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1.
Cell ; 165(4): 949-62, 2016 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-27114038

RESUMEN

Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA-DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for cleaving the non-target and target strands, respectively, and for jointly generating staggered DNA double-strand breaks. AsCpf1 recognizes the 5'-TTTN-3' protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1 and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox.


Asunto(s)
Acidaminococcus/química , Proteínas Bacterianas/química , ADN/química , Técnicas Genéticas , ARN Guía de Kinetoplastida/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Modelos Moleculares , Ácidos Nucleicos Heterodúplex/metabolismo , ARN Guía de Kinetoplastida/metabolismo
2.
Cell ; 164(5): 950-61, 2016 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-26875867

RESUMEN

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.


Asunto(s)
Proteínas Bacterianas/química , Sistemas CRISPR-Cas , Endonucleasas/química , Francisella/enzimología , Ingeniería Genética/métodos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Blastocisto/metabolismo , Proteína 9 Asociada a CRISPR , Cristalografía por Rayos X , Embrión de Mamíferos/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Ratones , Microinyecciones/métodos , Modelos Moleculares , ARN Guía de Kinetoplastida/genética
3.
Nat Methods ; 21(7): 1216-1221, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38862790

RESUMEN

Macromolecular structure determination by electron cryo-microscopy (cryo-EM) is limited by the alignment of noisy images of individual particles. Because smaller particles have weaker signals, alignment errors impose size limitations on its applicability. Here, we explore how image alignment is improved by the application of deep learning to exploit prior knowledge about biological macromolecular structures that would otherwise be difficult to express mathematically. We train a denoising convolutional neural network on pairs of half-set reconstructions from the electron microscopy data bank (EMDB) and use this denoiser as an alternative to a commonly used smoothness prior. We demonstrate that this approach, which we call Blush regularization, yields better reconstructions than do existing algorithms, in particular for data with low signal-to-noise ratios. The reconstruction of a protein-nucleic acid complex with a molecular weight of 40 kDa, which was previously intractable, illustrates that denoising neural networks will expand the applicability of cryo-EM structure determination for a wide range of biological macromolecules.


Asunto(s)
Microscopía por Crioelectrón , Procesamiento de Imagen Asistido por Computador , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Relación Señal-Ruido , Redes Neurales de la Computación , Sustancias Macromoleculares/química , Aprendizaje Profundo , Modelos Moleculares
4.
Nature ; 588(7838): 498-502, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32805734

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.


Asunto(s)
Microscopía por Crioelectrón , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestructura , Glicoproteína de la Espiga del Coronavirus/análisis , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Virión/química , Virión/ultraestructura , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Línea Celular Tumoral , Humanos , Modelos Moleculares , Docilidad , Conformación Proteica , Multimerización de Proteína , SARS-CoV-2/química , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación , Virión/aislamiento & purificación , Virión/metabolismo
5.
Nature ; 587(7832): 152-156, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33087931

RESUMEN

The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the ß3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.


Asunto(s)
Apoferritinas/química , Apoferritinas/ultraestructura , Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Receptores de GABA-A/química , Receptores de GABA-A/ultraestructura , Imagen Individual de Molécula/métodos , Animales , Microscopía por Crioelectrón/normas , Descubrimiento de Drogas , Humanos , Ratones , Modelos Moleculares , Polisacáridos/química , Polisacáridos/ultraestructura , Imagen Individual de Molécula/normas
6.
Mol Cell ; 65(6): 1109-1121.e3, 2017 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-28306506

RESUMEN

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Campylobacter jejuni/enzimología , Endonucleasas/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Proteínas Asociadas a CRISPR/química , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Endonucleasas/química , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato
7.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-35197289

RESUMEN

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.


Asunto(s)
Canales de Cloruro/química , Rayos Láser , Canales de Cloruro/metabolismo , Cristalografía , Citoplasma/metabolismo , Transporte Iónico , Luz , Conformación Proteica , Rayos X
8.
Nature ; 543(7643): 131-135, 2017 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-28219079

RESUMEN

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique µ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.


