RESUMO
While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16") connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures.
Assuntos
Impressão Tridimensional , Proteínas , Microscopia Crioeletrônica/métodos , Microscopia EletrônicaRESUMO
Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a 'nucleus', erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial ß/ß' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Fagos de Pseudomonas/enzimologia , Proteínas Virais/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Virais/metabolismoRESUMO
Support films are commonly used during cryo-EM specimen preparation to both immobilise the sample and minimise the exposure of particles at the air-water interface. Here we report preparation protocols for carbon and graphene supported single particle electron microscopy samples using a novel 3D-printed sample transfer block to facilitate the direct, wetted, movement of both carbon and graphene supports from the substrate on which they were generated to small volumes (10 µL) of sample. These approaches are simple and inexpensive to implement, minimise hydrophobic contamination of the support films, and are widely applicable to single particle studies. Our approach also allows the direct exchange of the sample buffer on the support film in cases in which it is unsuitable for vitrification, e.g. for samples from centrifugal density gradients that help to preserve sample integrity.
Assuntos
Carbono/química , Microscopia Crioeletrônica/métodos , Grafite/química , Manejo de Espécimes/métodos , VitrificaçãoRESUMO
Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, in which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study, we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.
Assuntos
Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Levivirus/metabolismo , RNA Viral/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/virologia , Modelos MolecularesRESUMO
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.
Assuntos
Imageamento Tridimensional , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Imagem Individual de Molécula/métodos , Algoritmos , Proteínas de Membrana/química , Razão Sinal-Ruído , SoftwareRESUMO
We present LAFTER, an algorithm for de-noising single particle reconstructions from cryo-EM. Single particle analysis entails the reconstruction of high-resolution volumes from tens of thousands of particle images with low individual signal-to-noise. Imperfections in this process result in substantial variations in the local signal-to-noise ratio within the resulting reconstruction, complicating the interpretation of molecular structure. An effective local de-noising filter could therefore improve interpretability and maximise the amount of useful information obtained from cryo-EM maps. LAFTER is a local de-noising algorithm based on a pair of serial real-space filters. It compares independent half-set reconstructions to identify and retain shared features that have power greater than the noise. It is capable of recovering features across a wide range of signal-to-noise ratios, and we demonstrate recovery of the strongest features at Fourier shell correlation (FSC) values as low as 0.144 over a 2563-voxel cube. A fast and computationally efficient implementation of LAFTER is freely available. We also propose a new way to evaluate the effectiveness of real-space filters for noise suppression, based on the correspondence between two FSC curves: 1) the FSC between the filtered and unfiltered volumes, and 2) Cref, the FSC between the unfiltered volume and a hypothetical noiseless volume, which can readily be estimated from the FSC between two half-set reconstructions.
Assuntos
Algoritmos , Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Razão Sinal-RuídoRESUMO
Bacteriophage ΦKZ (PhiKZ) is the founding member of a family of giant bacterial viruses. It has potential as a therapeutic as its host, Pseudomonas aeruginosa, kills tens of thousands of people worldwide each year. ΦKZ infection is independent of the host transcriptional apparatus; the virus forms a "nucleus", producing a proteinaceous barrier around the ΦKZ genome that excludes the host immune systems. It expresses its own non-canonical multi-subunit non-virion RNA polymerase (nvRNAP), which is imported into its "nucleus" to transcribe viral genes. The ΦKZ nvRNAP is formed by four polypeptides representing homologues of the eubacterial ß/ß' subunits, and a fifth that is likely to have evolved from an ancestral homologue to σ-factor. We have resolved the structure of the ΦKZ nvRNAP initiating transcription from its cognate promoter, p119L, including previously disordered regions. Our results shed light on the similarities and differences between ΦKZ nvRNAP mechanisms of transcription and those of canonical eubacterial RNAPs and the related non-canonical nvRNAP of bacteriophage AR9.
RESUMO
Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.
Assuntos
Carbono , Microscopia Crioeletrônica/instrumentação , Microscopia Eletrônica de Transmissão/instrumentaçãoRESUMO
The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumour suppressors, and mutations in them underlie the disease tuberous sclerosis. We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a central "body", with a "pincer" and a "tail" at the respective ends. The "body" is composed of a flexible TSC2 HEAT repeat dimer, along the surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a "pincer" is formed by the highly flexible N-terminal TSC1 core domains and a barbed "tail" makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1. Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.
Assuntos
Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Proteína 1 do Complexo Esclerose Tuberosa/química , Proteína 2 do Complexo Esclerose Tuberosa/química , Microscopia Crioeletrônica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes , Relação Estrutura-Atividade , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
BACKGROUND: The protein kinase Target Of Rapamycin (TOR) is a nexus for the regulation of eukaryotic cell growth. TOR assembles into one of two distinct signalling complexes, TOR complex 1 (TORC1) and TORC2 (mTORC1/2 in mammals), with a set of largely non-overlapping protein partners. (m)TORC1 activation occurs in response to a series of stimuli relevant to cell growth, including nutrient availability, growth factor signals and stress, and regulates much of the cell's biosynthetic activity, from proteins to lipids, and recycling through autophagy. mTORC1 regulation is of great therapeutic significance, since in humans many of these signalling complexes, alongside subunits of mTORC1 itself, are implicated in a wide variety of pathophysiologies, including multiple types of cancer, neurological disorders, neurodegenerative diseases and metabolic disorders including diabetes. METHODOLOGY: Recent years have seen numerous structures determined of (m)TOR, which have provided mechanistic insight into (m)TORC1 activation in particular, however the integration of cellular signals occurs upstream of the kinase and remains incompletely understood. Here we have collected and analysed in detail as many as possible of the molecular and structural studies which have shed light on (m)TORC1 repression, activation and signal integration. CONCLUSIONS: A molecular understanding of this signal integration pathway is required to understand how (m)TORC1 activation is reconciled with the many diverse and contradictory stimuli affecting cell growth. We discuss the current level of molecular understanding of the upstream components of the (m)TORC1 signalling pathway, recent progress on this key biochemical frontier, and the future studies necessary to establish a mechanistic understanding of this master-switch for eukaryotic cell growth.