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 GNAT (GCN5-related N-acetyltransferase) superfamily comprises enzymes with a conserved fold and diverse catalytic activities, including primarily acyl transfer, with a few examples of decarboxylation. EryM, a GNAT from Saccharopolyspora erythraea, has been implicated in both erythromycin and erythrochelin biosynthesis, with dual functionality as an acetyltransferase and a decarboxylase. Despite an historical association with malonyl-coenzyme A decarboxylation activity, this dual activity has remained enigmatic as its close homologs were identified with only acyl transfer activity. Here, functional assays demonstrate that EryM catalyzes acyl transfer but lacks decarboxylation activity, challenging long-standing assumptions about its biosynthetic role. Crystal structures of EryM and an acetyl-CoA complex and comparison with homologs in siderophore pathways reveal a conserved catalytic pocket with an essential His and identically positioned side chains common to GNAT enzymes for N-acyl transfer from CoA to primary hydroxylamine substrates. Bioinformatic analysis defines a large GNAT subfamily broadly distributed in the microbial world.

Bacteria are rich sources of pharmaceutically valuable natural products, many crafted by modular polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). PKS and NRPS systems typically contain a thioesterase (TE) to offload a linear or cyclized product from a carrier protein, but alternative chemistry is needed for products with a terminal amide. Several pathways with amidated products also possess an uncharacterized 400-amino acid terminal domain. We present the characterization and structure of this putative terminal amidation domain (TAD). TAD binds NAD with the nicotinamide near an invariant cysteine that is also accessible to an intermediate on a carrier protein, indicating a catalytic role. The TAD structure resembles cyanobacterial acyl-ACP reductase (AAR), which binds NADPH near an analogous catalytic cysteine. Bioinformatic analysis reveals that TADs are broadly distributed across bacterial phyla and often occur at the end of terminal NRPS modules, suggesting many amidated products may yet be discovered.

X-ray crystallography remains the dominant method of determining the three-dimensional structure of proteins. Nevertheless, this resource-intensive process may be hindered by the unintended crystallization of contaminant proteins from the expression source. Here, the serendipitous discovery of two novel crystal forms and one new, high-resolution structure of carbonic anhydrase 2 (CA2) from Escherichia coli that arose during a crystallization campaign for an unrelated target is reported. By comparing unit-cell parameters with those in the PDB, contaminants such as CA2 can be identified, preventing futile molecular-replacement attempts. Crystallographers can use these new lattice parameters to diagnose CA2 contamination in similar experiments.

Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the terminal PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TEDAP) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TEDAP, the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 & Aring; from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide insights to facilitate TE engineering and optimization.

The biosynthetic dogma of ribosomally synthesized and posttranslationally modified peptides (RiPP) involves enzymatic intermolecular modification of core peptide motifs in precursor peptides. The plant-specific BURP-domain protein family, named after their four founding members, includes autocatalytic peptide cyclases involved in the biosynthesis of side-chain-macrocyclic plant RiPPs. Here we show that AhyBURP, a representative of the founding Unknown Seed Protein-type BURP-domain subfamily, catalyzes intramolecular macrocyclizations of its core peptide during the sequential biosynthesis of monocyclic lyciumin I via glycine-tryptophan crosslinking and bicyclic legumenin via glutamine-tyrosine crosslinking. X-ray crystallography of AhyBURP reveals the BURP-domain fold with two type II copper centers derived from a conserved stapled-disulfide and His motif. We show the macrocyclization of lyciumin-C(sp3)-N-bond formation followed by legumenin-C(sp3)-O-bond formation requires dioxygen and radical involvement based on enzyme assays in anoxic conditions and isotopic labeling. Our study expands enzymatic intramolecular modifications beyond catalytic moiety and chromophore biogenesis to RiPP biosynthesis. BURP-domain proteins are an unexplored family of plant-specific, copper-dependent peptide cyclases. Here the authors show that a BURP-domain protein has a particular protein fold, investigate its mechanism and provide evidence for intramolecular modification in RiPP biosynthesis.

