correlative-microscopy-for-3d-structural-analysis-dynamic

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

University of Pittsburgh School of Medicine

Although most pesticides sprayed on terrestrial plants remain on their leaf surfaces, the relationship between leaf-associated microbial populations and pesticide degradation remains unclear. Here we examined changes in the bacterial community composition in the rape phyllosphere after treatment with dichlorvos, an organophosphorus pesticide. Results indicate that the bacterial community showed marked changes after treatment. We evaluated the rate of dichlorvos degradation by a natural microbial community on rape leaves and found that more dichlorvos was degraded on microbial-population-inhabited leaves than on surface-sterilized leaves. Six dichlorvos-degrading bacteria with 16S rRNA gene sequences that are most similar to those of members of the genera Pseudomonas, Xanthomonas, Sphingomonas, Acidovorax, Agrobacterium and Chryseobacterium were isolated from the natural community. We report for the first time that three of these epiphytic bacterial species, from the genera Sphingomonas, Acidovorax and Chryseobacterium, can degrade organophosphorus compounds. Collectively, these results provide direct evidence that bacteria on leaves can degrade organophosphate pesticides, and demonstrate that phyllosphere bacteria have great potential for the bioremediation of pesticides in situ, where the environment is hostile to nonepiphytic bacteria.

Functional assembly of bacterial communities with activity for the biodegradation of an organophosphorus pesticide in the rape phyllosphere.

Bien que la plupart des pesticides pulv&eacute;ris&eacute;s sur les plantes terrestres restent sur leur surface foliaire, la relation entre les populations microbiennes associ&eacute;es aux feuilles et de la d&eacute;gradation de pesticides reste peu claire. Ici, nous avons examin&eacute; les changements dans la composition des communaut&eacute;s bact&eacute;riennes dans la phyllosph&egrave;re de viol apr&egrave;s traitement avec le dichlorvos, un pesticide organophosphor&eacute;. Les r&eacute;sultats indiquent que la population bact&eacute;rienne a montr&eacute; des changements marqu&eacute;s apr&egrave;s traitement. Nous avons &eacute;valu&eacute; la vitesse de d&eacute;gradation du dichlorvos par une communaut&eacute; microbienne naturelle sur les feuilles de colza et trouv&eacute; que plus dichlorvos a &eacute;t&eacute; d&eacute;grad&eacute; sur les feuilles microbienne-population-habit&eacute;e que sur les feuilles st&eacute;rilis&eacute;es en surface. Six des bact&eacute;ries d&eacute;gradant le dichlorvos avec les s&eacute;quences de g&egrave;ne ARNr 16 s qui sont tr&egrave;s semblables &agrave; ceux des membres du genre Pseudomonas, Xanthomonas, Sphingomonas, Acidovorax, Agrobacterium et Chryseobacterium ont &eacute;t&eacute; isol&eacute;es de la communaut&eacute; naturelle. Nous pr&eacute;sentons pour la premi&egrave;re fois que trois de ces esp&egrave;ces bact&eacute;riennes &eacute;piphytes, des genres Sphingomonas, Acidovorax et Chryseobacterium, peuvent d&eacute;grader les compos&eacute;s organophosphor&eacute;s. Collectivement, ces r&eacute;sultats constituent une preuve directe que des bact&eacute;ries sur les feuilles peuvent d&eacute;grader les pesticides organophosphor&eacute;s et d&eacute;montrer que phyllosph&egrave;re bact&eacute;ries ont un grand potentiel pour la biorestauration des pesticides in situs, o&ugrave; l&#39;environnement est hostile aux bact&eacute;ries nonepiphytic.

Assembl&eacute;e fonctionnelle des communaut&eacute;s bact&eacute;riennes avec activit&eacute; pour la biod&eacute;gradation d&#39;un pesticide organophosphor&eacute; dans la phyllosph&egrave;re de viol.

