Biology subjects

Williams, H. M.

Publications and source records attributed to Williams, H. M..

8 recordsLinked to original sources

bioRxiv2025

Microsecond Time-Resolved Cryo-EM Based on Jet Vitrification

Understanding and ultimately predicting the function of proteins is one of the frontiers in structural biology. This will only be possible if it becomes feasible to routinely observe proteins on the fast timescales on which they perform their tasks. Recently, laser flash melting and revitrification experiments have improved the time resolution of cryo-electron microscopy (cryo-EM) to microseconds, rendering it fast enough to observe the domain motions of proteins that are frequently linked to function. However, observations have been limited to a time window of just a few hundred microseconds. Here, we introduce time-resolved cryo-EM experiments based on jet vitrification that combine microsecond resolution with an observation window of up to seconds. We use a short laser pulse to initiate protein dynamics, and as they unfold, vitrify the sample with a jet of a liquid cryogen to arrest the dynamics at that point in time. We demonstrate that our approach affords near-atomic spatial resolution and a time resolution of 21 {micro}s. This allows us to observe the photoinduced dynamics of the light-driven sodium pump ErNaR on the microsecond to millisecond timescale. Our experiments significantly expand the ability of cryo-EM to observe protein dynamics across multiple timescales.

biophysics↗

bioRxiv2025

Overcoming Preferred Orientation in Cryo-EM With Ultrasonic Excitation During Vitrification

Preferred particle orientation remains a frequently encountered problem in cryo-electron microscopy that arises when proteins adsorb to the air-water interface in only a limited number of orientations. This issue can significantly increase the data acquisition time required to reach a desired resolution or even make it impossible to obtain a reconstruction altogether. Here, we show that preferred orientation can be overcome by continuously exciting the sample with ultrasonic waves during vitrification. Our experiments suggest that mechanical oscillations induced in the sample support continuously shake proteins loose from the air-water interface, thereby scrambling their orientations. The simple, physical nature of this mechanism should make it applicable to a wide range of proteins. Since our method can be easily implemented in existing vitrification devices, we expect it to find widespread adoption.

biophysics↗

bioRxiv2025

Crystal structures of human SP-D complexed with synthetic oligosaccharides suggest a role for phosphorylated inner core LPS saccharides in host-pathogen interactions

The innate immune protein human surfactant protein D (SP-D) recognises pathogens in the lungs via binding to carbohydrate surface structures. SP-D has been shown to target gram-negative bacterial lipopolysaccharide via calcium-dependent binding, preferentially to the inner core heptose (HepI). To further investigate this recognition, we have determined the high-resolution crystal structures of a trimeric recombinant fragment of human SP-D complexed with synthetic di- and trisaccharides, HepI-Kdo, HepIII-HepII-HepI, and HepII-HepI phosphorylated at either HepI or HepII, inner core motifs common to the lipopolysaccharide of many gram-negative bacteria. In contrast to acid-hydrolysed lipopolysaccharide used in several previous studies, these synthetic saccharides allow presentation of both the innermost Kdo in its natural pyranose form and heptose phosphorylation. The structures confirm the flexibility of SP-D to adopt an alternative binding mode when the preferred epitope is not available, reveal a preference for recognition of the reducing terminal heptose (HepI) via the glyceryl group, indicate that a single Kdo attached to HepI does not have a significant role in ligand recognition, and provide evidence that recognition of phosphorylated inner core diheptosyl ligands varies dependent on whether HepI or HepII is phosphorylated. The disaccharide with HepII O4 phosphorylation binds via the preferred HepI glyceryl-hydroxyls, while HepI O4 phosphorylation reveals HepII binding via the pyranose ring O3 and O4 hydroxyls. The ability of the HepII O4 phosphate to prevent preferred HepI recognition suggests a role for heptose phosphorylation in shielding the bacterial LPS inner core from immune recognition.

