The Advanced Photon Source (APS) has undergone a comprehensive upgrade to replace the original electron storage ring with a new storage ring (APS-U) that will increase the X-ray brightness 500 times compared to the APS. The National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility at the Advanced Photon Source (GM/CA@APS) operates a national user facility for structural biology. The facility includes canted-undulator beamlines, 23ID-B, and 23ID-D. GM/CA users have been very productive, resulting in over 2100 publications and almost 3600 protein data bank deposits. Our micro-crystallography developments supported the research of Brian Kobilka, who was awarded the 2012 Nobel Prize in Chemistry for studies of G-protein-coupled receptors (GPCRs). During the year-long shutdown, the GM/CA beamlines and infrastructure were almost completely rebuilt to exploit the high brightness of the APS-U. New state-of-the-art focusing optics (mirrors and Compound Refractive Lenses (CRLs)) will provide extremely intense, clean, stable, and rapidly adjustable beam sizes between 1-50 microns. The maximum energy on 23-ID-D was increased to 35 keV to minimize radiation damage. The new high-stability end station table will support both the CRL translocator and sample environment. The new goniometer will allow data collection on crystals as small as one micron and provide rapid scanning of random or periodic fixed target samples. A Dectris Eiger2 16M CdTe detector will allow high-speed, high-efficiency X-ray detection on 23-ID-D. The new pyBluIce GUI and beamline control software will enable sophisticated data collection routines such as 3D-rastering and helical data collection, fully automated (unattended) data collection, and routine serial crystallography data collection from fixed target and injector-based sample delivery systems. We anticipate first X-rays this summer and will begin commissioning the beamlines. We look forward to welcoming users back in the fall of 2024. Here, we will present the new designs and game-changing opportunities for structural biology research enabled by these small, ultra- intense, high-energy beams. GM/CA@APS has been funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396). The Eiger 16M detector was funded by an NIH, High-End Instrumentation Grant (S10OD012289). 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 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.

Multicopper oxidases (MCOs) represent a diverse family of enzymes that catalyze the oxidation of either an organic or a metal substrate with concomitant reduction of dioxygen to water. These enzymes contain variable numbers of cupredoxin domains, two, three or six per subunit, and rely on four copper ions, a single type I copper and three additional copper ions organized in a trinuclear cluster (TNC), with one type II and two type III copper ions, to catalyze the reaction. Here, two crystal structures and the enzymatic characterization of Marinithermus hydrothermalis MCO, a two-domain enzyme, are reported. This enzyme decolorizes Congo Red dye at 70°C in the presence of high halide concentrations and may therefore be useful in the detoxification of industrial waste that contains dyes. In two distinct crystal structures, MhMCO forms the trimers seen in other two-domain MCOs, but differs from these enzymes in that four trimers interact to create a dodecamer. This dodecamer of MhMCO forms a closed ball-like structure and has implications for the sequestration of bound divalent metal ions as well as substrate accessibility. In each subunit of the dodecameric structures, a Trp residue, Trp351, located between the type I and TNC sites exists in two distinct conformations, consistent with a potential role in facilitating electron transfer in the enzyme.

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).

Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth (`pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A 2A adenosine receptor (A 2A AR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A 2A AR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.

Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.

With the recent developments in microcrystal handling, synchrotron microdiffraction beamline instrumentation and data analysis, microcrystal crystallography with crystal sizes of less than 10 µm is appealing at synchrotrons. However, challenges remain in sample manipulation and data assembly for robust microcrystal synchrotron crystallography. Here, the development of micro-sized polyimide well-mounts for the manipulation of microcrystals of a few micrometres in size and the implementation of a robust data-analysis method for the assembly of rotational microdiffraction data sets from many microcrystals are described. The method demonstrates that microcrystals may be routinely utilized for the acquisition and assembly of complete data sets from synchrotron microdiffraction beamlines.

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.

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A 2A adenosine receptor (A 2A AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A 2A AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A 2A AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.

The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals. Moreover, the use of TEM allowed (i) comparison of lattice quality among crystals from different conditions in crystallization screens; (ii) the detection of crystal pathologies that could contribute to poor X-ray diffraction, including crystal lattice defects, anisotropic diffraction and crystal contamination by heavy protein aggregates and nanocrystal nuclei; (iii) the qualitative estimation of crystal solvent content to explore the effect of lattice dehydration on diffraction and (iv) the selection of high-quality crystal fragments for microseeding experiments to generate reproducibly larger sized crystals. Applications to X-ray free-electron laser (XFEL) and micro-electron diffraction (microED) experiments are also discussed.