Anche se la maggior parte dei pesticidi irrorati sulle piante terrestri rimangono sulle loro superfici di foglia, il rapporto tra popolazioni microbiche associate a foglia e degradazione dei pesticidi rimane poco chiaro. Qui abbiamo esaminato le modifiche nella composizione comunit&agrave; batteriche nella fillosfera stupro dopo trattamento con diclorvos, un pesticida organofosforiche. I risultati indicano che la comunit&agrave; batterica ha mostrato cambiamenti contrassegnati dopo il trattamento. Abbiamo valutato il tasso di degradazione diclorvos da una comunit&agrave; microbica naturale su foglie di stupro e ha trovato che pi&ugrave; diclorvos fu degradata su foglie abitate da popolazione microbica rispetto su foglie di superficie-sterilizzato. Sei batteri degradanti diclorvos con sequenze di geni rRNA 16S che sono pi&ugrave; simili a quelli dei membri dei generi Pseudomonas, Xanthomonas, Sphingomonas, Acidovorax, Agrobacterium e Chryseobacterium sono stati isolati dalla comunit&agrave; naturale. Riportiamo per la prima volta che tre di queste specie batteriche epifite, dei generi Sphingomonas, Acidovorax e Chryseobacterium, possono degradare composti organofosforici. Collettivamente, questi risultati forniscono la prova diretta che batteri sulle foglie possono degradare pesticidi organofosfati e dimostrare che fillosfera batteri hanno un grande potenziale per il biorisanamento di pesticidi in situ, dove l&#39;ambiente &egrave; ostile ai batteri nonepiphytic.

Assemblaggio funzionale delle comunit&agrave; batteriche con attivit&agrave; per la biodegradazione di un antiparassitari organofosforici nella fillosfera di stupro.

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.

Protease cleavage leads to formation of mature trimer interface in HIV-1 capsid.

Au cours de la maturation de particule r&eacute;trovirus, la polyprot&eacute;ine Gag assembl&eacute; est cliv&eacute; par la prot&eacute;ase virale dans la matrice (MA), capside (CA) et les prot&eacute;ines nucl&eacute;ocapside (NC). Pour former la capside virale mature, CA transpose, r&eacute;sultant dans un r&eacute;seau compos&eacute; d&#39;unit&eacute;s de CA hexam&eacute;rique et pentam&eacute;rique. R&eacute;centes &eacute;tudes structurales de l&#39;assembl&eacute; du VIH-1 CA a r&eacute;v&eacute;l&eacute; plusieurs interfaces inter-subunit dans le r&eacute;seau de la capside, y compris une triple interface interhexamer qui est essentielle pour la stabilit&eacute; de la capside appropri&eacute;e. Une architecture g&eacute;n&eacute;rale des particules immatures ont &eacute;t&eacute; donn&eacute;es par les &eacute;tudes tomographie cryo-&eacute;lectronique, les d&eacute;tails de la particule immature et la voie de maturation demeurent inconnues. Ici, nous avons utilis&eacute; la cryo microscopie &eacute;lectronique (cryoEM) pour d&eacute;terminer la structure des assemblages tubulaires de la prot&eacute;ine de HIV-1 CA-SP1-NC. Par rapport &agrave; la structure de CA Assembl&eacute;e mature, nous avons observ&eacute; une nette diff&eacute;rence de conformation dans la position du CA-CTD par rapport &agrave; l&#39;ATN dans l&#39;Assembl&eacute;e de CA-SP1-NC, impliquant la charni&egrave;re flexible reliant les deux domaines. Cette diff&eacute;rence a &eacute;t&eacute; v&eacute;rifi&eacute; par l&#39;interm&eacute;diaire de r&eacute;ticulation disulfure machin&eacute;, r&eacute;v&eacute;lant qu&#39;inter-hexam&egrave;re contacte, en particulier ceux de la pseudo axe triple, sont alt&eacute;r&eacute;es dans les assemblys de CA-SP1-NC par rapport aux assemblys CA. Les r&eacute;sultats des analyses de la r&eacute;ticulation des particules de VIH-1 matures et immatures, contenant les m&ecirc;mes substitutions Cys dans la prot&eacute;ine Gag sont conformes &agrave; ces recommandations. En outre nous montrent que le clivage CA-SP1-NC pr&eacute;mont&eacute;e de prot&eacute;ase du VIH-1 in vitro entra&icirc;ne la lib&eacute;ration de SP1 et NC sans d&eacute;montage du treillis. Collectivement, nos r&eacute;sultats indiquent que le clivage prot&eacute;olytique de Gag conduit &agrave; une r&eacute;organisation structurelle du polypeptide et cr&eacute;e l&#39;interface interhexamer triple, important pour la formation de particules infectieuses de VIH-1.

Clivage de prot&eacute;ase conduit &agrave; la formation d&#39;interface trim&egrave;re matures dans la capside du VIH-1.