immunology↗

bioRxiv2023

Structural snapshots of phenuivirus cap-snatching and transcription

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by stealing host mRNA caps via a process known as cap-snatching. Here, we establish an in vitro cap-snatching assay and present three high-quality electron cryo-microscopy (cryo-EM) structures of the SFTSV L protein in biologically relevant, transcription-specific states. In a priming-state structure, we show capped RNA bound to the L protein cap-binding domain (CBD). The L protein conformation in this priming structure is significantly different from published replication-state structures, in particular the N- and C-terminal domains. The capped-RNA is positioned in a way that it can feed directly into the RNA-dependent RNA polymerase (RdRp) ready for elongation. We also captured the L protein in an early-elongation state following primer-incorporation demonstrating that this priming conformation is retained at least in the very early stages of primer extension. This structural data is complemented by in vitro biochemical and cell-based assays. Together, these insights further our mechanistic understanding of how SFTSV and other bunyaviruses incorporate stolen host mRNA fragments into their viral transcripts thereby allowing the virus to hijack host cell translation machinery.

biochemistry↗

bioRxiv2023

An RNA to rule them all: Critical steps in Lassa virus ribonucleoparticle assembly and recruitment

Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming the ribonucleoprotein complexes (RNPs) together with the viral RNA and the L protein. RNPs have to be continuously restructured during viral genome replication and transcription. The Z protein is important for membrane recruitment of RNPs, viral particle assembly and budding, and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for disassembly of ring-like NP trimers, a storage form, into monomers to subsequently form higher order RNA-bound NP assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of RNP assembly, recruitment and release in Lassa virus.

biophysics↗

bioRxiv2022

Structural insights into viral genome replication by the Severe fever with thrombocytopenia syndrome virus L protein

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 [A]. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early- and late-stages of elongation with the RdRp core accommodating a 9-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.

biochemistry↗

bioRxiv2021

Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

Lassa virus, which causes annual outbreaks in West Africa with increasing case numbers in recent years, is recognized by the WHO R&D blueprint as a significant threat for public health with high epidemic potential and no effective countermeasures. The viral large (L) protein, which contains the RNA-dependent RNA polymerase, is a key player for transcription of viral mRNA and genome replication. Here we present nine cryo-EM structures of Lassa virus L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved genomic 3 and 5 promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and elongation states, the endonuclease is inhibited by the binding of two distinct L protein peptides in the active site, respectively, whereas in the pre-initiation state, the endonuclease is uninhibited. In the stalled, early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide. In this configuration, the full C-terminal region of the L protein, including the putative cap-binding domain, is highly ordered. The structural data are complemented by in vitro and cell-based studies testing a broad range of L protein mutants to probe functional relevance. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated and will underpin antiviral drug development targeting the arenavirus L protein.

biochemistry↗

bioRxiv2020

The Alzheimer's disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function.

Recent genome-wide association studies of Alzheimers disease (AD) have identified variants implicating immune pathways in disease development. A rare coding variant of PLCG2, which encodes PLC{gamma}2, shows a significant protective effect for AD (rs72824905, P522R, P=5.38x10-10, Odds Ratio = 0.68). Molecular dynamic modelling of the PLC{gamma}2-R522 variant, situated within the auto-inhibitory domain of PLC{gamma}2, suggests a structural change to the protein. Through CRISPR-engineering we have generated novel PLCG2-R522 harbouring human induced pluripotent cell lines (hiPSC) and a mouse knockin model, neither of which exhibits alterations in endogenous PLCG2 expression. Mouse microglia and macrophages and hiPSC-derived microglia-like cells with the R522 mutation, all demonstrate a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This signalling alteration manifests as enhanced cellular Ca2+ store release ([~]20-40% increase) in response to physiologically-relevant stimuli (e.g. Fc receptor ligation and A{beta} oligomers). This hyperfunctionality resulted in increased PIP2 depletion in the cells with the PLC{gamma}2-R522 variant after exposure to stimuli and reduced basal detection of PIP2 levels in vivo. These PLC{gamma}2-R522 associated abnormalities resulted in impairments to phagocytosis (fungal and bacterial particles) and enhanced endocytosis (A{beta} oligomers and dextran). PLC{gamma}2 sits downstream of disease relevant pathways, such as TREM2 and CSF1R and alterations in its activity, direct impacts cell function, which in the context of the inherent drugability of enzymes such as PLC{gamma}2, raise the prospect of manipulation of PLC{gamma}2 as a therapeutic target in Alzheimers Disease.

neuroscience↗