RESUMO
Rosalind Franklin provided the key data for deriving the double helix structure of DNA. The English chemist also pioneered structural studies of colloids, viruses, and RNA. To celebrate the 100th anniversary of Franklin's birth, I summarize her work, which shaped the emerging discipline of molecular biology.
Assuntos
Biologia Molecular/história , Biografias como Assunto , DNA/química , História do Século XX , RNA/química , Vírus/química , Difração de Raios XRESUMO
X-ray free-electron lasers provide femtosecond-duration pulses of hard X-rays with a peak brightness approximately one billion times greater than is available at synchrotron radiation facilities. One motivation for the development of such X-ray sources was the proposal to obtain structures of macromolecules, macromolecular complexes, and virus particles, without the need for crystallization, through diffraction measurements of single noncrystalline objects. Initial explorations of this idea and of outrunning radiation damage with femtosecond pulses led to the development of serial crystallography and the ability to obtain high-resolution structures of small crystals without the need for cryogenic cooling. This technique allows the understanding of conformational dynamics and enzymatics and the resolution of intermediate states in reactions over timescales of 100 fs to minutes. The promise of more photons per atom recorded in a diffraction pattern than electrons per atom contributing to an electron micrograph may enable diffraction measurements of single molecules, although challenges remain.
Assuntos
Elétrons , Substâncias Macromoleculares/ultraestrutura , Fótons , Vírion/ultraestrutura , Difração de Raios X/métodos , Cristalização/instrumentação , Cristalização/métodos , Cristalografia por Raios X/história , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , História do Século XX , História do Século XXI , Lasers/história , Síncrotrons/instrumentação , Difração de Raios X/história , Difração de Raios X/instrumentação , Raios XRESUMO
Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.
Assuntos
Bacteriorodopsinas/ultraestrutura , Lasers , Prótons , Retinaldeído/química , Difração de Raios X/métodos , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Cristalografia/instrumentação , Cristalografia/métodos , Halobacterium salinarum/química , Halobacterium salinarum/metabolismo , Transporte de Íons , Modelos Moleculares , Conformação Proteica , Retinaldeído/metabolismo , Síncrotrons/instrumentação , Raios XRESUMO
Dozens of proteins are known to convert to the aggregated amyloid state. These include fibrils associated with systemic and neurodegenerative diseases and cancer, functional amyloid fibrils in microorganisms and animals, and many denatured proteins. Amyloid fibrils can be much more stable than other protein assemblies. In contrast to globular proteins, a single protein sequence can aggregate into several distinctly different amyloid structures, termed polymorphs, and a given polymorph can reproduce itself by seeding. Amyloid polymorphs may be the molecular basis of prion strains. Whereas the Protein Data Bank contains some 100,000 globular protein and 3,000 membrane protein structures, only a few dozen amyloid protein structures have been determined, and most of these are short segments of full amyloid-forming proteins. Regardless, these amyloid structures illuminate the architecture of the amyloid state, including its stability and its capacity for formation of polymorphs.
Assuntos
Proteínas Amiloidogênicas/química , Proteínas Priônicas/química , Agregação Patológica de Proteínas/metabolismo , Motivos de Aminoácidos , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Microscopia Crioeletrônica , Expressão Gênica , Humanos , Ressonância Magnética Nuclear Biomolecular , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Desnaturação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Difração de Raios XRESUMO
The ring-shaped cohesin complex brings together distant DNA domains to maintain, express, and segregate the genome. Establishing specific chromosomal linkages depends on cohesin recruitment to defined loci. One such locus is the budding yeast centromere, which is a paradigm for targeted cohesin loading. The kinetochore, a multiprotein complex that connects centromeres to microtubules, drives the recruitment of high levels of cohesin to link sister chromatids together. We have exploited this system to determine the mechanism of specific cohesin recruitment. We show that phosphorylation of the Ctf19 kinetochore protein by a conserved kinase, DDK, provides a binding site for the Scc2/4 cohesin loading complex, thereby directing cohesin loading to centromeres. A similar mechanism targets cohesin to chromosomes in vertebrates. These findings represent a complete molecular description of targeted cohesin loading, a phenomenon with wide-ranging importance in chromosome segregation and, in multicellular organisms, transcription regulation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Saccharomyces cerevisiae/metabolismo , Centrômero/metabolismo , Proteínas do Citoesqueleto/metabolismo , Complexos Multiproteicos/metabolismo , Fosforilação , Filogenia , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Difração de Raios X , CoesinasRESUMO
Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.
