Stephen Corcoran

Radiation damage in protein crystals is reduced with a micron-sized X-ray beam

Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 μm) to the smallest (0.84 μm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-μm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 μm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.

A 7 µm mini-beam improves diffraction data from small or imperfect crystals of macromolecules

An X-ray mini-beam of 8 × 6 µm cross-section was used to collect diffraction data from protein microcrystals with volumes as small as 150–300 µm3. The benefits of the mini-beam for experiments with small crystals and with large inhomogeneous crystals are investigated.

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation.

In this proof-of-principle study, the feasibility of structure determination of several proteins using serial millisecond crystallography (SMX) has been evaluated. The first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported.

Micro‐Crystallography Developments at GM/CA‐CAT at the APS

Recently, several important structures have been solved using micro‐crystallographic techniques that previously could not have been solved with conventional crystallography. At GM/CA‐CAT we continue to develop micro‐crystallographic capabilities for difficult problems such as small crystals of large macromolecular complexes or membrane proteins grown in the lipidic cubic phase. This paper will describe three major upgrades to our arsenal of tools, “mini‐beam” collimators, active beamstop, and an improved goniostat. Our “mini‐beam” collimators have evolved to a new triple‐collimator fabricated from molybdenum as a uni‐body. This has significantly improved the robustness, ease of initial alignment, and reduction of background. More recently, two prototypes of a quad‐collimator have been developed and fabricated to provide a selection of mini‐beams of 5, 10, 20 μm and a 300 μm scatter‐guard on a single body. The smaller beams and samples have increased the demand on the tolerances of our goniostat. To meet t...

One‐Micron Beams for Macromolecular Crystallography at GM/CA‐CAT

GM/CA‐CAT has developed a 1‐μm beam for challenging micro‐diffraction experiments with macromolecular crystals (e.g. small crystals) and for radiation damage studies. Reflective (Kirkpatrick‐Baez mirrors) and diffractive (Fresnel zone plates) optics have been used to focus the beam. Both cases are constrained by the need to maintain a small beam convergence. Using two different zone plates, 1.0×1.0 and 0.8×0.9 μm2 (V×H,FWHM) beams were created at 15.2 keV and 18.5 keV, respectively. Additionally, by introducing a vertical focusing mirror upstream of the zone plate, a line focus at 15.2 keV was created (28×1.4 μm2 V×H,FWHM) with the line oriented perpendicular to the X‐ray polarization and the crystal rotation axis. Crystal‐mounting stages with nanometer resolution have been assembled to profile these beams and to perform diffraction experiments.

Optical Performance of the GM/CA‐CAT Canted Undulator Beam lines for Protein Crystallography

A new macromolecular crystallographic facility developed by GM/CA‐CAT is operational at the Advanced Photon Source (APS). The facility consists of three beamlines: two lines based on the first “hard” dual canted undulators and one bending magnet beamline. The ID lines are operational, and the BM line is being commissioned. Both insertion device (ID) beamlines are independently tunable over a wide energy range. The inboard ID lines have been upgraded with a new insertion device to provide enhanced performance for MAD phasing experiments near the selenium and bromine K‐edges. The ID line monochromators’ crystals are indirectly, cryogenically cooled for improved performance and reliability. Focusing is achieved by long bimorph mirrors in a Kirkpatrick‐Baez geometry. This paper describes the design of the beam lines and the optical characterization of the mirrors and monochromators.

Design and Performance of the Compact YAG Imaging System for Diagnostics at GMCA Beamlines at APS

A compact YAG (Chromium Doped Yttrium Aluminum Garnet — Cr4+:YAG) imaging system has been designed as a diagnostic tool for monochromatic x‐rays emanating from the first “Hard” x‐ray dual‐canted undulator at the Advanced Photon Source at Argonne National Laboratory. This imaging system consists of a flat YAG crystal, right angle prism/mirror, video camera and monitor. A flat YAG crystal with a diameter of 10 mm has been installed in vacuum and positioned downstream of the monochromator of the insertion device beamline. Another 20 mm diameter YAG crystal has been installed in vacuum after the horizontal deflecting mirrors of the second insertion device beamline. CCD cameras are mounted in air close to the window of the vacuum ports to image the fluorescence of the YAG crystals. An additional 25 mm diameter YAG crystal has been used for K‐B (Kirkpatrick‐Baez) mirror focusing and beamline alignment. These YAG imaging systems have greatly facilitated beamline commissioning as well as sample alignment to the x...