Venugopalan Nagarajan

Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam

Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 µm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.

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.

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...

Alignment protocol for effective use of hard x-ray quad collimator for micro-crystallography

In October 2009, a quad, mini-beam collimator was implemented at GM/CA CAT that allowed users to select between a 5, 10, or 20 micron mini-beam or a 300 micron scatter guard for macromolecular crystallography. Initial alignment of each pinhole to the optical axis of each path through the mini-beam collimator is performed under an optical microscope using an alignment jig. Next, the pre-aligned collimator and its kinematic mount are moved to the beamline and attached to a pair of high precision translation stages attached to an on-axis-visualization system for viewing the protein crystal under investigation. The collimator is aligned to the beam axis by two angular and two translational motions. The pitch and yaw adjustments are typically only done during initial installation, and therefore are not motorized. The horizontal and vertical positions are adjusted remotely with high precision translational stages. Final alignment of the collimator is achieved using several endstation components, namely, a YAG crystal at the sample position to visualize the mini-beam, a CCD detector to record an X-ray background image, and a PIN diode to record the mini-beam intensity. The alignment protocol and its opto-mechanical instrumentation design will be discussed in detail.

Remote Access Capabilities at the GM/CA Beamlines at the APS

The GM/CA facility consists of two undulator source beamlines and a bending magnet beamline at the Advanced Photon Source (APS). Access to the operation of these beamlines is accomplished through visits by investigators who are either on-site, remote or a combination of the two. In all modes of access, user operations are controlled by the experimenter. The control and capabilities of the GM/CA beamlines are identical for remote and on-site users. Remote access to the beamlines is through NX or Teamviewer to local computers [1]. Once communication has been established, experienced GM/CA experimenters are greeted by our familiar JBluIce, the graphical user interface/control program[2] responsible for all operations from sample handling through data collection and reduction. Although investigators always see a familiar interface, both software and hardware on the beamlines are continually improving. Recent hardware upgrades include a shift of the optical focusing mirrors on the ID-B beamline closer to the sample to provide a significant increase in flux, and installation of a new Pilatus3 6M detector on ID-D, the second undulator beamline. The JBluIce program has incorporated new detector controls for shutterless operation while continuing to expand the features of rastering, vector (helical) data collection, strategy tools and data analysis. These tools have been essential to investigators working on membrane crystal samples, e.g. GPCRs, as well as for samples that decay quickly or require data to be collected from multiple crystals. The presentation will provide an overview of beamline remote control as well as an update of the equipment that it operates at GM/CA.