Molecular basis of global promoter sensing and nucleosome capture by the SWR1 chromatin remodeler.

The SWR1 chromatin remodeling complex is recruited to +1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the +1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes.

Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation.

Pyrenoids are subcompartments of algal chloroplasts that increase the efficiency of Rubisco-driven CO2 fixation. Diatoms fix up to 20% of global CO2, but their pyrenoids remain poorly characterized. Here, we used in vivo photo-crosslinking to identify pyrenoid shell (PyShell) proteins, which we localized to the pyrenoid periphery of model pennate and centric diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. In situ cryo-electron tomography revealed that pyrenoids of both diatom species are encased in a lattice-like protein sheath. Single-particle cryo-EM yielded a 2.4-Å-resolution structure of an in vitro TpPyShell1 lattice, which showed how protein subunits interlock. T. pseudonana TpPyShell1/2 knockout mutants had no PyShell sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with reduced photosynthetic efficiency and impaired growth under standard atmospheric conditions. The structure and function of the diatom PyShell provide a molecular view of how CO2 is assimilated in the ocean, a critical ecosystem undergoing rapid change.

The variable structural flexibility of the Bacillus circulans ß-galactosidase isoforms determines their unique functionalities.

ß-Galactosidase from Bacillus circulans ATCC 31382 (BgaD) is a biotechnologically important enzyme for the synthesis of ß-galactooligosaccharides (GOS). Among its four isoforms, isoform A (BgaD-A) has distinct synthetic properties. Here, we present cryoelectron microscopy (cryo-EM) structures of BgaD-A and compare them with the known X-ray crystal structure of isoform D (BgaD-D), revealing substantial structural divergences between the two isoforms. In contrast to BgaD-D, BgaD-A features a flexible Big-4 domain and another enigmatic domain. The newly identified flexible region in BgaD-A is termed as "barrier domain 8," and serves as a barricade, obstructing the access of longer oligosaccharide substrates into the active site of BgaD-A. The transgalactosylation reactions catalyzed by both isoforms revealed that BgaD-A has a higher selectivity than BgaD-D in the earlier stages of the reaction and is prevailingly directed to shorter galactooligosaccharides. This study improves our understanding of the structural determinants governing ß-galactosidase catalysis, with implications for tailored GOS production.

Structural insights into KSHV-GPCR constitutive activation and CXCL1 chemokine recognition.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a viral G protein-coupled receptor, KSHV-GPCR, that contributes to KSHV immune evasion and pathogenesis of Kaposi's sarcoma. KSHV-GPCR shares a high similarity with CXC chemokine receptors CXCR2 and can be activated by selected chemokine ligands. Like other herpesvirus-encoded GPCRs, KSHV-GPCR is characterized by its constitutive activity by coupling to various G proteins. We investigated the structural basis of ligand-dependent and constitutive activation of KSHV-GPCR, obtaining high-resolution cryo-EM structures of KSHV-GPCR-Gi complexes with and without the bound CXCL1 chemokine. Analysis of the apo-KSHV-GPCR-Gi structure (2.81 Å) unraveled the involvement of extracellular loop 2 in constitutive activation of the receptor. In comparison, the CXCL1-bound KSHV-GPCR-Gi structure (3.01 Å) showed a two-site binding mode and provided detailed information of CXCL1 binding to a chemokine receptor. The dual activation mechanism employed by KSHV-GPCR represents an evolutionary adaptation for immune evasion and contributes to the pathogenesis of Kaposi's sarcoma. Together with results from functional assays that confirmed the structural models, these findings may help to develop therapeutic strategies for KSHV infection.

Development of a liquid-helium free cryogenic sample holder with mK temperature control for autonomous electron microscopy.

The automated and autonomous cryogenic transmission electron microscopy (Cryo-EM) demands a sample holder capable of maintaining temperatures below 10 K with precise control, long holding times, and minimal helium use. Rising to this challenge, we initiated an ambitious project to develop a novel closed-cycle cryocooler-based cryogenic sample holder that operates without the use of liquid helium and the consumption of gaseous helium. This article presents the design, construction, and experimental testing of the initial prototype, which achieves an ultimate temperature of 5.6 K with exceptional stability close to 1mK, while providing a wide temperature control range from 295 K to 5.6 K, marking a clear advancement in cryo-EM holder development. While the prototype was not designed for atomic resolution imaging and thus lacks a sturdy support system to mitigate mechanical vibrations from the cryocooler's pulsed tube, this innovative approach successfully demonstrates proof of concept. It offers unprecedented capabilities for state-of-the-art cryogenic microscopy and microanalysis in materials and biological sciences.

Roodmus: a toolkit for benchmarking heterogeneous electron cryo-microscopy reconstructions.

