The Advanced Photon Source (APS) will shut down in April 2023 for a year-long upgrade (APS-U) to replace the storage ring.The APS-U will start up in April 2024 and be up to 500-times brighter than the current APS.The GM/CA@APS beamlines also will be upgraded with new X-ray optics that will focus the full beam to 5 microns with mirrors (JTEC Corp. mirrors and benders built by AXILON AG) or ~1-micron with Compound Refractive Lenses (RXOPTICS GmbH & Co. and a transfocator built by AXILON AG).Growing evidence suggests that radiation damage can be reduced by using X-ray energies well above the usual 12 keV, and with micron-sized beams.To exploit this, we plan to purchase a DECTRIS Eiger2 detector with a CdTe-sensor that has high efficiency for energies well above the beamline maximum energy of 35 keV.The sample capacity of our automounters will be increased to support higher throughput.The recently-released PyBluIce control software will incorporate additional advanced features, including automated data collection, while retaining the power and flexibility of the previous control software, JBluIce.Analytical software developments and coordination with the Argonne Advanced Leadership Computing Facility will provide increased speed of data analysis.These upgrades will provide enhanced room-temperature and cryo-cooled serial crystallography capabilities, whether with viscous flow jets [1] or fixed-target [2] sample delivery systems.For cryo-cooled samples, efforts are in progress to minimize the movement of the sample as the pin-base thermally equilibrates after transfer from the Dewar to the goniometer.Once the X-ray beam is available in April 2024, we will begin recommissioning the beamlines, and after a few months, start ramping up the user program.Researchers are encouraged to apply for GM/CA@APS beamtime, whether for advanced techniques such as room-temperature serial crystallography, or for 'standard' and high-throughput crystallography.We look forward to welcoming you back to the beamlines to experience some exciting new capabilities.

The Advanced Photon Source (APS) is rapidly approaching its yearlong shutdown for the upgrade to the APS-U starting in April of 2023.The GM/CA beamlines at Sector 23 have begun to prepare for the higher brightness of the new source.Installation of new optical focusing elements is underway.

Algorithms and procedures to fully automate retuning of synchrotron radiation beamlines over wide energy ranges are discussed. The discussion is based on the implementation at the National Institute of General Medical Sciences and the National Cancer Institute Structural Biology Facility at the Advanced Photon Source. When a user selects a new beamline energy, software synchronously controls the beamline monochromator and undulator to maintain the X-ray beam flux after the monochromator, preserves beam attenuation by determining a new set of attenuator foils, changes, as needed, mirror reflecting stripes and the undulator harmonic, preserves beam focal distance of compound refractive lens focusing by changing the In/Out combination of lenses in the transfocator, and, finally, restores beam position at the sample by on-the-fly scanning of either the Kirkpatrick-Baez mirror angles or the transfocator up/down and inboard/outboard positions. The sample is protected from radiation damage by automatically moving it out of the beam during the energy change and optimization.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

The General Medical Sciences and Cancer Institutes’ structural biology facility at the Advanced Photon Source (GM/[email protected]) operates a national user facility for crystallographic structure determination of biological macromolecules, with an emphasis on challenging, high-impact projects. The facility includes two undulator beamlines, 23ID-B and 23ID-D, that provide stable, intense X-ray beams of user-selectable size down to 5-micron diameter, an intuitive user interface for experiment control, and an automated pipeline for data processing. The beamlines are equipped with high capacity (288 samples) automounters and Dectris PADs allowing 100Hz data collection. This presentation will give an overview of recent developments at the beamlines, including advances in the implementation of serial crystallography experiments with viscous-jet or fixed target mounting systems. These efforts align well with the planned APS storage ring upgrade (APS-U) that increases the brightness of the X-ray beam by 100 to 1000 fold. Researchers who seek beam time for traditional crystallography or interested in pursuing novel structures via serial crystallography at GM/[email protected] are encouraged to contact us, https://www.gmca.aps.anl.gov/userprogram/applying.html, to help enable their experiments. Acknowledgment: GM/[email protected] has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The Eiger 16M detector was funded by an NIH-Office of Research Infrastructure Programs, High-End Instrumentation Grant (1S10OD012289-01A1).

