Biology subjects

Rambo, R.

Publications and source records attributed to Rambo, R..

5 recordsLinked to original sources

bioRxiv2026

Carbonara: a SAXS-guided seeding framework for exploring protein solution-state dynamics

Proteins in solution often populate conformational ensembles that differ from the static states captured by crystallography or AI-based structure prediction. Conventional molecular dynamics (MD) simulations often fail to cross the energy barriers separating these states on accessible timescales, and statistical reweighting cannot recover conformations never sampled. Here we present Carbonara, a framework that uses experimental small-angle X-ray scattering (SAXS) data to predict alternative physically plausible protein conformations. Carbonara builds on Wiggle, a standalone C-based SAXS forward model validated against explicit-solvent calculations and experimental benchmarks. Using two case studies, an AI-predicted multi-domain helicase (SMAR-CAL1) and a crystallographic antibody fragment (ChiLob7/4 IgG2), we show how seeding MD simulations from Carbonara conformations enables efficient exploration of solution-state conformational landscapes. In both cases, MD ensembles initiated from available models either fail to match the SAXS data or do so only after discarding nearly all sampled conformations, whereas Carbonara-seeded ensembles reach agreement while retaining the majority of conformations. Our modelling framework provides a route from static structural models of flexible multi-domain proteins and multimeric assemblies to solution-state ensembles.

biophysics↗

bioRxiv2023

Novel topological methods for identifying surprising protein tertiary structure relationships.

We present fast and simple-to-implement measures of the entanglement of protein tertiary structures which are appropriate for highly flexible structure comparison. These quantities are based on the writhing and crossing numbers heavily utilised in DNA topology studies which and which have shown some promising results when applied to proteins recently. Here we show how they can be applied in a novel manner across various scales of the proteins backbone to identify similar topologies which can be missed by more common RMSD, secondary structure or primary sequence based comparison methods. We derive empirical bounds on the entanglement implied by these measures and show how they can be used to constrain the search space of a protein for solution scattering, a method highly suited to determining the likely structure of proteins in solution where crystal structure or machine learning based predictions often fail to match experimental data. In addition we identify large scale helical geometries present in a large array of proteins, which are consistent across a number of different protein structure types and sequences. This is used in one specific case to demonstrate significant structural similarity between Rossmann fold and TIM Barrel proteins, a link which is potentially significant as attempts to engineer the latter have in the past produced the former. Finally we provide the SWRITHE python notebook to calculate these metrics. Author summaryThere is much interest in developing quantitative methods to compare different protein structures or identify common sub-structures across protein families. We present novel methods for studying and comparing protein structures based on the entanglement of their amino-acid backbone and demonstrate a number of their critical properties. First, they are shown to be especially useful in identifying similar protein entanglement for structures which may be seen as distinct via more established methods. Second, by studying the distribution of entanglement across a wide sample of proteins, we show that there exists a minimum expected amount (a lower bound) of entanglement given the proteins length. This bound is shown to be useful in ensuring realistic predictions from experimental structural determination methods. Third, using fundamental properties of this entanglement measure, we identify two common classes of protein sub-structure. The first are large scale helices, which provide stability to the structure. These helical structures indicate strong structural similarity of two protein families usually regarded as differing significantly. The second class of substructure is one which, though complex, has a small net entanglement. This configuration is physically useful in other disciplines, but its function in proteins is not yet clear. Finally, we provide an interactive python notebook to compute these measures for a given protein.

bioinformatics↗

bioRxiv2022

Crystal structure and solution state of the C-terminal head region of the narmovirus receptor binding protein

