Biology subjects

Gomez-Blanco, J.

Publications and source records attributed to Gomez-Blanco, J..

8 recordsLinked to original sources

bioRxiv2025

Structural determinants of rotavirus proteolytic activation

The infectivity of rotavirus (RV), the leading cause of childhood diarrhea, hinges on the activation of viral particles through the proteolysis of the spike protein by trypsin-like proteases in the host intestinal lumen. Despite comprehensive structural characterization of the virus particle, the structural rationale behind the necessity of trypsin digestion of the VP4 protein for infectivity remains poorly understood. In this study, using cryo-electron microscopy (cryo-EM) and advanced image processing techniques, we compared uncleaved and cleaved RV virions and found that the conformation of the non-proteolyzed spike is constrained by the position of loops that surround its structure, linking the lectin domains of the spike head to its body. The proteolysis of these loops removes this structural constraint, thereby enabling the spike to undergo the necessary conformational changes required for cell membrane penetration. Thus, these loops function as regulatory elements to ensure that the spike protein is activated precisely when and where it is needed to facilitate a successful infection.

microbiology↗

bioRxiv2021

Structural basis for DNA targeting by the Tn7 transposon

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7-family utilize a AAA+ adaptor (TnsC) to coordinates target-site selection with transposase activation and prevent insertions at sites already containing a Tn7 element. Due to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. We find that TnsC forms an asymmetric ring on target DNA that segregates target-site selection and transposase recruitment to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP-hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.

biophysics↗

bioRxiv2020

Near Atomic Structure of an Atadenovirus Reveals a Conserved Capsid-Binding Motif and Intergenera Variations in Cementing Proteins

Little is known about the basic biology of non-human adenoviruses, which could be alternative vectors free of issues posed by preexisting immunity to human adenoviruses. We present the cryo-EM structure of a lizard atadenovirus, LAdV-2, at 3.4 [A] resolution. This is the first high resolution structure of an adenovirus with non-mammalian host, and of an adenovirus not belonging to the Mastadenovirus genus. Atadenovirus capsids contain genus specific proteins LH3, p32k, and LH2, and are more thermostable than the more studied human adenoviruses. We find a large conformational difference in the internal vertex protein IIIa between mast- and atadenoviruses, induced by the presence of an extended polypeptide in the region. This polypeptide, as well as -helical clusters located beneath the icosahedral facet, likely correspond to proteins LH2 and p32k. The external genus specific protein LH3, with a trimeric {beta}-helix fold typical of bacteriophage host attachment proteins, contacts the hexon shell surface via a triskelion structure identical to that used by protein IX in human AdV, revealing a conserved capsid-binding motif and a possible gene duplication event. Altogether, this work shows how the network of minor coat proteins differs between AdV genera and relates to virus evolution and capsid stability properties.

microbiology↗

bioRxiv2020

DeepEMhacer: a deep learning solution for cryo-EM volume post-processing

Cryo-EM maps are valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed to improve their interpretability. Most popular approaches, based on B-factor correction, suffer from limitations. For instance, they ignore the heterogeneity in the map local quality that reconstructions tend to exhibit. Aiming to overcome these problems, we present DeepEMhancer, a deep learning approach designed to perform automatic post-processing of cryo-EM maps. Trained on a dataset of pairs of experimental maps and maps sharpened using their respective atomic models, DeepEMhancer has learned how to post-process experimental maps performing masking-like and sharpening-like operations in a single step. DeepEMhancer was evaluated on a testing set of 20 different experimental maps, showing its ability to obtain much cleaner and more detailed versions of the experimental maps. Additionally, we illustrated the benefits of DeepEMhancer on the structure of the SARS-CoV-2 RNA polymerase.

bioinformatics↗

bioRxiv2020

Local computational methods to improve the interpretability and analysis of cryo-EM maps

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, over-sharpening due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.

biophysics↗

bioRxiv2020

Alternative Conformations and Motions Adopted by 30S Ribosomal Subunits Visualized by Cryo-Electron Microscopy.

It is only after recent advances in cryo-electron microscopy that is now possible to describe at high resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between the "active" and "inactive" conformations. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains unsolved. Here we used cryo-electron microscopy to obtain the structure of the inactive conformation of the 30S subunit to 3.6[A] resolution and study its motions. In the inactive conformation, three nucleotides at the 3 end of the 16S rRNA cause the region of helix 44 forming the decoding center to adopt an unlatched conformation and the 3 end of the 16S rRNA positions similarly to the mRNA during translation. Incubation of inactive 30S subunits at 42 {degrees}C reverts these structural changes. The position adopted by helix 44 dictates the most prominent motions of the 30S subunit. We found that extended exposures to low magnesium concentrations induces unfolding of large rRNA structural domains. The air-water interface to which ribosome subuints are exposed during sample preparation also peel off some ribosomal proteins. Overall this study provides new insights about the conformational space explored by the 30S ribosomal subunit when the ribosomal particles are free in solution.

molecular biology↗

bioRxiv2019

Role of Era in Assembly and Homeostasis of the Ribosomal Small Subunit

To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains were able to fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Eras role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.

molecular biology↗

bioRxiv2016

Quantitative analysis of 3D alignment quality: itsimpact on soft-validation, particle pruning andhomogeneity analysis

Single Particle Analysis using cryo-electron microscopy is a structural biology technique to capture the three-dimensional conformation of biological macromolecules. The projection images used to construct the 3D density map are characterized by a very low signal-to-noise ratio to minimize radiation damage in the samples. As a consequence, the 3D alignment process is a challenging and error prone task and this job usually determines the success or failure of the macromolecule reconstruction. In this work, we present a soft-alignment validation approach, which can quantify the alignment precision and accuracy as well as the data homogeneity of the single particles when they are confronted with the resultant reconstructed 3DEM map. We have also applied this method to data homogeneity analysis and particle pruning, improving the data quality and as a consequence the final map resolution.

molecular biology↗