Nagarajan Venugopalan

JBluIce–EPICS control system for macromolecular crystallography

The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline component. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments, especially in the field of microcrystallography.

Fast fluorescence techniques for crystallography beamlines.

This paper reports on several developments of X-ray fluorescence techniques for macromolecular crystallography recently implemented at the National Institute of General Medical Sciences and National Cancer Institute beamlines at the Advanced Photon Source. These include (i) three-band on-the-fly energy scanning around absorption edges with adaptive positioning of the fine-step band calculated from a coarse pass; (ii) on-the-fly X-ray fluorescence rastering over rectangular domains for locating small and invisible crystals with a shuttle-scanning option for increased speed; (iii) fluorescence rastering over user-specified multi-segmented polygons; and (iv) automatic signal optimization for reduced radiation damage of samples.

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.

Tissue specific specialization of the nanoscale architecture of Arabidopsis.

The Arabidopsis stem is composed of five tissues - the pith, xylem, phloem, cortex and epidermis - each of which fulfills specific roles in support of the growth and survival of the organism. The lignocellulosic scaffolding of cell walls is specialized to provide optimal support for the diverse functional roles of these layers, but little is known about this specialization. X-ray scattering can be used to study this tissue-specific diversity because the cellulosic components of the cell walls give rise to recognizable scattering features interpretable in terms of the underlying molecular architecture and distinct from the largely unoriented scatter from other constituents. Here we use scanning X-ray microdiffraction from thin sections to characterize the diversity of molecular architecture in the Arabidopsis stem and correlate that diversity to the functional roles the distinct tissues of the stem play in the growth and survival of the organism.

Amyloid structure exhibits polymorphism on multiple length scales in human brain tissue

Aggregation of Aβ amyloid fibrils into plaques in the brain is a universal hallmark of Alzheimer’s Disease (AD), but whether plaques in different individuals are equivalent is unknown. One possibility is that amyloid fibrils exhibit different structures and different structures may contribute differentially to disease, either within an individual brain or between individuals. However, the occurrence and distribution of structural polymorphisms of amyloid in human brain is poorly documented. Here we use X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid. Our observations indicate that (i) tissue derived from subjects with different clinical histories may contain different ensembles of fibrillar structures; (ii) plaques harboring distinct amyloid structures can coexist within a single tissue section and (iii) within individual plaques there is a gradient of fibrillar structure from core to margins. These observations have immediate implications for existing theories on the inception and progression of AD.

Tightly integrated single- and multi-crystal data collection strategy calculation and parallelized data processing in JBluIce beamline control system

Single- and multi-crystal data collection strategy and automated data processing have been tightly integrated into the JBluIce graphical user interface. Grid Engine is used to distribute these processes into multiple workstations to make efficient use of all available computing resources.

Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody

The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules), ClfA (clumping factor A) is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9), and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab) complex. The epitope for tefibazumab is located to the “top” of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC50 value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.

Monochromatic and polychromatic serial crystallography at the Advanced Photon Source

Photon Source R.F. Fischetti, J. Martin‐Garcia, N. Zatsepin, N. Stander, L. Zhu, G. Subramanian, G. Nelson, J. Coe N. Nagaratnam, S. Roy‐Chowdhury, D. Kissick, A. Ishchenko, C. Conrad, G. Ketawala, D. James, J. Zook, C. Ogata, N. Venugopalan, S. Xu, A. Meents, V. Srajer, R. Henning, H. Chapman, J. Spence, U. Weierstall, V. Cherezov, P. Fromme, W. Liu Advanced Photon Source, Argonne National Laboratory, Chicago, IL, USA Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ, USA School of Molecular Sciences, Arizona State University, Tempe, AZ, USA Department of Physics, Arizona State University, Tempe, AZ, USA Department of Chemistry, University of Southern California, Los Angeles, CA, USA Paul Scherrer Institute, Villigen, Switzerland Centre for Free‐Electron Laser Science, Deutsches Elektronen‐Synchrotron, Hamburg, Germany BioCARS, University of Chicago, IL, USA

Rapid in situ X-ray position stabilization via extremum seeking feedback.

X-ray beam stability is crucial for acquiring high-quality data at synchrotron beamline facilities. When the X-ray beam and defining apertures are of similar dimensions, small misalignments driven by position instabilities give rise to large intensity fluctuations. This problem is solved using extremum seeking feedback control (ESFC) for in situ vertical beam position stabilization. In this setup, the intensity spatial gradient required for ESFC is determined by phase comparison of intensity oscillations downstream from the sample with pre-existing vertical beam oscillations. This approach compensates for vertical position drift from all sources with position recovery times <6 s and intensity stability through a 5 µm aperture measured at 1.5% FWHM over a period of 8 hours.

High Brightness Beam for Microcrystallography at GM/CA@APS

GM/CA is a world leader in the development of microcrystallography capabilities for biological macromolecules. The combination of the GM/CA-developed quad-mini-beam collimator and advanced rastering and vector collect software tools have revolutionized microcrystallography. Recently, beamline 23ID-B was reconfigured by shifting the focusing optics 3.8 m downstream. This significantly increased the source demagnification and led to a 4-fold increase in the 5and 10-micron beam intensity. Beamline 23IDD is also being upgraded. A Pilatus3-6M with a 1.0 mm thick X-ray sensor was commissioned in January 2014 allowing shutterless data collection with high S/N. The detector specifications include 100 Hz frame rate, 10 MHz/pixel count rate, and high X-ray efficiency. The beamline optics and endstation are also being upgraded to provide a high intensity beam whose size can be variable rapidly in the range of 1 20 micron, a new air bearing goniometer with a sphere-of-confusion (SOC) of ~100 nm, a miniature sample XYZ stage that allows centering and scanning of a micron-sized crystal, and a new on-axis-visualization system that provides high resolution optical images of sample crystals. Plans are being developed to upgrade the Advance Photon Source storage ring with a Multi-Bend Achromat lattice. The source properties will be dramatically improved primarily by reducing the horizontal source size to be comparable to the vertical source size, resulting in a 2-3 orders of magnitude increase in source brightness. Both beamlines will be significantly improved by the source upgrade. Moreover the new microfocusing optics for 23ID-D will fully exploit the new source and could deliver a 500 nm (FWHM) beam with >2e13 photons/sec. This unprecedented flux density will provide new opportunities and challenges, and allow the study some of the most important problems in biology. Details of these developments will be presented.