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1.
Commun Chem ; 7(1): 4, 2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-38172567

RESUMO

In situ structures of Platinum (Pt) nanoparticles (NPs) can be determined with graphene liquid cell transmission electron microscopy. Atomic-scale three-dimensional structural information about their physiochemical properties in solution is critical for understanding their chemical function. We here analyze eight atomic-resolution maps of small (<3 nm) colloidal Pt NPs. Their structures are composed of an ordered crystalline core surrounded by surface atoms with comparatively high mobility. 3D reconstructions calculated from cumulative doses of 8500 and 17,000 electrons/pixel, respectively, are characterized in terms of loss of atomic densities and atomic displacements. Less than 5% of the total number of atoms are lost due to dissolution or knock-on damage in five of the structures analyzed, whereas 10-16% are lost in the remaining three. Less than 5% of the atomic positions are displaced due to the increased electron irradiation in all structures. The surface dynamics will play a critical role in the diverse catalytic function of Pt NPs and must be considered in efforts to model Pt NP function computationally.

3.
Sci Rep ; 13(1): 1814, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36725868

RESUMO

Determining the 3D atomic structures of multi-element nanoparticles in their native liquid environment is crucial to understanding their physicochemical properties. Graphene liquid cell (GLC) TEM offers a platform to directly investigate nanoparticles in their solution phase. Moreover, exploiting high-resolution TEM images of single rotating nanoparticles in GLCs, 3D atomic structures of nanoparticles are reconstructed by a method called "Brownian one-particle reconstruction". We here introduce a 3D atomic structure determination method for multi-element nanoparticle systems. The method, which is based on low-pass filtration and initial 3D model generation customized for different types of multi-element systems, enables reconstruction of high-resolution 3D Coulomb density maps for ordered and disordered multi-element systems and classification of the heteroatom type. Using high-resolution image datasets obtained from TEM simulations of PbSe, CdSe, and FePt nanoparticles that are structurally relaxed with first-principles calculations in the graphene liquid cell, we show that the types and positions of the constituent atoms are precisely determined with root mean square displacement values less than 24 pm. Our study suggests that it is possible to investigate the 3D atomic structures of synthesized multi-element nanoparticles in liquid phase.

4.
Nano Lett ; 21(2): 1175-1183, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33416334

RESUMO

Active sites and catalytic activity of heterogeneous catalysts is determined by their surface atomic structures. However, probing the surface structure at an atomic resolution is difficult, especially for solution ensembles of catalytic nanocrystals, which consist of heterogeneous particles with irregular shapes and surfaces. Here, we constructed 3D maps of the coordination number (CN) and generalized CN (CN_) for individual surface atoms of sub-3 nm Pt nanocrystals. Our results reveal that the synthesized Pt nanocrystals are enclosed by islands of atoms with nonuniform shapes that lead to complex surface structures, including a high ratio of low-coordination surface atoms, reduced domain size of low-index facets, and various types of exposed high-index facets. 3D maps of CN_ are directly correlated to catalytic activities assigned to individual surface atoms with distinct local coordination structures, which explains the origin of high catalytic performance of small Pt nanocrystals in important reactions such as oxygen reduction reactions and CO electro-oxidation.

5.
Sci Adv ; 7(5)2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33514557

RESUMO

Analysis of the three-dimensional (3D) structures of nanocrystals with solution-phase transmission electron microscopy is beginning to reveal their unique physiochemical properties. We developed a "one-particle Brownian 3D reconstruction method" based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Projection images of differently rotated nanocrystals are acquired using a direct electron detector with high temporal (<2.5 ms) resolution and analyzed to obtain an ensemble of 3D reconstructions. Here, we introduce computational methods required for successful atomic-resolution 3D reconstruction: (i) tracking of the individual particles throughout the time series, (ii) subtraction of the interfering background of the graphene liquid cell, (iii) identification and rejection of low-quality images, and (iv) tailored strategies for 2D/3D alignment and averaging that differ from those used in biological cryo-electron microscopy. Our developments are made available through the open-source software package SINGLE.