Conformational heterogeneity of biological macromolecules is a challenge in single-particle averaging (SPA). Current standard practice is to employ classification and filtering methods that may allow a discrete number of conformational states to be reconstructed. However, the conformation space accessible to these molecules is continuous and, therefore, explored incompletely by a small number of discrete classes. Recently developed heterogeneous reconstruction algorithms (HRAs) to analyse continuous heterogeneity rely on machine-learning methods that employ low-dimensional latent space representations. The non-linear nature of many of these methods poses a challenge to their validation and interpretation and to identifying functionally relevant conformational trajectories. These methods would benefit from in-depth benchmarking using high-quality synthetic data and concomitant ground truth information. We present a framework for the simulation and subsequent analysis with respect to the ground truth of cryo-EM micrographs containing particles whose conformational heterogeneity is sourced from molecular dynamics simulations. These synthetic data can be processed as if they were experimental data, allowing aspects of standard SPA workflows as well as heterogeneous reconstruction methods to be compared with known ground truth using available utilities. The simulation and analysis of several such datasets are demonstrated and an initial investigation into HRAs is presented.

Structure of the CUL1-RBX1-SKP1-FBXO4 SCF ubiquitin ligase complex.

Cullin-RING E3 ubiquitin ligases (CRLs) constitute the largest family of ubiquitin ligase and play important roles in regulation of proteostasis. Here we presented the cryo-EM structure of CRL1FBXO4, a member of Cullin-1 E3 ligase. CRL1FBXO4 adopts a homodimer architecture. Structural analysis revealed that in the CRL1FBXO4 protomer, the substrate recognition subunit FBXO4 interacts both the adaptor protein SKP1, and the scaffold protein CUL1 via hydrophobic and electrostatic interactions. Two FBXO4 forms a domain-swapped dimer in the CRL1FBXO4 structure, which constitutes the basis for the dimerization of CRL1FBXO4. Inspired by the cryo-EM density, we modeled the architecture of whole CRL1FBXO4 as a symmetrical dimer, which provides insights into CRL1FBXO4-medaited turnover of oncogene proteins.

Expression, purification, and biochemical analysis of the RNA-DNA hybrid helicase Sen1/SETX from Chaetomium thermophilum.

Yeast Sen1 and its vertebrate ortholog Senataxin (also known as SETX) are RNA-DNA resolving helicases. Sen1 and SETX are implicated in multiple critical nuclear functions not limited to but including DNA replication and repair, RNA processing, and transcription. These> 200 kDa helicases have a two-domain architecture with an N-terminal regulatory helical repeat array linked to an SF1b helicase motor core via a variable sized central linker of low complexity sequence. Given the size of these proteins, production of milligram quantities of protein that is suitable for biochemical, biophysical, and protein structural analysis has been challenging. To overcome these limitations, we developed a robust selectable high-yield YFP-fusion protein expression method for Sen1 production in mammalian cells, followed by purification on a high-affinity YFP-binding camelid nanobody support. Herein, we detail methods and protocols for the expression and purification of recombinant Sen1 from the thermophilic fungus Chaetomium thermophilum, and the quantitative characterization of its RNA-DNA duplex resolution activity.

TRPML1 gating modulation by allosteric mutations and lipids.

Transient Receptor Potential Mucolipin 1 (TRPML1) is a lysosomal cation channel whose loss-of-function mutations directly cause the lysosomal storage disorder mucolipidosis type IV (MLIV). TRPML1 can be allosterically regulated by various ligands including natural lipids and small synthetic molecules and the channel undergoes a global movement propagated from ligand-induced local conformational changes upon activation. In this study, we identified a functionally critical residue, Tyr404, at the C-terminus of the S4 helix, whose mutations to tryptophan and alanine yield gain- and loss-of-function channels, respectively. These allosteric mutations mimic the ligand activation or inhibition of the TRPML1 channel without interfering with ligand binding and both mutant channels are susceptible to agonist or antagonist modulation, making them better targets for screening potent TRPML1 activators and inhibitors. We also determined the high-resolution structure of TRPML1 in complex with the PI(4,5)P2 inhibitor, revealing the structural basis underlying this lipid inhibition. In addition, an endogenous phospholipid likely from sphingomyelin is identified in the PI(4,5)P2-bound TRPML1 structure at the same hotspot for agonists and antagonists, providing a plausible structural explanation for the inhibitory effect of sphingomyelin on agonist activation.

Selective ligand recognition and activation of somatostatin receptors SSTR1 and SSTR3.