Assuntos
Fertilização , Invertebrados/fisiologia , Vertebrados/fisiologia , Sequência de Aminoácidos , Animais , Evolução Biológica , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Humanos , Invertebrados/química , Invertebrados/genética , Masculino , Modelos Moleculares , Mucoproteínas/química , Mucoproteínas/metabolismo , Óvulo/química , Óvulo/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Espermatozoides/química , Espermatozoides/metabolismo , Vertebrados/genética , Difração de Raios X , Glicoproteínas da Zona Pelúcida/química , Glicoproteínas da Zona Pelúcida/metabolismoRESUMO
Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Leptotrichia/imunologia , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Proteínas Associadas a CRISPR/ultraestrutura , Leptotrichia/química , Leptotrichia/metabolismo , Leptotrichia/virologia , Modelos Moleculares , Processamento Pós-Transcricional do RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/ultraestrutura , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/ultraestrutura , RNA Viral/química , Difração de Raios XRESUMO
Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Meiose/genéticaRESUMO
The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications.
Assuntos
Anticorpos Neutralizantes/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/química , Anticorpos Neutralizantes/ultraestrutura , Epitopos , Proteína gp120 do Envelope de HIV/ultraestrutura , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação Proteica , Difração de Raios XRESUMO
The universally conserved GTPase elongation factor G (EF-G) catalyzes the translocation of tRNA and mRNA on the ribosome after peptide bond formation. Despite numerous studies suggesting that EF-G undergoes extensive conformational rearrangements during translocation, high-resolution structures exist for essentially only one conformation of EF-G in complex with the ribosome. Here, we report four atomic-resolution crystal structures of EF-G bound to the ribosome programmed in the pre- and posttranslocational states and to the ribosome trapped by the antibiotic dityromycin. We observe a previously unseen conformation of EF-G in the pretranslocation complex, which is independently captured by dityromycin on the ribosome. Our structures provide insights into the conformational space that EF-G samples on the ribosome and reveal that tRNA translocation on the ribosome is facilitated by a structural transition of EF-G from a compact to an elongated conformation, which can be prevented by the antibiotic dityromycin.
Assuntos
Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Thermus thermophilus/metabolismo , Depsipeptídeos/farmacologia , Escherichia coli/química , Escherichia coli/metabolismo , Modelos Moleculares , RNA de Transferência/química , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Thermus thermophilus/química , Difração de Raios XRESUMO
Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery.
Assuntos
Complexo Mediador/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Porinas/química , Porinas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Porinas/genética , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Polimerase II/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Transcriptoma , Difração de Raios XRESUMO
Intrinsically disordered proteins (IDPs) and IDP regions fail to form a stable structure, yet they exhibit biological activities. Their mobile flexibility and structural instability are encoded by their amino acid sequences. They recognize proteins, nucleic acids, and other types of partners; they accelerate interactions and chemical reactions between bound partners; and they help accommodate posttranslational modifications, alternative splicing, protein fusions, and insertions or deletions. Overall, IDP-associated biological activities complement those of structured proteins. Recently, there has been an explosion of studies on IDP regions and their functions, yet the discovery and investigation of these proteins have a long, mostly ignored history. Along with recent discoveries, we present several early examples and the mechanisms by which IDPs contribute to function, which we hope will encourage comprehensive discussion of IDPs and IDP regions in biochemistry textbooks. Finally, we propose future directions for IDP research.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Animais , Calcineurina/química , Caseínas/química , Biologia Computacional , Espectroscopia de Ressonância de Spin Eletrônica , Fibrina/química , Fibrinogênio/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fosvitina/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Solubilidade , Tripsina/química , Tripsinogênio/química , Difração de Raios XRESUMO
The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.
Assuntos
Actinina/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/química , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios XRESUMO
Protein folding in the cell relies on the orchestrated action of conserved families of molecular chaperones, the Hsp70 and Hsp90 systems. Hsp70 acts early and Hsp90 late in the folding path, yet the molecular basis of this timing is enigmatic, mainly because the substrate specificity of Hsp90 is poorly understood. Here, we obtained a structural model of Hsp90 in complex with its natural disease-associated substrate, the intrinsically disordered Tau protein. Hsp90 binds to a broad region in Tau that includes the aggregation-prone repeats. Complementarily, a 106-Å-long substrate-binding interface in Hsp90 enables many low-affinity contacts. This allows recognition of scattered hydrophobic residues in late folding intermediates that remain after early burial of the Hsp70 sites. Our model resolves the paradox of how Hsp90 specifically selects for late folding intermediates but also for some intrinsically disordered proteins-through the eyes of Hsp90 they look the same.
