Kathryn Janzen

08B1-1: an automated beamline for macromolecular crystallography experiments at the Canadian Light Source

Beamline 08B1-1 is a recently commissioned bending-magnet beamline at the Canadian Light Source. The beamline is designed for automation and remote access. Together with the undulator-based beamline 08ID-1, they constitute the Canadian Macromolecular Crystallography Facility. This paper describes the design, specifications, hardware and software of beamline 08B1-1. A few scientific results using data obtained at the beamline will be highlighted.

MxDC and MxLIVE: software for data acquisition, information management and remote access to macromolecular crystallography beamlines.

An integrated computer software system for on-site and remote collection of macromolecular crystallography (MX) data at the Canadian Light Source (CLS) is described. The system consists of an integrated graphical user interface for data collection and beamline control [MX Data Collector (MxDC)] which provides experiment-focused control of beamline devices, and a laboratory information management system [MX Laboratory Information Virtual Environment (MxLIVE)] for managing sample and experiment information through a web browser. The system allows remote planning and transmission of sample and experiment parameters to the beamline through MxLIVE, on-site or remote data collection through MxDC guided by information from MxLIVE, and remote monitoring and download of experimental results through MxLIVE. The system is deployed and in use on both MX beamlines at the CLS which constitute the Canadian Macromolecular Crystallography Facility.

Celebrating the 10th anniversary of CMCF operations

The Canadian Macromolecular Crystallography Facility (CMCF) supports more than 70 protein crystallographers from across Canada. It consists of an insertion device beamline (CMCF-ID) and a bending magnet beamline (CMCF-BM). The first diffraction image was acquired at the CMCF-ID beamline on March 18, 2006, and the first paper containing data collected at CMCF-ID was published in December 2006 [1]. CMCF-BM was commissioned in 2011, and is a general-purpose crystallography beamline having characteristics that support the protein and chemical crystallography communities. Both beamlines are fully automatized and operate mainly through remote access to the CMCF facility [2]. Up to 25% of available beamtime is reserved for commercial users. To date, more than 450 peer reviewed articles used data collected at the CMCF. Additionally, some 860 PDB deposits and 6 patents have originated from the CMCF. Since 2010, we have been running the Annual CLS Mx Data Collection School which has trained more than 140 participants (http://cmcf.lightsource.ca/school/). After a successful 10 years of operation, the CMCF-ID beamline will undergo an upgrade to establish micro-beam capabilities [3]. After completion, the focused beam at the sample will have a much smaller size of 50 × 5 μm^2 (H x V) with flux at 12 keV of ~10^13 [photons/s], allowing measurement of X-ray diffraction patterns from much smaller crystals than possible presently. The beamline will be equipped with a very fast sample changer and an ultra-low noise photon counting detector, allowing shutterless operation of the beamline. The first phase of the upgrade will include installation of a new PILATUS 3 6M detector that will become operational in May 2017, while an ISARA robot will be installed at the beamline in the autumn of 2017. For the second phase of the upgrade, a new longer in-vacuum undulator and new optics will be installed in approximately 3 years. To substantially increase the flux produced by the CMCF-BM beamline, we will additionally be upgrading its double crystal monochromator with multilayers.

AutoProcess:Automated strategy calculation, data processing & structure solution

Two critical aspect of macromolecular crystallography experiments are (1) Determining the optimal parameters and strategy for collecting good quality data and (2) Optimal processing of the collected data to obtain to facilitate structure determination. These tasks can be daunting to inexperienced crystallographers and often lead to inefficiencies as valuable beam-time is used up. To support automation, remote access and high-throughput crystallography, we have developed a software system for automation of all data processing tasks required at the synchrotron. AutoProcess, is layered on the XDS data processing package and makes use of other utilities such as BEST from the European Molecular Biology Laboratory (EMBL), CCP4 utilities and SHELX. The software can be used from the command line as a standalone application but can also be run as a service on a high-performance computing cluster, and integrated into beamline control and information management systems such as MxDC and MxLIVE to allows users to determine the optimal strategy for data collection, and/or process full datasets with the click of a button. Users are presented with a graphical data processing report as well as reflection output files in popular formats automatically. For small molecule and peptide structures, an unrefined initial structure with an electron density map is automatically generated using only the raw diffraction images and the chemical composition of the molecule. Future developments will include sub-structure solution for MAD/SAD/SIRAS data. The software is freely available under an open-source license from the authors. The Canadian Light Source is supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan.

The State of the Canadian Macromolecular Crystallography Facility

The Canadian Macromolecular Crystallography Facility (CMCF) at the Canadian Light Source consists of two macromolecular crystallography beamlines for structure determination using x-ray diffraction. The equipment at the CMCF beamlines have undergone or will undergo changes and improvements to better meet the needs of the most challenging experiments users may present. Among these improvements are: 1) Automounter improvements; 2) Better goniometry on 08ID-1 with the addition of a Huber air-bearing goniometer; 3) Added beam size capabilities on 08ID-1 with the addition of a multiple beam defining aperture holder; 4) XAFS capability on 08B1-1; 5) Improved low energy S-SAD data collection with the addition of a Helium path; 6) Improvements to the data collection and data management software; 7) A vacuum path for scattering experiments with detector distances up to 1 m; 8) A comprehensive beamline upgrade project on the 08ID-1 beamline; and 9) Service crystallography services.

Future developments for macromolecular crystallography at the CLS

The Canadian Macromolecular Crystallography Facility (CMCF) at the Canadian Light Source (CLS) is a suite of fully automated beamlines, 08ID-1 and 08B1-1 [1]. It serves over 60 Canadian groups plus academic and commercial users in the US. Besides remote data collection, we offer Mail-In service where data are collected by CMCF staff. Beamline 08B1-1 has been in operation since 2011 and beamline 08ID-1 since 2006. When beamline 08ID-1 was designed, over 10 years ago, small crystals were defined as having sizes of 50-100 μm. Today, the most challenging experiments require more intense X-ray beams that can be focused to accommodate much smaller crystal sizes of less than 5 μm with flux on the order of 10^11 photons/s. To reach these stringent parameters, a new more powerful source of X-rays will be required, which will be provided by a longer small-gap in-vacuum undulator (SGU). To accommodate the higher power levels and to focus X-rays to a smaller focal spot with a high degree of spatial and temporal stability, the existing X-ray optical elements need to be upgraded. The remaining components of the project include a 5-axis alignment table for improving alignment of small samples with the microbeam, a high-efficiency robotic sample-changer and a single-photon X-ray detector. Several options for the new design will be discussed. These developments are consistent with the current direction of structural biology research at the CLS [2]. Since 2006 over 225 (240) papers and 400 (444) PDB deposits reported data collected at beamline 08ID-1. Parentheses indicate the total number for the CMCF. Many of these have been published in very high impact journals such as N. Engl. J. Med., Nature, Cell, Science, PNAS, among others (http://cmcf.lightsource.ca/publications/).

XAS at the Canadian Macromolecular Crystallography Facility

Recent additions to the Canadian Macromolecular Crystallography Facility have expanded the capabilities of its bending magnet beamline. It is now possible to perform x-ray absorption spectroscopy (XAS) on crystals. A wide range of biologically relevant metals can be further studied, supplementing diffraction data. XAS can be used to determine if metalloproteins are photoreducing during diffraction data collection. The geometries of metal complexes can also be inferred with near-edge and EXAFS data, often more accurately than crystallography. CMCF-BM can be employed to do the abovementioned techniques on powder and solution samples that contain a metal of interest. One XAS-based technique that shows promise is single crystal plane polarized EXAFS. This technique combines crystallographic data with the findings from XAS to yield a high resolution three dimensional atomic model. More recently a number of the procedural steps required for the acquisition of XAS-based data have been automated in the MxDC software suite. These changes to data collection make it easier for users new to these disciplines to run the XAS-based experiments. By having the necessary equipment to do XAS at a protein crystallography facility, researchers who may not have had the opportunity delve into the field of XAS now can do so with minimal risk in terms of materials, funds and time.