AbstractZAP is an antiviral protein that binds to and depletes viral RNA, which is often distinguished from vertebrate host RNA by its elevated CpG content. Two ZAP cofactors, TRIM25 and KHNYN, have activities that are poorly understood. Here, we show that functional interactions between ZAP, TRIM25 and KHNYN involve multiple domains of each protein, and that the ability of TRIM25 to multimerize via its RING domain augments ZAP activity and specificity. We show that KHNYN is an active nuclease that acts in a partly redundant manner with its homolog N4BP1. The ZAP N-terminal RNA binding domain constitutes a minimal core that is essential for antiviral complex activity, and we present a crystal structure of this domain that reveals contacts with the functionally required KHNYN C-terminal domain. These contacts are remote from the ZAP CpG binding site and would not interfere with RNA binding. Based on our dissection of ZAP, TRIM25 and KHNYN functional anatomy, we could design artificial chimeric antiviral proteins that reconstitute the antiviral function of the intact authentic proteins, but in the absence of protein domains that are otherwise required for activity. Together, these results suggest a model for the RNA recognition and action of ZAP-containing antiviral protein complexes.

Detecting viral infection is a key role of the innate immune system. The genomes of some RNA viruses have a high CpG dinucleotide content relative to most vertebrate cell RNAs, making CpGs a molecular marker of infection. The human zinc-finger antiviral protein (ZAP) recognizes CpG, mediates clearance of the foreign CpG-rich RNA, and causes attenuation of CpG-rich RNA viruses. While ZAP binds RNA, it lacks enzymatic activity that might be responsible for RNA degradation and thus requires interacting cofactors for its function. One of these cofactors, KHNYN, has a predicted nuclease domain. Using biochemical approaches, we found that the KHNYN NYN domain is a single-stranded RNA ribonuclease that does not have sequence specificity and digests RNA with or without CpG dinucleotides equivalently in vitro. We show that unlike most KH domains, the KHNYN KH domain does not bind RNA. Indeed, a crystal structure of the KH region revealed a double-KH domain with a negatively charged surface that accounts for the lack of RNA binding. Rather, the KHNYN C-terminal domain (CTD) interacts with the ZAP RNA-binding domain (RBD) to provide target RNA specificity. We define a minimal complex composed of the ZAP RBD and the KHNYN NYN-CTD and use a fluorescence polarization assay to propose a model for how this complex interacts with a CpG dinucleotide-containing RNA. In the context of the cell, this module would represent the minimum ZAP and KHNYN domains required for CpG-recognition and ribonuclease activity essential for attenuation of viruses with clusters of CpG dinucleotides.

ABSTRACTLimited ancestral diversity has impaired our ability to detect risk variants more prevalent in non-European ancestry groups in genome-wide association studies (GWAS). We constructed and analyzed a multi-ancestry GWAS dataset in the Alzheimer’s Disease (AD) Genetics Consortium (ADGC) to test for novel shared and ancestry-specific AD susceptibility loci and evaluate underlying genetic architecture in 37,382 non-Hispanic White (NHW), 6,728 African American, 8,899 Hispanic (HIS), and 3,232 East Asian individuals, performing within-ancestry fixed-effects meta-analysis followed by a cross-ancestry random-effects meta-analysis. We identified 13 loci with cross-ancestry associations including known loci at/nearCR1,BIN1,TREM2,CD2AP,PTK2B,CLU,SHARPIN,MS4A6A,PICALM,ABCA7,APOEand two novel loci not previously reported at 11p12 (LRRC4C) and 12q24.13 (LHX5-AS1). Reflecting the power of diverse ancestry in GWAS, we observed theSHARPINlocus using 7.1% the sample size of the original discovering single-ancestry GWAS (n=788,989). We additionally identified three GWS ancestry-specific loci at/near (PTPRK(P=2.4×10-8) andGRB14(P=1.7×10-8) in HIS), andKIAA0825(P=2.9×10-8in NHW). Pathway analysis implicated multiple amyloid regulation pathways (strongest withPadjusted=1.6×10-4) and the classical complement pathway (Padjusted=1.3×10-3). Genes at/near our novel loci have known roles in neuronal development (LRRC4C, LHX5-AS1, andPTPRK) and insulin receptor activity regulation (GRB14). These findings provide compelling support for using traditionally-underrepresented populations for gene discovery, even with smaller sample sizes.

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.

Limited ancestral diversity has impaired our ability to detect risk variants more prevalent in non-European ancestry groups in genome-wide association studies (GWAS). We constructed and analyzed a multi-ancestry GWAS dataset in the Alzheimer Disease (AD) Genetics Consortium (ADGC) to test for novel shared and ancestry-specific AD susceptibility loci and evaluate underlying genetic architecture in 37,382 non-Hispanic White (NHW), 6,728 African American, 8,899 Hispanic (HIS), and 3,232 East Asian individuals, performing within-ancestry fixed-effects meta-analysis followed by a cross-ancestry random-effects meta-analysis. We identified 13 loci with cross-ancestry associations including known loci at/near CR1, BIN1, TREM2, CD2AP, PTK2B, CLU, SHARPIN, MS4A6A, PICALM, ABCA7, APOE and two novel loci not previously reported at 11p12 (LRRC4C) and 12q24.13 (LHX5-AS1). Reflecting the power of diverse ancestry in GWAS, we observed the SHARPIN locus using 7.1% the sample size of the original discovering single-ancestry GWAS (n=788,989). We additionally identified three GWS ancestry-specific loci at/near (PTPRK (P=2.4E10-8) and GRB14 (P=1.7E10-8) in HIS), and KIAA0825 (P=2.9E10-8 in NHW). Pathway analysis implicated multiple amyloid regulation pathways (strongest with Padjusted=1.6E10-4) and the classical complement pathway (Padjusted=1.3E10-3). Genes at/near our novel loci have known roles in neuronal development (LRRC4C, LHX5-AS1, and PTPRK) and insulin receptor activity regulation (GRB14). These findings provide compelling support for using traditionally-underrepresented populations for gene discovery, even with smaller sample sizes.

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes’ innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.

A commentary on Wayne Hendrickson’s article ‘Facing the phase problem’.

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.

Polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) are essential enzymes responsible for the assembly of natural products, such as the common antibiotic erythromycin and the siderophore erythrochelin.Most bacterial PKS and NRPS biosynthetic pathways are encoded by gene located in a cluster.Interestingly, the enzyme EryM, encoded by a gene apart from any PKS or NRPS gene cluster, was identified to be crucial to producing erythrochelin and erythromycin.1EryMwas first identified as a malonyl-CoA decarboxylase.3Later studies led to the proposal that EryM is an acetyltransferase where the decarboxylation product, acetyl-CoA, is the acetyl group donor.4The NRPS gene cluster for the erythrochelin pathway lacks an acetyltransferase to generate acetyl-hydroxyl-ornithine as the starter unit, thus EryM provides the missing link.Yet another study demonstrated that eryM is essential for erythromycin biosynthesis.The erythromycin PKS gene cluster lacks any gene for an enzyme to generate the starter unit, propionyl-CoA.Thus, EryM is proposed to form this starter unit by decarboxylation of methylmalonyl-CoA.However, it is still unknown how or whether EryM performs both malonyland methylmalonyl-CoA decarboxylation as well as acetyl transfer.We used structural and biochemical techniques to probe the biological function of EryM.We found that EryM is composed of an N-terminal uncharacterized domain and a C-terminal domain containing the acetyltransferase active site.Purified EryM catalyzed decarboxylation of malonyl-CoA and subsequently used the intermediate acetyl-CoA as a substrate for acetyl transfer.These results suggest that EryM is a bifunctional enzyme that synthesizes the substrate for downstream natural product biosynthesis.

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.

Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.

Installation of methyl groups can significantly improve the binding of small-molecule drugs to protein targets; however, site-selective methylation often presents a significant synthetic challenge. Metal- and S-adenosyl-methionine (SAM)-dependent methyltransferases (MTs) in natural-product biosynthetic pathways are powerful enzymatic tools for selective or chemically challenging C-methylation reactions. Each of these MTs selectively catalyzes one or two methyl transfer reactions. Crystal structures and biochemical assays of the Mn2+-dependent monomethyltransferase from the saxitoxin biosynthetic pathway (SxtA MT) revealed the structural basis for control of methylation extent. The SxtA monomethyltransferase was converted to a dimethyltransferase by modification of the metal binding site, addition of an active site base, and an amino acid substitution to provide space in the substrate pocket for two methyl substituents. A reciprocal change converted a related dimethyltransferase into a monomethyltransferase, supporting our hypothesis that steric hindrance can prevent a second methylation event. A novel understanding of MTs will accelerate the development of MT-based catalysts and MT engineering for use in small-molecule synthesis.