6.
J Struct Biol X ; 4: 100040, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33294840

RESUMO

We here introduce the third major release of the SIMPLE (Single-particle IMage Processing Linux Engine) open-source software package for analysis of cryogenic transmission electron microscopy (cryo-EM) movies of single-particles (Single-Particle Analysis, SPA). Development of SIMPLE 3.0 has been focused on real-time data processing using minimal CPU computing resources to allow easy and cost-efficient scaling of processing as data rates escalate. Our stream SPA tool implements the steps of anisotropic motion correction and CTF estimation, rapid template-based particle identification and 2D clustering with automatic class rejection. SIMPLE 3.0 additionally features an easy-to-use web-based graphical user interface (GUI) that can be run on any device (workstation, laptop, tablet or phone) and supports a remote multi-user environment over the network. The new project-based execution model automatically records the executed workflow and represents it as a flow diagram in the GUI. This facilitates meta-data handling and greatly simplifies usage. Using SIMPLE 3.0, it is possible to automatically obtain a clean SP data set amenable to high-resolution 3D reconstruction directly upon completion of the data acquisition, without the need for extensive image processing post collection. Only minimal standard CPU computing resources are required to keep up with a rate of ∼300 Gatan K3 direct electron detector movies per hour. SIMPLE 3.0 is available for download from simplecryoem.com.

8.
Science ; 368(6486): 60-67, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32241943

RESUMO

Precise three-dimensional (3D) atomic structure determination of individual nanocrystals is a prerequisite for understanding and predicting their physical properties. Nanocrystals from the same synthesis batch display what are often presumed to be small but possibly important differences in size, lattice distortions, and defects, which can only be understood by structural characterization with high spatial 3D resolution. We solved the structures of individual colloidal platinum nanocrystals by developing atomic-resolution 3D liquid-cell electron microscopy to reveal critical intrinsic heterogeneity of ligand-protected platinum nanocrystals in solution, including structural degeneracies, lattice parameter deviations, internal defects, and strain. These differences in structure lead to substantial contributions to free energies, consequential enough that they must be considered in any discussion of fundamental nanocrystal properties or applications.

9.
Bioinformatics ; 36(7): 2237-2243, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31790146

RESUMO

MOTIVATION: No rigorous statistical tests for detecting point-group symmetry in three-dimensional (3D) charge density maps obtained by electron microscopy (EM) and related techniques have been developed. RESULTS: We propose a method for determining the point-group symmetry of 3D charge density maps obtained by EM and related techniques. Our ab initio algorithm does not depend on atomic coordinates but utilizes the density map directly. We validate the approach for a range of publicly available single-particle cryo-EM datasets. In straightforward cases, our method enables fully automated single-particle 3D reconstruction without having to input an arbitrarily selected point-group symmetry. When pseudo-symmetry is present, our method provides statistics quantifying the degree to which the 3D density agrees with the different point-groups tested. AVAILABILITY AND IMPLEMENTATION: The software is freely available at https://github.com/hael/SIMPLE3.0.


Assuntos
Algoritmos , Software , Microscopia Crioeletrônica , Imageamento Tridimensional
10.
J Struct Biol ; 204(2): 172-181, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30092280

RESUMO

Cryogenic electron microscopy (cryo-EM) and single-particle analysis enables determination of near-atomic resolution structures of biological molecules. However, large computational requirements limit throughput and rapid testing of new image processing tools. We developed PRIME, an algorithm part of the SIMPLE software suite, for determination of the relative 3D orientations of single-particle projection images. PRIME has primarily found use for generation of an initial ab initio 3D reconstruction. Here we show that the strategy behind PRIME, iterative estimation of per-particle orientation distributions with stochastic hill climbing, provides a competitive approach to near-atomic resolution single-particle 3D reconstruction. A number of mathematical techniques for accelerating the convergence rate are introduced, leading to a speedup of nearly two orders of magnitude. We benchmarked our developments on numerous publicly available data sets and conclude that near-atomic resolution ab initio 3D reconstructions can be obtained with SIMPLE in a matter of hours, using standard over-the-counter CPU workstations.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Software , Algoritmos , Microscopia Crioeletrônica
11.
Structure ; 26(7): 948-959.e5, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29804823

RESUMO

Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.


Assuntos
Clostridium botulinum/enzimologia , Hidroliases/química , Hidroliases/metabolismo , Ácido Pirúvico/metabolismo , Alquilação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clostridium botulinum/química , Cristalografia por Raios X , Cisteína/química , Estabilidade Enzimática , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
12.
Protein Sci ; 27(1): 51-61, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28795512

RESUMO

Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. We developed the SIMPLE open-source image-processing suite for analysing cryo-EM images of single-particles. A core component of SIMPLE is the probabilistic PRIME algorithm for identifying clusters of images in 2D and determine relative orientations of single-particle projections in 3D. Here, we extend our previous work on PRIME and introduce new stochastic optimization algorithms that improve the robustness of the approach. Our refined method for identification of homogeneous subsets of images in accurate register substantially improves the resolution of the cluster centers and of the ab initio 3D reconstructions derived from them. We now obtain maps with a resolution better than 10 Å by exclusively processing cluster centers. Excellent parallel code performance on over-the-counter laptops and CPU workstations is demonstrated.