Macromolecular crystallography has advanced from using macroscopic crystals, which might be >1 mm on a side, to crystals that are essentially invisible to the naked eye, or even under a standard laboratory microscope. As crystallography requires recognizing crystals when they are produced, and then placing them in an X-ray, electron, or neutron beam, this provides challenges, particularly in the case of advanced X-ray sources, where beams have very small cross sections and crystals may be vanishingly small. Methods for visualizing crystals are reviewed here, and examples of different types of cases are presented, including: standard crystals, crystals grown in mesophase, in situ crystallography, and crystals grown for X-ray Free Electron Laser or Micro Electron Diffraction experiments. As most techniques have limitations, it is desirable to have a range of complementary techniques available to identify and locate crystals. Ideally, a given technique should not cause sample damage, but sometimes it is necessary to use techniques where damage can only be minimized. For extreme circumstances, the act of probing location may be coincident with collecting X-ray diffraction data. Future challenges and directions are also discussed.

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.

Synchronous digitization, in which an optical sensor is probed synchronously with the firing of an ultrafast laser, was integrated into an optical imaging station for macromolecular crystal positioning prior to synchrotron X-ray diffraction. Using the synchronous digitization instrument, second-harmonic generation, two-photon-excited fluorescence and bright field by laser transmittance were all acquired simultaneously with perfect image registry at up to video-rate (15 frames s(-1)). A simple change in the incident wavelength enabled simultaneous imaging by two-photon-excited ultraviolet fluorescence, one-photon-excited visible fluorescence and laser transmittance. Development of an analytical model for the signal-to-noise enhancement afforded by synchronous digitization suggests a 15.6-fold improvement over previous photon-counting techniques. This improvement in turn allowed acquisition on nearly an order of magnitude more pixels than the preceding generation of instrumentation and reductions of well over an order of magnitude in image acquisition times. These improvements have allowed detection of protein crystals on the order of 1 µm in thickness under cryogenic conditions in the beamline. These capabilities are well suited to support serial crystallography of crystals approaching 1 µm or less in dimension.

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.

Second order nonlinear optical imaging of chiral crystals (SONICC), based on femtosecond laser scanning microscopy, has been implemented at GM/CA@APS undulator beamline 23ID-B for rapid protein crystal localization and centering. The technique is based on infrared laser light impinging on non-centrosymmetric crystals of proteins, which selectively may yield a frequency-doubled, visible signal generated by the anharmonic response of the electron cloud of the protein in response to the laser field. One aim of this method is to locate small crystals grown in opaque crystallization media for centering in X-ray beams of only a few microns or less in cross-section [1]. The optical system implemented at the beamline includes `trans' and `epi' detection of Second Harmonic Generation (SHG) signals [2]. In addition, scanning visible laser light across the sample and detecting two-photon excited UV fluorescence (TPE-UVF) provides complementary contrast based on the native fluorescence of proteins. An update on progress towards offering a user-friendly system to users will be provided. Different factors that influence imaging signals and the practice of successfully locating and accurately positioning a crystal via SONICC will be discussed.

Significance Exposure to high-energy X-rays, experienced during X-ray diffraction experiments for determining atomic structures, can potentially create large local electric fields from photoelectron ejection. Under cryogenic conditions, these fields may persist indefinitely. Mapping of electric fields is important for a complete understanding of damage mechanisms involved in X-ray measurements, for potential effects on the quality and interpretation of X-ray data, and for possible direct impact on diffraction resolution through the piezoelectric effect. Mapping of electric fields may also be useful in retaining a record of locations of X-ray exposure. In this paper, electric-field induced second harmonic generation imaging is explored in simulation and experiment. It provides insight into the position and distribution of local electric fields within an X-ray exposed sample.

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 5- and 10-micron beam intensity. Beamline 23ID-D 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.

The calculation of single- and multi-crystal data collection strategies and a data processing pipeline have been tightly integrated into the macromolecular crystallographic data acquisition and beamline control software JBluIce. Both tasks employ wrapper scripts around existing crystallographic software. JBluIce executes scripts through a distributed resource management system to make efficient use of all available computing resources through parallel processing. The JBluIce single-crystal data collection strategy feature uses a choice of strategy programs to help users rank sample crystals and collect data. The strategy results can be conveniently exported to a data collection run. The JBluIce multi-crystal strategy feature calculates a collection strategy to optimize coverage of reciprocal space in cases where incomplete data are available from previous samples. The JBluIce data processing runs simultaneously with data collection using a choice of data reduction wrappers for integration and scaling of newly collected data, with an option for merging with pre-existing data. Data are processed separately if collected from multiple sites on a crystal or from multiple crystals, then scaled and merged. Results from all strategy and processing calculations are displayed in relevant tabs of JBluIce.

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼10 3 –10 4 -fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.