Increased viral surveillance has led to the isolation and identification of numerous uncharacterized paramyxoviruses, rapidly expanding our understanding of paramyxoviral diversity beyond the bounds of known genera. Despite this diversity, a key feature that unites paramyxoviruses is the presence of a receptor-binding protein, RBP, which facilitates host-cell attachment and plays a fundamental role in determining host-range. Here, we study the RBP presented on the surface of rodent-borne paramyxoviruses Mossman and Nariva (MosV and NarV, respectively), viruses that constitute founding members of the recently defined Narmovirus genus within the Paramyxoviridae family. Crystallographic analysis of the C-terminal head region of the dimeric MosV and NarV RBPs demonstrates that while these glycoproteins retain the canonical six-bladed {beta}-propeller fold found in other paramyxoviral RBPs, they lack the structural motifs associated with established paramyxovirus host-cell receptor entry pathways. Consistent with MosV-RBP and NarV-RBP undergoing a distinct entry pathway from other characterized paramyxoviruses, structure-based phylogenetic analysis demonstrates that these six-bladed {beta}-propeller head domains form a singular structural class that is distinct from other paramyxoviral RBPs. Additionally, using an integrated crystallographic and small angle X-ray scattering analysis, we confirm that MosV-RBP and NarV-RBP form homodimeric arrangements that are distinct from those adopted by other paramyxovirus RBPs. Altogether, this investigation provides a molecular-level blueprint of the narmovirus RBP that broadens our understanding of the structural space and functional diversity available to paramyxovirus RBPs. ImportanceGenetically diverse paramyxoviruses are united in their presentation of a receptor-binding protein (RBP), which works in concert with the fusion protein to facilitate host-cell entry. The C-terminal head region of the paramyxoviral RBP, a primary determinant of host-cell tropism and inter-species transmission potential, forms structurally distinct classes dependent upon protein and glycan receptor specificity. Here, we reveal the architecture of the C-terminal head region of the RBPs from Nariva virus (NarV) and Mossman virus (MosV), two archetypal rodent-borne paramyxoviruses within the recently established genus Narmovirus, family Paramyxoviridae. Our analysis reveals that while narmoviruses retain the general architectural features associated with paramyxoviral RBPs, namely a six-bladed {beta}-propeller fold, they lack the structural motifs associated with known receptor-mediated host-cell entry pathways. This investigation indicates that the RBPs of narmoviruses exhibit pathobiological features that are distinct from those of other paramyxoviruses.

biochemistry↗

bioRxiv2022

Biophysical characterisation of the structure of a SARS-CoV-2 self-amplifying - RNA (saRNA) vaccine

The current SARS-Covid-2 pandemic has led to an acceleration of messenger - ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA) has required concomitant development of analytical characterisation techniques. Characterising the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This paper describes the biophysical characterisation of the Imperial College London Self-amplifying viral RNA vaccine (IMP-1) developed for SARS-CoV-2. A variety of analytical techniques have been used to characterise the IMP-1 RNA molecule. In this paper we use UV spectroscopy, dynamic light scattering (DLS), size-exclusion chromatography small angle scattering (SEC-SAXS) and circular dichroism (CD) to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule. Statement of significanceThis paper is highly significant as it provides a prescient biophysical characterisation of an efficacious Sars-Cov-2 vaccine self-amplifying (sa)RNA molecule. RNA vaccines have been a major scientific breakthrough of the Covid-19 pandemic. saRNA is a further development of conventional mRNA vaccines, amplifying the RNA of interest in the cell, allowing the vaccine to be administered at lower dosages. These new biologics are distinct from previous biologics and have required distinct analytical characterisation. The analytics described herein provide detailed information on the size, shape, and structure of the RNA molecule. This paper is therefore an important step in characterising large saRNA biological relevant molecules.

biophysics↗

bioRxiv2019

Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) molecules which act as a second messenger, signalling infection and potentiating a powerful immune response by activating a range of downstream effector proteins that can lead to viral clearance, cell dormancy or death. Only one type of effector enzyme has been studied - the Csm6/Csx1 ribonuclease domain, and the mechanism of cOA activation is not understood at a molecular level. Here we describe the structure and mechanism of a novel cOA-activated CRISPR defence DNA endonuclease, Can1 (\"CRISPR ancillary nuclease 1\"). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) CARF domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.

biochemistry↗