Ultrashort pulses in structural analysis of diamond layers with angstrom resolution.

X-ray crystallography is commonly used to determine crystal structures, whether continuous or ultrashort x rays are used. In this paper, it is shown that using only ultrashort pulses, it is possible to determine interplanar spacing in diamond layers, the distance between which can be only a few angstroms. The results obtained can be extended, with further development of the presented theory, to determine 3D objects in the crystal structure, the dimensions of which can be only a few angstroms.

Ecological Determinants of Altruism in Prokaryote Antivirus Defense.

Prokaryote evolution is driven in large part by the incessant arms race with viruses. Genomic investments in antivirus defense can be coarsely classified into two categories, immune systems that abrogate virus reproduction resulting in clearance, and altruistic programmed cell death (PCD) systems. Prokaryotic defense systems are enormously diverse, as revealed by an avalanche of recent discoveries, but the basic ecological determinants of defense strategy remain poorly understood.

CRISPR in mobile genetic elements: counter-defense, inter-element competition and RNA-guided transposition.

CRISPR are adaptive immunity systems that protect bacteria and archaea from viruses and other mobile genetic elements (MGE) via an RNA-guided interference mechanism. However, in the course of the host-parasite co-evolution, CRISPR systems have been recruited by MGE themselves for counter-defense or other functions. Some bacteriophages encode fully functional CRISPR systems that target host defense systems, and many others recruited individual components of CRISPR systems, such as single repeat units that inhibit host CRISPR systems and CRISPR mini-arrays that target related viruses contributing to inter-virus competition.

COG database update 2024.

The Clusters of Orthologous Genes (COG) database, originally created in 1997, has been updated to reflect the constantly growing collection of completely sequenced prokaryotic genomes. This update increased the genome coverage from 1309 to 2296 species, including 2103 bacteria and 193 archaea, in most cases, with a single representative genome per genus. This set covers all genera of bacteria and archaea that included organisms with 'complete genomes' as per NCBI databases in November 2023.

Biochemical plasticity of the Escherichia coli CRISPR Cascade revealed by in vitro reconstitution of Cascade activities from purified Cas proteins.

The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E.

Beyond bacterial paradigms: uncovering the functional significance and first biogenesis machinery of archaeal lipoproteins.

Lipoproteins are major constituents of prokaryotic cell surfaces. In bacteria, lipoprotein attachment to membrane lipids is catalyzed by prolipoprotein diacylglyceryl transferase (Lgt). However, no Lgt homologs have been identified in archaea, suggesting the unique archaeal membrane lipids require distinct enzymes for lipoprotein lipidation.

Long range segmentation of prokaryotic genomes by gene age and functionality.

Bacterial and archaeal genomes encompass numerous operons that typically consist of two to five genes. On larger scales, however, gene order is poorly conserved through the evolution of prokaryotes. Nevertheless, non-random localization of different classes of genes on prokaryotic chromosomes could reflect important functional and evolutionary constraints.

Long range segmentation of prokaryotic genomes by gene age and functionality.

Bacterial and archaeal genomes encompass numerous operons that typically consist of two to five genes. On larger scales, however, gene order is poorly conserved through the evolution of prokaryotes. Nevertheless, non-random localization of different classes of genes on prokaryotic chromosomes could reflect important functional and evolutionary constraints.

Diversity, Origin and Evolution of the ESCRT Systems.

Endosomal Sorting Complexes Required for Transport (ESCRT) play key roles in protein sorting between membrane-bounded compartments of eukaryotic cells. Homologs of many ESCRT components are identifiable in various groups of archaea, especially in Asgardarchaeota, the archaeal phylum that is currently considered to include the closest relatives of eukaryotes, but not in bacteria. We performed a comprehensive search for ESCRT protein homologs in archaea and reconstructed ESCRT evolution using the phylogenetic tree of Vps4 ATPase (ESCRT IV) as a scaffold, using sensitive protein sequence analysis and comparison of structural models to identify previously unknown ESCRT proteins.

Quantum beam splitter based on free charged particles.