Assuntos
Proteínas tau/química , Doença de Alzheimer/tratamento farmacológico , Sequência de Aminoácidos , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X , Proteínas tau/metabolismoRESUMO
MDA5, a viral double-stranded RNA (dsRNA) receptor, shares sequence similarity and signaling pathways with RIG-I yet plays essential functions in antiviral immunity through distinct specificity for viral RNA. Revealing the molecular basis for the functional divergence, we report here the crystal structure of MDA5 bound to dsRNA, which shows how, using the same domain architecture, MDA5 recognizes the internal duplex structure, whereas RIG-I recognizes the terminus of dsRNA. We further show that MDA5 uses direct protein-protein contacts to stack along dsRNA in a head-to-tail arrangement, and that the signaling domain (tandem CARD), which decorates the outside of the core MDA5 filament, also has an intrinsic propensity to oligomerize into an elongated structure that activates the signaling adaptor, MAVS. These data support a model in which MDA5 uses long dsRNA as a signaling platform to cooperatively assemble the core filament, which in turn promotes stochastic assembly of the tandem CARD oligomers for signaling.
Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , RNA de Cadeia Dupla/metabolismo , Sequência de Aminoácidos , Humanos , Helicase IFIH1 Induzida por Interferon , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/química , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Alinhamento de Sequência , Difração de Raios XRESUMO
p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin reuptake inhibitors (SSRIs). We show that SMARCA3, a chromatin-remodeling factor, is a target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer. Formation of this complex increases the DNA-binding affinity of SMARCA3 and its localization to the nuclear matrix fraction. In the dentate gyrus, both p11 and SMARCA3 are highly enriched in hilar mossy cells and basket cells. The SSRI fluoxetine induces expression of p11 in both cell types and increases the amount of the ternary complex of p11/annexin A2/SMARCA3. SSRI-induced neurogenesis and behavioral responses are abolished by constitutive knockout of SMARCA3. Our studies indicate a central role for a chromatin-remodeling factor in the SSRI/p11 signaling pathway and suggest an approach to the development of improved antidepressant therapies. PAPERCLIP:
Assuntos
Anexina A2/metabolismo , Proteínas de Ligação a DNA/metabolismo , Giro Denteado/metabolismo , Proteínas S100/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Feminino , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Fibras Musgosas Hipocampais/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Fatores de Transcrição/química , Difração de Raios XRESUMO
Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.
Assuntos
Transporte Ativo do Núcleo Celular , HIV-1/química , RNA Mensageiro/química , RNA Viral/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Células HEK293 , HIV-1/genética , Humanos , Dados de Sequência Molecular , Poro Nuclear/metabolismo , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Inorganic-organic hybrid materials represent a large share of newly reported structures, owing to their simple synthetic routes and customizable properties1. This proliferation has led to a characterization bottleneck: many hybrid materials are obligate microcrystals with low symmetry and severe radiation sensitivity, interfering with the standard techniques of single-crystal X-ray diffraction2,3 and electron microdiffraction4-11. Here we demonstrate small-molecule serial femtosecond X-ray crystallography (smSFX) for the determination of material crystal structures from microcrystals. We subjected microcrystalline suspensions to X-ray free-electron laser radiation12,13 and obtained thousands of randomly oriented diffraction patterns. We determined unit cells by aggregating spot-finding results into high-resolution powder diffractograms. After indexing the sparse serial patterns by a graph theory approach14, the resulting datasets can be solved and refined using standard tools for single-crystal diffraction data15-17. We describe the ab initio structure solutions of mithrene (AgSePh)18-20, thiorene (AgSPh) and tethrene (AgTePh), of which the latter two were previously unknown structures. In thiorene, we identify a geometric change in the silver-silver bonding network that is linked to its divergent optoelectronic properties20. We demonstrate that smSFX can be applied as a general technique for structure determination of beam-sensitive microcrystalline materials at near-ambient temperature and pressure.
Assuntos
Elétrons , Prata , Cristalografia por Raios X , Lasers , Difração de Raios XRESUMO
Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends on the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold.
Assuntos
Azoarcus/enzimologia , Azoarcus/genética , Dobramento de RNA , RNA Catalítico/química , RNA Catalítico/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , RNA Catalítico/genética , Espalhamento a Baixo Ângulo , Termodinâmica , Difração de Raios XRESUMO
Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR.