Sean McSweeney

ELECTRON-DENSITY MAPS OF LYSOZYME CALCULATED USING SYNCHROTRON LAUE DATA COMPRISING SINGLES AND DECONVOLUTED MULTIPLES

We have used lysozyme as a test case to illustrate the application of a new method of estimating the intensities of Laue multiples reflection data in protein crystallography. Hen egg-white lysozyme, an enzyme with a single polypeptide chain of 129 amino acids, crystallizes in space group P43212 with cell parametersa=b=79·1 Å,c=37·9 Å. Laue image plate data were collected using synchrotron radiation on station 9·5 at Daresbury with a total exposure time of 0·95 seconds. The data processed were separated into two data sets comprising the singles and the combined singles and deconvoluted multiples. The method of deconvolution was that of Campbell and Hao (1993), which utilizes the intensity variation of theλ-curve. Electron density maps (2Fo−Fc) based on the two data sets are then compared. This comparison shows that the deconvoluted multiples do indeed contribute usefully to the continuity of the maps due to the improved completeness of the data. A number of map sections along the polypeptide chain, based on the two Laue data sets, are shown for comparison. These include Arg 5, His 15, Phe 38, Asp 52, Tyr 53, Pro 70, Trp 108 and the four disulphide bridges.

NSLS-II biomedical beamlines for micro-crystallography, FMX, and for highly automated crystallography, AMX: New opportunities for advanced data collection

We present the final design of the x-ray optics and experimental stations of two macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source-II. The microfocusing FMX beamline will deliver a flux of ∼5×1012 ph/s at 1 A into a 1 – 20 µm spot, its flux density surpassing current MX beamlines by up to two orders of magnitude. It covers an energy range from 5 – 30 keV. The highly automated AMX beamline is optimized for high throughput, with beam sizes from 4 – 100 µm, an energy range of 5 – 18 keV and a flux at 1 A of ∼1013 ph/s. A focus in designing the beamlines lay on achieving high beam stability, for example by implementing a horizontal bounce double crystal monochromator at FMX. A combination of compound refractive lenses and bimorph mirror optics at FMX supports rapid beam size changes. Central components of the in-house developed experimental stations are horizontal axis goniometers with a target sphere of confusion of 100 nm, piezo-slits for dynamic beam size changes during...

The emergence of the synchrotron Laue method for rapid data collection from protein crystals

Synchrotron X-radiation (SR) is intense, polychromatic and collimated. It is widely exploited, in macromolecular crystallography, particularly using a monochromatized short wavelength beam. The spectral curve of SR, however, ideally lends itself to use of Laue geometry, i. e. the original diffraction experimental arrangement based on a stationary crystal and a polychromatic X-ray beam. Rapid exposure times and time-resolved crystallography studies, e. g. of enzymes, are now possible. Historical objections to the use of Laue diffraction data, particularly the multiplicity distribution, have been found not to be as limiting as once thought. The credentials of the Laue method have been established through a variety of Laue crystal structure analyses, involving photographic film as detector. Recently a three-dimensional arrangement of films, known as a toast-rack, has been used to alleviate problems with spatially overlapping spots. This paper provides a review of these results and then reports several developments. In particular, one of the first Laue analyses using an image plate as detector, namely of a cobalt substituted concanavalin A crystal, is discussed. Recent experimental developments, also at the Daresbury synchrotron, are then described. First, a large toast-rack has been used to record Laue data from a protein crystal. Secondly, a transmission X-ray mirror has been constructed from thin mylar (1.5 μm) and used to provide a λmax filter instead of using aluminium foils. Thirdly, since the Laue method suffers from poor sampling of the low resolution data, a new method (known as LOT) has been introduced.

Comments on the use of asymmetric monochromators for x‐ray diffraction on a synchrotron source

Crystals of germanium or silicon provide monochromatic radiation on synchrotron radiation beamlines. By using asymmetrically cut crystals, it is possible to alter the properties of the beam in position, angle, wavelength space. The results obtained from using two types of asymmetrically cut monochromators are compared with theory. For single crystal focusing monochromators, the asymmetry results in a dispersion term and a resultant broadening of the focal spot. The expression for the broadening given here differs from that given previously for these type of monochromators and has implications for the design of beamlines where the x‐ray source has a small size. Asymmetric channel cut monochromators are not commonly used for x‐ray diffraction on a synchrotron. However, we show here that, compared with a symmetric monochromator, intensity gains of three or more can be obtained with a simple compact design.

Synchrotron biosciences at National Synchrotron Light Source II: a biomedical technology research resource

We have developed a powerful and readily accessible suite of structural biology tools for the lifescience research community. With the main focus on macromolecular crystallography (MX) and xray scattering, there is also a user program in fluorescence imaging of metals in biological materials. The facilities are operated by the NIHand DOE-funded Life Science Biomedical Technology Research Resource: LSBR.

Automation for BioSAXS experiments at BM29 of the ESRF

Small Angle X-ray Scattering (SAXS) experiments of macromolecules (proteins, nucleic acids, ...) in solution can provide a wealth of information concerning structure, dynamics and therefore function. However, correct interpretation of results depends critically on data quality. To achieve this, well defined acquisition protocols and immediate feedback are essential. At beamline BM29 at ESRF, BioSAXS data are acquired in a temperature controlled flow through capillary. Samples and buffers can be loaded into the capillary via a robotic liquid handling sample changer (SC) [1] or by connecting the capillary to the outlet of a size exclusion chromatography (SEC) setup [2]. Samples loaded by the SC can be passed through the beam to avoid unwanted effects caused by radiation damage. All recorded data are immediately analyzed to give user-oriented feedback on estimated molecular weight, size, possible radiation damage and inter-particle effects, quality of background correction, etc. Subsequent downstream analysis, also carried out autmatically, results in the calculation of molecular envelopes for solution structures. The information management system ISPyBB (Information System for Protein crystallography Beamlines and BioSAXS) logs and presents all information on data acquisition and analysis and allows users to easily compare the results of different data acquisitions and to decide whether further measurements or changes of acquisition parameters are necessary. The combination of these tools allows non-expert users to carry out BioSAXS experiments autonomously, thereby making the technique accessible to a broader community.

Reply to ‘‘Comment on ‘Comments on the use of asymmetric monochromators for x‐ray diffraction on a synchrotron source’ ’’ [Rev. Sci. Instrum. 66, 2174 (1995)]

The issue raised in Nave et al. [Rev. Sci. Instrum. 66, 2174 (1995)] is the description and results given by Sanchez del Rio and Cerrina [Nucl. Instrum. Methods A 301, 589 (1991)] and has no bearing on our understanding or otherwise of the SHADOW code (we do not refer to SHADOW in our article). The problem is whether Sanchez del Rio and Cerrina demonstrate that a demagnified image is obtained from asymmetric monochromators when operating in the white beam on a synchrotron source.