Asunto(s)
Cristalografía/métodos , Electrones , Rayos Láser , Luz , Oxígeno/química , Oxígeno/efectos de la radiación , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/efectos de la radiación , Biocatálisis/efectos de la radiación , Cianobacterias/química , Transporte de Electrón/efectos de la radiación , Análisis de Fourier , Manganeso/química , Manganeso/metabolismo , Modelos Moleculares , Proteínas de Hierro no Heme/química , Proteínas de Hierro no Heme/metabolismo , Proteínas de Hierro no Heme/efectos de la radiación , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Protones , Temperatura , Factores de Tiempo , Agua/química , Agua/metabolismo
9.
Biochem J ; 478(24): 4169-4185, 2021 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-34783343

RESUMEN

We describe new tools for the processing of electron cryo-microscopy (cryo-EM) images in the fourth major release of the RELION software. In particular, we introduce VDAM, a variable-metric gradient descent algorithm with adaptive moments estimation, for image refinement; a convolutional neural network for unsupervised selection of 2D classes; and a flexible framework for the design and execution of multiple jobs in pre-defined workflows. In addition, we present a stand-alone utility called MDCatch that links the execution of jobs within this framework with metadata gathering during microscope data acquisition. The new tools are aimed at providing fast and robust procedures for unsupervised cryo-EM structure determination, with potential applications for on-the-fly processing and the development of flexible, high-throughput structure determination pipelines. We illustrate their potential on 12 publicly available cryo-EM data sets.


Asunto(s)
Microscopía por Crioelectrón/métodos , Proteínas/ultraestructura , Ribosomas/ultraestructura , Programas Informáticos , Algoritmos
10.
Nat Chem Biol ; 15(1): 18-26, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30510193

RESUMEN

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.


Asunto(s)
Subtipo EP4 de Receptores de Prostaglandina E/química , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Regulación Alostérica , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Caprilatos/química , Caprilatos/metabolismo , Cristalografía por Rayos X , Epoprostenol/análogos & derivados , Epoprostenol/química , Epoprostenol/metabolismo , Humanos , Ligandos , Membrana Dobles de Lípidos , Simulación del Acoplamiento Molecular , Naftalenos/química , Naftalenos/metabolismo , Éteres Fenílicos/química , Éteres Fenílicos/metabolismo , Fenilbutiratos/química , Fenilbutiratos/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/genética , Spodoptera/genética
11.
J Struct Biol ; 211(2): 107545, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32534144

RESUMEN

Single particle analysis has become a key structural biology technique. Experimental images are extremely noisy, and during iterative refinement it is possible to stably incorporate noise into the reconstruction. Such "over-fitting" can lead to misinterpretation of the structure and flawed biological results. Several strategies are routinely used to prevent over-fitting, the most common being independent refinement of two sides of a split dataset. In this study, we show that over-fitting remains an issue within regions of low local signal-to-noise, despite independent refinement of half datasets. We propose a modification of the refinement process through the application of a local signal-to-noise filter: SIDESPLITTER. We show that our approach can reduce over-fitting for both idealised and experimental data while maintaining independence between the two sides of a split refinement. SIDESPLITTER refinement leads to improved density, and can also lead to improvement of the final resolution in extreme cases where datasets are prone to severe over-fitting, such as small membrane proteins.


Asunto(s)
Imagenología Tridimensional , Proteínas de la Membrana/ultraestructura , Modelos Moleculares , Imagen Individual de Molécula/métodos , Algoritmos , Proteínas de la Membrana/química , Relación Señal-Ruido , Programas Informáticos
12.
Proc Natl Acad Sci U S A ; 114(51): 13357-13362, 2017 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-28835537

RESUMEN

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.


Asunto(s)
Protones , Proteínas de la Matriz Viral/química , Secuencias de Aminoácidos , Enlace de Hidrógeno , Activación del Canal Iónico , Simulación de Dinámica Molecular , Dominios Proteicos , Electricidad Estática , Temperatura , Proteínas de la Matriz Viral/metabolismo
13.
Proc Natl Acad Sci U S A ; 113(11): 2928-33, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26929369

RESUMEN

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.