Somatostatin receptors (SSTRs) exert critical biological functions such as negatively regulating hormone release and cell proliferation, making them popular targets for developing therapeutics to treat endocrine disorders, especially neuroendocrine tumors. Although several panagonists mimicking the endogenous ligand somatostatin are available, the development of more effective and safer somatostatinergic therapies is limited due to a lack of molecular understanding of the ligand recognition and regulation of divergent SSTR subtypes. Here, we report four cryoelectron microscopy structures of Gi-coupled SSTR1 and SSTR3 activated by distinct agonists, including the FDA-approved panagonist pasireotide as well as their selective small molecule agonists L-797591 and L-796778. Our structures reveal a conserved recognition pattern of pasireotide in SSTRs attributed to the binding with a conserved extended binding pocket, distinct from SST14, octreotide, and lanreotide. Together with mutagenesis analyses, our structures further reveal the dynamic feature of ligand binding pockets in SSTR1 and SSTR3 to accommodate divergent agonists, the key determinants of ligand selectivity lying across the orthosteric pocket of different SSTR subtypes, as well as the molecular mechanism underlying diversity and conservation of receptor activation. Our work provides a framework for rational design of subtype-selective SSTR ligands and may facilitate drug development efforts targeting SSTRs with improved therapeutic efficacy and reduced side effects.

Structural mechanism of HP1⍺-dependent transcriptional repression and chromatin compaction.

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. Here, we present the cryoelectron microscopy (cryo-EM) structure of an HP1α dimer bound to an H2A.Z-nucleosome, revealing two distinct HP1α-nucleosome interfaces. The primary HP1α binding site is located at the N terminus of histone H3, specifically at the trimethylated lysine 9 (K9me3) region, while a secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. Our study offers a model for HP1α-mediated heterochromatin maintenance and gene silencing. It also sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.

The structural landscape of Microprocessor-mediated processing of pri-let-7 miRNAs.

MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryoelectron microscopy (cryo-EM) and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "flipped U with paired N" (fUN) motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a non-canonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC motif and Drosha's Piwi/Argonaute/Zwille (PAZ)-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.

Structure of TnsABCD transpososome reveals mechanisms of targeted DNA transposition.

Tn7-like transposons are characterized by their ability to insert specifically into host chromosomes. Recognition of the attachment (att) site by TnsD recruits the TnsABC proteins to form the transpososome and facilitate transposition. Although this pathway is well established, atomic-level structural insights of this process remain largely elusive. Here, we present the cryo-electron microscopy (cryo-EM) structures of the TnsC-TnsD-att DNA complex and the TnsABCD transpososome from the Tn7-like transposon in Peltigera membranacea cyanobiont 210A, a type I-B CRISPR-associated transposon. Our structures reveal a striking bending of the att DNA, featured by the intercalation of an arginine side chain of TnsD into a CC/GG dinucleotide step. The TnsABCD transpososome structure reveals TnsA-TnsB interactions and demonstrates that TnsC not only recruits TnsAB but also directly participates in the transpososome assembly. These findings provide mechanistic insights into targeted DNA insertion by Tn7-like transposons, with implications for improving the precision and efficiency of their genome-editing applications.

ERK1/2 interaction with DHPS regulates eIF5A deoxyhypusination independently of ERK kinase activity.

This study explores a non-kinase effect of extracellular regulated kinases 1/2 (ERK1/2) on the interaction between deoxyhypusine synthase (DHPS) and its substrate, eukaryotic translation initiation factor 5A (eIF5A). We report that Raf/MEK/ERK activation decreases the DHPS-ERK1/2 interaction while increasing DHPS-eIF5A association in cells. We determined the cryoelectron microscopy (cryo-EM) structure of the DHPS-ERK2 complex at 3.5 Å to show that ERK2 hinders substrate entrance to the DHPS active site, subsequently inhibiting deoxyhypusination in vitro. In cells, impairing the ERK2 activation loop, but not the catalytic site, prolongs the DHPS-ERK2 interaction irrespective of Raf/MEK signaling. The ERK2 Ser-Pro-Ser motif, but not the common docking or F-site recognition sites, also regulates this complex. These data suggest that ERK1/2 dynamically regulate the DHPS-eIF5A interaction in response to Raf/MEK activity, regardless of its kinase function. In contrast, ERK1/2 kinase activity is necessary to regulate the expression of DHPS and eIF5A. These findings highlight an ERK1/2-mediated dual kinase-dependent and -independent regulation of deoxyhypusination.

A binding site for the antibiotic GE81112 in the ribosomal mRNA channel.

The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B and B1 with molecular masses of 643-658 Da). Previous biochemical and structural characterisation of GE81112 indicates that the primary mechanism of action of this antibiotic is to (1) prevent the initiator tRNA from binding correctly to the P-site and (2) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S pre/C state. In this study, using cryoEM, we have determined the binding site of GE81112 in initiation complexes (3.2-3.7Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel (E-site) but remote from the binding site of the initiation factors and initiator tRNA. This suggests that it acts allosterically to prevent the initiator tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.