Durante la maturazione delle particelle di retrovirus, il polyprotein Gag assemblato viene scisso dalla proteasi virale in matrix (MA), proteine del capside (CA) e proteine del nucleocapside (NC). Per formare il capside virale maturo, CA ridispone, risultante in un reticolo composto da unit&agrave; di CA esamerica e pentamerica. Recenti studi strutturali di CA di HIV-1 assemblato ha rivelato diverse interfacce inter-subunit nel reticolo del capside, compreso un triplice interfaccia interhexamer che &egrave; fondamentale per la stabilit&agrave; del capside corretta. Sebbene l&#39;architettura generale di particelle immaturi &egrave; stato fornito da studi tomografici cryo-elettrone, rimangono sconosciuti i dettagli strutturali della particella immatura e il percorso di maturazione. Qui, abbiamo utilizzato crio-microscopia elettronica (cryoEM) per determinare la struttura delle assemblee tubolare della proteina HIV-1 CA-SP1-NC. Relativo alla struttura CA matura assemblata, abbiamo osservato una marcata differenza di conformazione in posizione di CA-CTD rispetto il NTD in Assemblea CA-SP1-NC, che coinvolgono la cerniera flessibile che collega i due domini. Questa differenza &egrave; stata verificata mediante reticolazione disolfuro ingegnerizzati, rivelando che inter-hexamer contatti, in particolare quelli a pseudo asse triplice, sono alterati nelle assemblee CA-SP1-NC rispetto agli assembly CA. Risultati di analisi di reticolazione di mature e immature particelle di HIV-1 che contiene le stesse sostituzioni Cys in proteina Gag sono coerenti con questi risultati. Abbiamo inoltre mostrano che la scissione del CA-SP1-NC premontati da proteasi HIV-1 in vitro conduce al rilascio di SP1 e NC senza smontaggio del reticolo. Collettivamente, i nostri risultati indicano che il clivaggio proteolitico di Gag conduce ad una riorganizzazione strutturale del polipeptide e crea la triplice interfaccia interhexamer, importante per la formazione di particelle infettive HIV-1.

Scissione di proteasi conduce alla formazione dell&#39;interfaccia trimero maturo nel capside di HIV-1.

Tripartite motif protein isoform 5 alpha (TRIM5Î±) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5Î± recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5Î± induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5Î± PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5Î± PRY/SPRY, but not the human TRIM5Î± PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5Î± that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5Î±-mediated retroviral restriction.

Structural insight into HIV-1 capsid recognition by rhesus TRIM5Î±.

Motif tripartite prot&eacute;ine isoforme 5 alpha (TRIM5&alpha;) est une prot&eacute;ine antivirale puissante qui limite l&#39;infection par le VIH-1 et autres r&eacute;trovirus. TRIM5&alpha; reconna&icirc;t le treillis de la capside du r&eacute;trovirus par le biais de son domaine B30.2 (PRY/SPRY) d&#39;une mani&egrave;re sp&eacute;cifique &agrave; l&#39;esp&egrave;ce. Lors de la liaison, TRIM5&alpha; induit d&eacute;montage pr&eacute;matur&eacute; de la capside virale et active le syst&egrave;me immunitaire inn&eacute; en aval. Nous avons d&eacute;termin&eacute; la structure cristalline du domaine TRIM5&alpha; PRY/SPRY Rh&eacute;sus qui r&eacute;v&egrave;le des caract&eacute;ristiques essentielles pour la liaison de la capside. Combin&eacute; cryo-microscopie &eacute;lectronique et les donn&eacute;es biochimiques montrent que le monom&egrave;re rh&eacute;sus TRIM5&alpha; PRY/SPRY, mais pas le levier de TRIM5&alpha; humaine/SPRY, peut lier aux assembl&eacute;es de prot&eacute;ine de capside du VIH-1, sans provoquer de perturbations de la capside. Ceci sugg&egrave;re que le domaine PRY/SPRY seul constitue un &eacute;l&eacute;ment important de d&eacute;tection de mod&egrave;le de TRIM5&alpha; qui est capable d&#39;interagir avec des capsides virales des courbures diff&eacute;rentes. Nos r&eacute;sultats donnent un aper&ccedil;u mol&eacute;culaire sur les m&eacute;canismes de restriction retroviral induite par le TRIM5&alpha;.

Aper&ccedil;u structural reconnaissance de capside du VIH-1 par rh&eacute;sus TRIM5&alpha;.