In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometer-based X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.

Microcrystals present a significant impediment to the determination of macromolecular structures by X-ray diffraction methods. Although microfocus synchrotron beamlines and X-ray free-electron lasers (XFELs) can enable the collection of interpretable diffraction data from microcrystals, there is a need for efficient methods of harvesting small volumes (<2 µl) of microcrystals grown under common laboratory formats and delivering them to an X-ray beam source under native growth conditions. One approach that shows promise in overcoming the challenges intrinsic to microcrystal analysis is to pair so-called `fixed-target' sample-delivery devices with microbeam-based X-ray diffraction methods. However, to record weak diffraction patterns it is necessary to fabricate devices from X-ray-transparent materials that minimize background scattering. Presented here is the design of a new micro-diffraction device consisting of three layers fabricated from silicon nitride, photoresist and polyimide film. The chip features low X-ray scattering and X-ray absorption properties, and uses a customizable blend of hydrophobic and hydrophilic surface patterns to help localize microcrystals to defined regions. Microcrystals in their native growth conditions can be loaded into the chips with a standard pipette, allowing data collection at room temperature. Diffraction data collected from hen egg-white lysozyme microcrystals (10–15 µm) loaded into the chips yielded a complete, high-resolution (<1.6 Å) data set sufficient to determine a high-quality structure by molecular replacement. The features of the chip allow the rapid and user-friendly analysis of microcrystals grown under virtually any laboratory format at microfocus synchrotron beamlines and XFELs.

An emergent challenge in macromolecular crystallography is the identification of the substructure from native anomalous scatterers in crystals that diffract to low to moderate resolution. Increasing the multiplicity of data sets has been shown to make previously intractable phasing problems solvable and to increase the useful resolution in model refinement. For the West Nile virus nonstructural protein 1 (NS1), a protein of novel fold, the utility of exceptionally high multiplicity data is demonstrated both in solving the crystal structure from the anomalous scattering of the native S atoms and in extending the useful limits of resolution during refinement. A high-multiplicity data set from 18 crystals had sufficient anomalous signal to identify sulfur sites using data to 5.2 Å resolution. Phases calculated to 4.5 Å resolution and extended to 3.0 Å resolution were of sufficient quality for automated building of three-quarters of the final structure. Crystallographic refinement to 2.9 Å resolution proceeded smoothly, justifying the increase in resolution that was made possible by combining multiple data sets. The identification and exclusion of data from outlier crystals is shown to result in more robust substructure determination.

Flaviviruses, the human pathogens responsible for dengue fever, West Nile fever, tick-borne encephalitis, and yellow fever, are endemic in tropical and temperate parts of the world. The flavivirus nonstructural protein 1 (NS1) functions in genome replication as an intracellular dimer and in immune system evasion as a secreted hexamer. We report crystal structures for full-length, glycosylated NS1 from West Nile and dengue viruses. The NS1 hexamer in crystal structures is similar to a solution hexamer visualized by single-particle electron microscopy. Recombinant NS1 binds to lipid bilayers and remodels large liposomes into lipoprotein nanoparticles. The NS1 structures reveal distinct domains for membrane association of the dimer and interactions with the immune system and are a basis for elucidating the molecular mechanism of NS1 function.

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.

GM/CA at the APS has developed microcrystallography capabilities for structural biology applications. The robust, quad, mini-beam collimators, which enable users to rapidly select between a 5, 10 or 20 micron diameter beam or a scatter guard for the full focused beam, are coupled with several powerful automated software tools that are built into the beamline control system JBluIce-EPICS. Recent successes at beamlines around the world in solving structures from microcrystals (2 - 10 microns) have led to increased demand for high-intensity micro-focus beams. We have designed a new micro-focus endstation to increase the intensity in mini- and micro-beams at GM/CA by one to two orders of magnitude to meet this growing demand. The new optical design is based on the well-established approach of using two-stage demagnification. The existing bimorph mirrors, arranged in a Kirkpatrick-Baez geometry, focus the beam onto slits located upstream of the sample whereby the slit aperture defines a secondary source, that is reimaged with a second pair of mirrors. This design incorporates two focal modes: a mini-beam mode where the beam is focused to 20-micron diameter and a micro-beam mode where it is focused to 5-microns. The size of the secondary source aperture can be varied rapidly (seconds) to adjust the beam size at the sample position in two ranges 20 - 3 micron and 5 - 1 micron. The second set of mirrors will each have two super polished ellipses allowing quick (minutes) interchange between modes.