Assuntos
Algoritmos , Microscopia Crioeletrônica , Imageamento Tridimensional , Software
13.
Structure ; 24(6): 988-96, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27184214

RESUMO

A critical step in the analysis of novel cryogenic electron microscopy (cryo-EM) single-particle datasets is the identification of homogeneous subsets of images. Methods for solving this problem are important for data quality assessment, ab initio 3D reconstruction, and analysis of population diversity due to the heterogeneous nature of macromolecules. Here we formulate a stochastic algorithm for identification of homogeneous subsets of images. The purpose of the method is to generate improved 2D class averages that can be used to produce a reliable 3D starting model in a rapid and unbiased fashion. We show that our method overcomes inherent limitations of widely used clustering approaches and proceed to test the approach on six publicly available experimental cryo-EM datasets. We conclude that, in each instance, ab initio 3D reconstructions of quality suitable for initialization of high-resolution refinement are produced from the cluster centers.


Assuntos
Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Algoritmos , Análise por Conglomerados , Processamento de Imagem Assistida por Computador , Modelos Moleculares
14.
Nat Commun ; 7: 10588, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26841934

RESUMO

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming ß-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.


Assuntos
Complemento C9/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/ultraestrutura , Polímeros , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Estrutura Molecular
15.
Biochim Biophys Acta ; 1858(3): 475-86, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26607011

RESUMO

Pore Forming Toxins (PFTs) represent a key mechanism for permitting the passage of proteins and small molecules across the lipid membrane. These proteins are typically produced as soluble monomers that self-assemble into ring-like oligomeric structures on the membrane surface. Following such assembly PFTs undergo a remarkable conformational change to insert into the lipid membrane. While many different protein families have independently evolved such ability, members of the Membrane Attack Complex PerForin/Cholesterol Dependent Cytolysin (MACPF/CDC) superfamily form distinctive giant ß-barrel pores comprised of up to 50 monomers and up to 300Å in diameter. In this review we focus on recent advances in understanding the structure of these giant MACPF/CDC pores as well as the underlying molecular mechanisms leading to their formation. Commonalities and evolved variations of the pore forming mechanism across the superfamily are discussed. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.


Assuntos
Membrana Celular/metabolismo , Evolução Molecular , Perforina/classificação , Perforina/metabolismo , Animais , Membrana Celular/química , Humanos , Perforina/química , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
16.
Proc Natl Acad Sci U S A ; 112(50): 15360-5, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26627714

RESUMO

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and ß), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.


Assuntos
Venenos de Peixe/química , Perforina/química , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Colesterol/química , Complexo de Ataque à Membrana do Sistema Complemento/química , Cristalografia por Raios X , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Solubilidade , Homologia Estrutural de Proteína
17.
PLoS One ; 10(10): e0140219, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26470022

RESUMO

The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency.


Assuntos
Bacteriófagos/enzimologia , Enzimas/química , Streptococcus/virologia , Bacteriófagos/química , Domínio Catalítico , Cristalografia por Raios X/métodos , Enzimas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo
18.
PLoS Biol ; 13(2): e1002049, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25654333

RESUMO

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central ß-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane ß-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of ß-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into ß-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted ß-barrel. The intermediate structures of the MACPF domain during refolding into the ß-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.


Assuntos
Membrana Celular/química , Complexo de Ataque à Membrana do Sistema Complemento/química , Proteínas Fúngicas/química , Proteínas Hemolisinas/química , Pleurotus/química , Proteínas Recombinantes de Fusão/química , Animais , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Eritrócitos/química , Eritrócitos/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ovinos
19.
Elife ; 3: e04247, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25457051

RESUMO

Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.


Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Proteínas Hemolisinas/metabolismo , Perforina/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Sistemas Computacionais , Microscopia Crioeletrônica , Difusão , Dissulfetos/metabolismo , Cinética , Microscopia de Força Atômica , Modelos Moleculares , Coloração Negativa , Multimerização Proteica
20.
PLoS Comput Biol ; 10(8): e1003791, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25144725

RESUMO

Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200-300 Å) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three ß-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs.


Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Membrana Celular/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Membrana Celular/química , Colesterol , Simulação de Dinâmica Molecular , Conformação Proteica
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