Asunto(s)
Alcaligenes faecalis/enzimología , Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Nitrito Reductasas/química , Alcaligenes faecalis/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Cobre/química , Cristalografía por Rayos X/instrumentación , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Nitrito Reductasas/genética , Nitrito Reductasas/metabolismo , Nitritos/metabolismo , Oxidación-Reducción , Mutación Puntual , Conformación Proteica , Protones , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad
14.
Proc Natl Acad Sci U S A ; 113(46): 13039-13044, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27799539

RESUMEN

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/química , Cristalización , Cristalografía/métodos , Detergentes/química , Electrones , Halobacterium , Rayos Láser , Conformación Proteica , Ácidos Triyodobenzoicos/química
15.
J Synchrotron Radiat ; 24(Pt 5): 1086-1091, 2017 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-28862633

RESUMEN

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

16.
Int J Mol Sci ; 18(9)2017 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-28880248

RESUMEN

The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form.


Asunto(s)
Cristalografía por Rayos X/métodos , Rayos Láser , Proteínas Luminiscentes/química , Conformación Proteica , Temperatura
17.
Bioinformatics ; 31(1): 126-7, 2015 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-25183487

RESUMEN

SUMMARY: The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) resource provides tools for query, analysis and visualization of the 3D structures in the PDB archive. As the mobile Web is starting to surpass desktop and laptop usage, scientists and educators are beginning to integrate mobile devices into their research and teaching. In response, we have developed the RCSB PDB Mobile app for the iOS and Android mobile platforms to enable fast and convenient access to RCSB PDB data and services. Using the app, users from the general public to expert researchers can quickly search and visualize biomolecules, and add personal annotations via the RCSB PDB's integrated MyPDB service. AVAILABILITY AND IMPLEMENTATION: RCSB PDB Mobile is freely available from the Apple App Store and Google Play (http://www.rcsb.org).


Asunto(s)
Biología Computacional/métodos , Gráficos por Computador , Bases de Datos de Proteínas , Aplicaciones Móviles , Programas Informáticos , Investigación Biomédica , Humanos , Interfaz Usuario-Computador , Flujo de Trabajo
18.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 12): 2519-25, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26627659

RESUMEN

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Šis successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.


Asunto(s)
Cloro/química , Cristalografía por Rayos X/métodos , Muramidasa/química , Azufre/química , Secuencias de Aminoácidos , Animales , Pollos , Clara de Huevo/química , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/aislamiento & purificación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
19.
BMC Bioinformatics ; 15: 56, 2014 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-24564583

RESUMEN

BACKGROUND: Visualization of protein-ligand complex plays an important role in elaborating protein-ligand interactions and aiding novel drug design. Most existing web visualizers either rely on slow software rendering, or lack virtual reality support. The vital feature of macromolecular surface construction is also unavailable. RESULTS: We have developed iview, an easy-to-use interactive WebGL visualizer of protein-ligand complex. It exploits hardware acceleration rather than software rendering. It features three special effects in virtual reality settings, namely anaglyph, parallax barrier and oculus rift, resulting in visually appealing identification of intermolecular interactions. It supports four surface representations including Van der Waals surface, solvent excluded surface, solvent accessible surface and molecular surface. Moreover, based on the feature-rich version of iview, we have also developed a neat and tailor-made version specifically for our istar web platform for protein-ligand docking purpose. This demonstrates the excellent portability of iview. CONCLUSIONS: Using innovative 3D techniques, we provide a user friendly visualizer that is not intended to compete with professional visualizers, but to enable easy accessibility and platform independence.


Asunto(s)
Biología Computacional/métodos , Simulación del Acoplamiento Molecular/métodos , Proteínas/química , Proteínas/metabolismo , Programas Informáticos , Gráficos por Computador , Internet , Ligandos , Unión Proteica , Interfaz Usuario-Computador
20.
Nat Commun ; 15(1): 5518, 2024 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-38951539

RESUMEN

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.


Asunto(s)
Manganeso , Muramidasa , Muramidasa/química , Manganeso/química , Cristalografía por Rayos X , Porosidad , Complejos de Coordinación/química , Modelos Moleculares , Animales , Monóxido de Carbono/química , Factores de Tiempo , Pollos
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