Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

Plasmid targeting and destruction by the DdmDE bacterial defence system.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

Lipid droplets as substrates for protein phase separation.

Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by reducing dimensionality from three to two dimensions, lipid bilayers can nucleate phase separation at far lower concentrations compared with those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that the model disordered domains FUS LC and LAF-1 RGG separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF-1 RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.

Type III-B CRISPR-Cas cascade of proteolytic cleavages.

The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from Haliangium ochraceum that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide-based antiphage signaling system (CBASS). Cyclic tri-adenosine monophosphate (AMP)-induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase. Subsequently, activated PCaspase cleaves a multitude of proteins, which results in a strong interference phenotype in vivo in Escherichia coli. Taken together, our findings reveal how a CRISPR-Cas-based detection of a target RNA triggers a cascade of caspase-associated proteolytic activities.

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes.

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 19,000 XLs, we identified 4,757 unique amino acid interactions among 1,143 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease.

Structural biology of an organ.

Su et al.1 use a build-and-retrieve approach to both identify and determine structures of ten macromolecular machines in the human liver. The authors' method will launch researchers forward in understanding the structural biology of the cell (or organ).

Type III CRISPR-Cas complexes act as protein-assisted ribozymes during target RNA cleavage.

CRISPR-Cas systems are an adaptive immune system in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes; however, the mechanism has remained enigmatic6,7. Here, we determine the structures of the Synechocystis type III-Dv complex, an evolutionary intermediate in type III effectors8,9, in pre- and post-cleavage states, which show metal ion coordination in the active sites. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we reveal the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Thus, type III CRISPR-Cas complexes function as protein-assisted ribozymes, and their programmable nature has important implications for how these complexes could be repurposed for applications.

Invasive pulmonary aspergillosis presenting with tracheopleural fistula in a pediatric patient with a history of rhabdomyosarcoma.

Invasive aspergillosis is a known complication in patients with hematologic malignancies. Tracheopleural fistulas are very rare and reported in immunocompromised adults. We present a case of invasive pulmonary aspergillosis with tracheopleural fistula in a pediatric patient with a history of rhabdomyosarcoma and macrophage activation syndrome. This case highlights the importance of recognizing life-threatening fungal infections and coordinating surgical subspecialities for patient care.

Does AlphaFold2 model proteins' intracellular conformations? An experimental test using cross-linking mass spectrometry of endogenous ciliary proteins.

A major goal in structural biology is to understand protein assemblies in their biologically relevant states. Here, we investigate whether AlphaFold2 structure predictions match native protein conformations. We chemically cross-linked proteins in situ within intact Tetrahymena thermophila cilia and native ciliary extracts, identifying 1,225 intramolecular cross-links within the 100 best-sampled proteins, providing a benchmark of distance restraints obeyed by proteins in their native assemblies. The corresponding structure predictions were highly concordant, positioning 86.2% of cross-linked residues within Cɑ-to-Cɑ distances of 30 Å, consistent with the cross-linker length. 43% of proteins showed no violations. Most inconsistencies occurred in low-confidence regions or between domains. Overall, AlphaFold2 predictions with lower predicted aligned error corresponded to more correct native structures. However, we observe cases where rigid body domains are oriented incorrectly, as for ciliary protein BBC118, suggesting that combining structure prediction with experimental information will better reveal biologically relevant conformations.

Structural snapshots of R-loop formation by a type I-C CRISPR Cascade.

Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various stages of double-stranded (ds)DNA target capture, revealing mechanisms that underpin PAM recognition and Cascade allosteric activation. We uncover an interesting mechanism of non-target strand (NTS) DNA stabilization via stacking interactions with the "belly" subunits, securing the NTS in place. This "molecular seatbelt" mechanism facilitates efficient R-loop formation and prevents dsDNA reannealing. Additionally, we provide structural insights into how two anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning. These observations form a structural basis for directional R-loop formation and reveal how different Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.

RNA targeting unleashes indiscriminate nuclease activity of CRISPR-Cas12a2.

Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection1. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of 'aromatic clamp' residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.

A single 2'-O-methylation of ribosomal RNA gates assembly of a functional ribosome.

RNA modifications are widespread in biology and abundant in ribosomal RNA. However, the importance of these modifications is not well understood. We show that methylation of a single nucleotide, in the catalytic center of the large subunit, gates ribosome assembly. Massively parallel mutational scanning of the essential nuclear GTPase Nog2 identified important interactions with rRNA, particularly with the 2'-O-methylated A-site base Gm2922. We found that methylation of G2922 is needed for assembly and efficient nuclear export of the large subunit. Critically, we identified single amino acid changes in Nog2 that completely bypass dependence on G2922 methylation and used cryoelectron microscopy to directly visualize how methylation flips Gm2922 into the active site channel of Nog2. This work demonstrates that a single RNA modification is a critical checkpoint in ribosome biogenesis, suggesting that such modifications can play an important role in regulation and assembly of macromolecular machines.

Integrative modeling reveals the molecular architecture of the intraflagellar transport A (IFT-A) complex.

Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.

Constructing next-generation CRISPR-Cas tools from structural blueprints.

Clustered regularly interspaced short palindromic repeats - CRISPR-associated protein (CRISPR-Cas) systems are a critical component of the bacterial adaptive immune response. Since the discovery that they can be reengineered as programmable RNA-guided nucleases, there has been significant interest in using these systems to perform diverse and precise genetic manipulations. Here, we outline recent advances in the mechanistic understanding of CRISPR-Cas9, how these findings have been leveraged in the rational redesign of Cas9 variants with altered activities, and how these novel tools can be exploited for biotechnology and therapeutics. We also discuss the potential of the ubiquitous, yet often-overlooked, multisubunit CRISPR effector complexes for large-scale genomic deletions. Furthermore, we highlight how future structural studies will bolster these technologies.

The protein organization of a red blood cell.

Red blood cells (RBCs) (erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we define a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validate protein complexes through electron microscopy and chemical crosslinking and, with these data, build 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-like compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research.

Structural basis for broad anti-phage immunity by DISARM.

In the evolutionary arms race against phage, bacteria have assembled a diverse arsenal of antiviral immune strategies. While the recently discovered DISARM (Defense Island System Associated with Restriction-Modification) systems can provide protection against a wide range of phage, the molecular mechanisms that underpin broad antiviral targeting but avoiding autoimmunity remain enigmatic. Here, we report cryo-EM structures of the core DISARM complex, DrmAB, both alone and in complex with an unmethylated phage DNA mimetic. These structures reveal that DrmAB core complex is autoinhibited by a trigger loop (TL) within DrmA and binding to DNA substrates containing a 5' overhang dislodges the TL, initiating a long-range structural rearrangement for DrmAB activation. Together with structure-guided in vivo studies, our work provides insights into the mechanism of phage DNA recognition and specific activation of this widespread antiviral defense system.

Structural rearrangements allow nucleic acid discrimination by type I-D Cascade.

CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.