It is well known that the beam splitter is an integral part of many classical and quantum devices. The use of beam splitters in quantum technologies is currently particularly relevant. The emergence of new types of beam splitters provides new statistical characteristics of the separated photon beam and their control and new possibilities for use in various devices.

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote iled-il clease andems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes.

Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.

In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses.

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector.

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread.

Widespread photosynthesis reaction centre barrel proteins are necessary for haloarchaeal cell division.

Cell division is fundamental to all cellular life. Most archaea depend on either the prokaryotic tubulin homologue FtsZ or the endosomal sorting complex required for transport for division but neither system has been robustly characterized. Here, we show that three of the four photosynthesis reaction centre barrel domain proteins of Haloferax volcanii (renamed cell division proteins B1/2/3 (CdpB1/2/3)) play important roles in cell division.

Diversity, origin, and evolution of the ESCRT systems.

Endosomal sorting complexes required for transport (ESCRT) play key roles in protein sorting between membrane-bounded compartments of eukaryotic cells. Homologs of many ESCRT components are identifiable in various groups of archaea, especially in Asgardarchaeota, the archaeal phylum that is currently considered to include the closest relatives of eukaryotes, but not in bacteria. We performed a comprehensive search for ESCRT protein homologs in archaea and reconstructed ESCRT evolution using the phylogenetic tree of Vps4 ATPase (ESCRT IV) as a scaffold and using sensitive protein sequence analysis and comparison of structural models to identify previously unknown ESCRT proteins.

Crystal structure of GTP-dependent dephospho-coenzyme A kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The biosynthesis pathways of coenzyme A (CoA) in most archaea involve several unique enzymes including dephospho-CoA kinase (DPCK) that converts dephospho-CoA to CoA in the final step of CoA biosynthesis in all domains of life. The archaeal DPCK is unrelated to the analogous bacterial and eukaryotic enzymes and shows no significant sequence similarity to any proteins with known structures. Unusually, the archaeal DPCK utilizes GTP as the phosphate donor although the analogous bacterial and eukaryotic enzymes are ATP-dependent kinases.

Computational analysis of genes with lethal knockout phenotype and prediction of essential genes in archaea.

The identification of microbial genes essential for survival as those with lethal knockout phenotype (LKP) is a common strategy for functional interrogation of genomes. However, interpretation of the LKP is complicated because a substantial fraction of the genes with this phenotype remains poorly functionally characterized. Furthermore, many genes can exhibit LKP not because their products perform essential cellular functions but because their knockout activates the toxicity of other genes (conditionally essential genes).

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail.

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes.

Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering.

Microbial systems underpin many biotechnologies, including CRISPR, but the exponential growth of sequence databases makes it difficult to find previously unidentified systems. In this work, we develop the fast locality-sensitive hashing-based clustering (FLSHclust) algorithm, which performs deep clustering on massive datasets in linearithmic time. We incorporated FLSHclust into a CRISPR discovery pipeline and identified 188 previously unreported CRISPR-linked gene modules, revealing many additional biochemical functions coupled to adaptive immunity.

Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12.

The TnpB proteins are transposon-associated RNA-guided nucleases that are among the most abundant proteins encoded in bacterial and archaeal genomes, but whose functions in the transposon life cycle remain unknown. TnpB appears to be the evolutionary ancestor of Cas12, the effector nuclease of type V CRISPR-Cas systems. We performed a comprehensive census of TnpBs in archaeal and bacterial genomes and constructed a phylogenetic tree on which we mapped various features of these proteins.

CoCoNuTs: A diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote CoCoNuTs (coiled-coil nuclease tandems) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with 3 distinct types and multiple subtypes.

Widespread CRISPR-derived RNA regulatory elements in CRISPR-Cas systems.

CRISPR-cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR-cas loci produce distinct cr-like RNAs implicated in regulatory or other functions.

Search for Origins of Anti-CRISPR Proteins by Structure Comparison.

Many bacterial and archaeal viruses encode anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas systems via various mechanisms. The majority of the Acrs are small, non-enzymatic proteins that abrogate CRISPR activity by binding to Cas effector proteins. The Acrs evolve fast, due to the arms race with the respective CRISPR-Cas systems, which hampers the elucidation of their evolutionary origins by sequence comparison.