Motivo tripartito proteina isoforma 5 alfa (TRIM5&alpha;) &egrave; una proteina antivirale potente che limita l&#39;infezione da HIV-1 e altri retrovirus. TRIM5&alpha; riconosce il reticolo del capside retrovirus attraverso il dominio B30.2 (PRY/SPRY) in modo specie-specifiche. Dell&#39;associazione, TRIM5&alpha; induce prematuro smontaggio delle proteine del capside virale e attiva la risposta immunitaria innata a valle. Abbiamo determinato la struttura cristallina del dominio TRIM5&alpha; PRY/SPRY rhesus che rivela caratteristiche essenziali per l&#39;associazione di proteine del capside. Combinato crio-microscopia elettronica e dati biochimici mostrano che il rhesus monomerico TRIM5&alpha; PRY/SPRY, ma non l&#39;umana TRIM5&alpha; PRY/SPRY, pu&ograve; associare agli assembly di proteina del capside HIV-1 senza causare interruzioni delle proteine del capside. Questo suggerisce che il dominio PRY/SPRY da sola costituisce una componente importante modello di rilevamento del TRIM5&alpha; che &egrave; in grado di interagire con capsidi virali di diverse curvature. I nostri risultati forniscono approfondimenti molecolare dei meccanismi di restrizione retrovirale TRIM5&alpha;-mediata.

Riconoscimento del capside HIV-1 da rhesus TRIM5&alpha; visione strutturale.

Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a 'fullerene cone' model, in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven Î±-helices and a Î²-hairpin, a carboxy-terminal domain (CTD) comprising four Î±-helices, and a flexible linker with a 310-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8â€‰Ã… resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention.

Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics.

Prot&eacute;ines de la capside retroviral sont conserv&eacute;s structurellement mais assemblent dans diff&eacute;rentes morphologies. La mature 1-virus de l&#39;immunod&eacute;ficience humaine (VIH-1) capside est mieux d&eacute;crit par un mod&egrave;le de &laquo; c&ocirc;ne de fuller&egrave;ne &raquo;, dans lequel hexam&egrave;res de la capside prot&eacute;ique sont li&eacute;es pour former un r&eacute;seau hexagonal de surface qui est ferm&eacute; en int&eacute;grant 12-la capside pentam&egrave;res. Le VIH-1 de la capside contient un domaine amino-terminale (NTD) comprenant sept &alpha;-h&eacute;lices et un &beta;-&eacute;pingle &agrave; cheveux, un domaine carboxy-terminal (CTD) comprenant quatre h&eacute;lices-&alpha; et un &eacute;diteur de liens souple avec une h&eacute;lice 310 reliant les deux domaines structuraux. On a d&eacute;termin&eacute; les structures des unit&eacute;s d&#39;assemblage des capside diffraction des rayons x ; Toutefois, les informations structurelles concernant la capside Assembl&eacute;e et les contacts entre les unit&eacute;s d&#39;assemblage sont incompl&egrave;tes. Nous pr&eacute;sentons la structure de microscopie de cryo-&eacute;lectron d&#39;une Assembl&eacute;e la capside du VIH-1 tubulaire 8 &Aring; de r&eacute;solution et de la structure tridimensionnelle d&#39;un noyau natif de VIH-1 par la tomographie cryo-&eacute;lectronique. La structure de l&#39;assemblage tubulaire montre, &agrave; l&#39;interface triple, un faisceau de trois h&eacute;lices avec des interactions hydrophobes critiques. &Eacute;tudes de mutagen&egrave;se confirment que les r&eacute;sidus hydrophobes dans le centre du faisceau de trois h&eacute;lices sont cruciales pour la stabilit&eacute; et l&#39;assemblage de la capside et pour l&#39;infectivit&eacute; virale. Les structures de la cryo-microscopie &eacute;lectronique permettent la mod&eacute;lisation par simulation de dynamique mol&eacute;culaire &agrave; grande &eacute;chelle, aboutissant &agrave; des mod&egrave;les de tout-atome pour les &eacute;l&eacute;ments hexam&egrave;re-de-hexam&egrave;re et pentam&egrave;re-de-hexam&egrave;re ainsi que pour l&#39;ensemble de la capside. Incorporation de pentam&egrave;res se traduit par des contacts plus &eacute;troits de trim&egrave;re et induit la courbure aigu&euml; de surface. Le mod&egrave;le de capside du VIH-1 atomique complet fournit une plate-forme pour poursuivre ses &eacute;tudes de fonction capside et pour une intervention pharmacologique cibl&eacute;e.

Jiying Ning

Department of Structural Biology

University of Pittsburgh School of Medicine

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Jiying Ning

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Structural Biology

University of Pittsburgh School of Medicine

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Department of Structural Biology