Host Tropism and Structural Biology of ABC Toxin Complexes.

ABC toxin complexes are a class of protein toxin translocases comprised of a multimeric assembly of protein subunits. Each subunit displays a unique composition, contributing to the formation of a syringe-like nano-machine with natural cargo carrying, targeting, and translocation capabilities. Many of these toxins are insecticidal, drawing increasing interest in agriculture for use as biological pesticides. The A subunit (TcA) is the largest subunit of the complex and contains domains associated with membrane permeation and targeting. The B and C subunits, TcB and TcC, respectively, package into a cocoon-like structure that contains a toxic peptide and are coupled to TcA to form a continuous channel upon final assembly. In this review, we outline the current understanding and gaps in the knowledge pertaining to ABC toxins, highlighting seven published structures of TcAs and how these structures have led to a better understanding of the mechanism of host tropism and toxin translocation. We also highlight similarities and differences between homologues that contribute to variations in host specificity and conformational change. Lastly, we review the biotechnological potential of ABC toxins as both pesticides and cargo-carrying shuttles that enable the transport of peptides into cells.

The Arthropoda-specific Tramtrack group BTB protein domains use previously unknown interface to form hexamers.

BTB (bric-a-brack, Tramtrack, and broad complex) is a diverse group of protein-protein interaction domains found within metazoan proteins. Transcription factors contain a dimerizing BTB subtype with a characteristic N-terminal extension. The Tramtrack group (TTK) is a distinct type of BTB domain, which can multimerize. Single-particle cryo-EM microscopy revealed that the TTK-type BTB domains assemble into a hexameric structure consisting of three canonical BTB dimers connected through a previously uncharacterized interface. We demonstrated that the TTK-type BTB domains are found only in Arthropods and have undergone lineage-specific expansion in modern insects. The Drosophila genome encodes 24 transcription factors with TTK-type BTB domains, whereas only four have non-TTK-type BTB domains. Yeast two-hybrid analysis revealed that the TTK-type BTB domains have an unusually broad potential for heteromeric associations presumably through a dimer-dimer interaction interface. Thus, the TTK-type BTB domains are a structurally and functionally distinct group of protein domains specific to Arthropodan transcription factors.

Structural Changes Likely Cause Chemical Differences between Empty and Full AAV Capsids.

Due to the success of adeno associated viruses (AAVs) in treating single-gene diseases, improved manufacturing technology is now needed to meet their demand. The largest challenge is creating a process to separate empty and full capsids. Patients received larger capsid doses than necessary due to the presence of empty capsids. By enabling the better separation of empty and full capsids, patients would receive the greatest therapeutic benefit with the least amount of virus capsids, thus limiting potential side effects from empty capsids. The two most common empty/full separation methods used in downstream processing are ultracentrifugation and anion exchange chromatography. Both processes have limitations, leading to a need for the identification of other structural differences that can be exploited to separate empty and full capsids. Here, we describe four possible theories of the structural changes that occur when AAV capsids envelop a genome. These theories include conformational changes occurring due to either the expansion or contraction of the capsid in the presence of nucleic acids, the constraining of the N-terminus into the five-fold pore when the genome is present, and the increased number of VP3 proteins in full capsids. These theories may reveal structural differences that can be exploited to separate full and empty capsids during manufacturing.

Quantum refinement in real and reciprocal space using the Phenix and ORCA software.

X-ray and neutron crystallography, as well as cryogenic electron microscopy (cryo-EM), are the most common methods to obtain atomic structures of biological macromolecules. A feature they all have in common is that, at typical resolutions, the experimental data need to be supplemented by empirical restraints, ensuring that the final structure is chemically reasonable. The restraints are accurate for amino acids and nucleic acids, but often less accurate for substrates, inhibitors, small-molecule ligands and metal sites, for which experimental data are scarce or empirical potentials are harder to formulate. This can be solved using quantum mechanical calculations for a small but interesting part of the structure. Such an approach, called quantum refinement, has been shown to improve structures locally, allow the determination of the protonation and oxidation states of ligands and metals, and discriminate between different interpretations of the structure. Here, we present a new implementation of quantum refinement interfacing the widely used structure-refinement software Phenix and the freely available quantum mechanical software ORCA. Through application to manganese superoxide dismutase and V- and Fe-nitrogenase, we show that the approach works effectively for X-ray and neutron crystal structures, that old results can be reproduced and structural discrimination can be performed. We discuss how the weight factor between the experimental data and the empirical restraints should be selected and how quantum mechanical quality measures such as strain energies should be calculated. We also present an application of quantum refinement to cryo-EM data for particulate methane monooxygenase and show that this may be the method of choice for metal sites in such structures because no accurate empirical restraints are currently available for metals.