging hydrogen atoms with deuterium atoms, the resulting one-dimensional neutron scattering length density (NSLD) profile can be systematically altered. The difference between the deuterated and nondeuterated NSLD profiles can then be utilized to accu- rately pinpoint the labeled moieties with a precision of better than 1 A ˚ (Bu ¨ ldt et al., 1978). Over the years, such experiments have yielded a number of important results, including the conformation of lipid fatty acid chains and headgroups (Bu ¨ ldt et al., 1979; Zaccai et al., 1979; Mihailescu et al., 2011), the orientation of macromolecules in lipid bilayers (Worcester & Franks, 1976; Pebay-Peyroula et al., 1994; Kucerka et al., 2010), and the organization of complex proteins in membranes (Krepkiy et al. , 2009). Since biological samples interact inti- mately with water, by exchanging common water with deut- erated water, a number of different external contrast conditions can be achieved. This simple yet powerful contrast variation approach has been widely used to determine the phases of structure factors, which are essential in the recon- struction of the one-dimensional NSLD profile (Worcester & Franks, 1976). Despite the scientific impact of neutron scattering, the technique, for the most part, has been overshadowed by synchrotron X-ray sources with their greater availability and much higher flux. However, with the advent of the short- pulsed high-flux Spallation Neutron Source (SNS) at Oak Ridge National Laboratory (ORNL), neutron scattering techniques are entering a new realm (Mason et al., 2006). In this regard, we are utilizing the Liquids Reflectometer (LR) at the SNS to develop methods of characterizing aligned multi- bilayer systems. Aside from the SNS’s high flux, a distinct advantage for diffraction experiments is the time-of-flight (TOF) capability, which makes use of the broad-energy inci- dent neutrons. A ‘white beam’ allows for a wide range of momentum transfers to be simultaneously recorded at fixed incident and detection angles, resulting in diffraction patterns equivalent to those obtained using traditional –2 scans at reactor-based steady state neutron sources (monochromatic neutrons) (Dura et al., 2006). We describe the first Bragg scattering experiments carried out at the LR instrument using a temperature- and relative humidity (RH)-controlled sample environment. Although diffraction experiments in the TOF mode have previously been carried out at other facilities (Salditt et al., 2003; Ryabova et al., 2010; Strobl et al., 2011), here we describe the specific details of the LR at SNS. Two well known lipid systems were studied, and a procedure used to evaluate their TOF scattering data is described. The capability was then applied to show how an antimicrobial peptide affects membranes with and without cholesterol. Depending on the types of samples LR offers the possibility of high-throughput data, enabling the study of kinetics (of the order of minutes) and of chemically labile systems (e.g. lipids with polyunsaturated fatty acid chains) that often deteriorate over extended data collection times.