Biology subjects

Danev, R.

Publications and source records attributed to Danev, R..

17 recordsLinked to original sources

bioRxiv2026

Structural basis of MK-97 positive allosteric modulation at the M4 mAChR

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M4 mAChR PAMs, like LY2033298, demonstrated proof-of-concept, second-generation compounds, such as MK-97, exhibit substantially improved potency and reduced species variability. Here we report the cryo-EM structure of the M4 mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 [A] resolution, revealing the molecular basis for improved M4 mAChR PAM activity. MK-97 adopts a distinctive boomerang-shaped conformation within the extracellular-facing allosteric binding site, with a central pyridine vertex, a lower cyclopentylmethylpyrazole arm extending toward the floor of the orthosteric site, and an upper isoindolinone arm projecting toward extracellular loop 2 (ECL2). This extended binding mode establishes a distributed interaction network across transmembrane helices TM2, TM3, TM5, TM6, and TM7, with key contacts including a hydrogen bond with Y922.64 and a {pi}-{pi} stacking interaction with W4357.35. Integration of structural data, molecular dynamics simulations, and mutagenesis validation reveals that the high affinity of MK-97 derives from optimized engagement across all three binding regions rather than dependence on any single critical contact. Insights from comprehensive structure-activity relationship (SAR) studies provide a molecular framework for the rational design of next-generation M4 mAChR PAMs with improved pharmacological properties. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723386v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@75cb90org.highwire.dtl.DTLVardef@1876c9corg.highwire.dtl.DTLVardef@1eba8d0org.highwire.dtl.DTLVardef@9818ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

Atomic resolution cryo-EM at 200 keV

Atomic resolution in cryo-electron microscopy was first demonstrated six years ago. This was accomplished using 300 kV electron microscopes equipped with new hardware that provided narrower energy spread, aberration correction, and energy filtering. Here, we report the achievement of 1.24 [A] atomic resolution on an upgraded 200 kV electron microscope featuring a cold field emission gun, a high-resolution objective lens polepiece, and an energy filter. These components transform the instrument into a cost-effective single particle cryo-EM platform with performance comparable to that of significantly more expensive 300 kV systems. The microscope can also be operated at 100 kV and by using a high-speed hybrid-pixel detector we were able to reach sub-2 [A] resolution.

molecular biology↗

bioRxiv2025

The Hitchdock domain in Kinesin-2 Tail Enables Adaptor Assembly and Cargo Binding

Intracellular transport relies on motor proteins like kinesins to deliver essential cargoes along microtubules, yet the mechanisms of cargo recognition remain unclear. Here, we present high-resolution cryo-electron microscopy structures of the heterotrimeric kinesin-2 complex (KIF3A/KIF3B/KAP3) bound to the adenomatous polyposis coli (APC) cargo. Our findings reveal a previously uncharacterized KIF3 tail motif, termed the "Hitchdock domain," which plays a pivotal role in mediating interactions with both the KAP3 adaptor and the APC cargo. In this domain, the KIF3A helical regions facilitate specific cargo binding, while the {beta}-hairpin region and KIF3B provide structural support. Mutagenesis and molecular dynamics simulations confirm the domains functional importance. Interestingly, the Hitchdock/KAP3 structure suggests a conserved structural basis for cargo recognition across molecular motors, including kinesin-1 and dynein, which utilize similar hook-like architectures, highlighting the potential universality of this mechanism. Furthermore, our findings provide insights into kinesin-2 cargo specificity and offer a molecular framework for understanding related diseases.

cell biology↗

bioRxiv2023

Smart Parallel Automated Cryo Electron tomography

In situ cryo electron tomography enables investigation of macromolecules in their native cellular environment. Data collection, however, requires an experienced operator and valuable microscope time to carefully select targets for tilt series acquisition. Here, we developed a workflow using machine learning approaches to fully automate the entire process, including lamella detection, biological feature segmentation, target selection, and tilt series acquisition, all without the need for human intervention.

biophysics↗

bioRxiv2022

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular level understanding of the general principals that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a well-validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. Here, we present high-resolution cryo-electron microscopy structures of the M4 mAChR bound to a cognate Gi1 protein and the high affinity agonist, iperoxo, in the absence and presence of two different positive allosteric modulators, LY2033298 or VU0467154. We have also determined the structure of the M4 mAChR-Gi1 complex bound to its endogenous agonist, acetylcholine (ACh). Structural comparisons, together with molecular dynamics, mutagenesis, and pharmacological validations, have provided in-depth insights into the role of structure and dynamics in orthosteric and allosteric ligand binding, global mechanisms of receptor activation, cooperativity, probe-dependence, and species variability; all key hallmarks underpinning contemporary GPCR drug discovery.

pharmacology and toxicology↗

bioRxiv2022

Parallel cryo electron tomography on in situ lamellae

In situ cryo electron tomography of cryo focused ion beam milled samples emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. The lamella-shaped samples, however, have a limited area and are created with a necessary pretilt. This severely limits the possibilities for recording tomographic tilt series in a high-throughput manner. Here, we utilise a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining sample areas conventionally used for tracking of specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows the collection of sample areas that were previously unreachable, thus maximising the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high-throughput and high-quality of PACE-tomo.

biophysics↗

bioRxiv2021

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins: insight into mechanisms of G protein selectivity

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here we used cryo-electron microscopy to determine structures of the CCK1R bound to the CCK peptide agonist, CCK-8 and two distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-5 helix bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket was essentially equivalent when Gs was bound, with the Gs 5 helix displaying a conformation that arises from "unwinding" of the far C-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein play critical roles in the promiscuous coupling of individual GPCRs. One-Sentence SummaryCryo-EM structures of the CCK-1R reveal key mechanisms for promiscuous G protein coupling.

pharmacology and toxicology↗

bioRxiv2021

Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation

The glucagon-like peptide-1 receptor (GLP-1R) has broad physiological roles and is a validated target for treatment of metabolic disorders. Despite recent advances in GLP-1R structure elucidation, detailed mechanistic understanding of how different peptides generate profound differences in G protein-mediated signalling is still lacking. Here we have combined cryo-electron microscopy, molecular dynamics simulations, receptor mutagenesis and pharmacological assays, to interrogate the mechanism and consequences of GLP-1R binding by four peptide agonists; glucagon-like peptide-1, oxyntomodulin, exendin-4 and exendin-P5. These data revealed that distinctions in peptide N-terminal interactions and dynamics with the GLP-1R transmembrane domain are reciprocally associated with differences in the allosteric coupling to G proteins. In particular, transient interactions with residues at the base of the binding cavity correlate with enhanced kinetics for G protein activation, providing a rationale for differences in G protein-mediated signalling efficacy from distinct agonists.

biochemistry↗

bioRxiv2021

Structural and Functional Diversity among Agonist-Bound States of the GLP-1 Receptor

Recent advances in G protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multi-dimensional signal propagation mediated by these receptors is, in part, dependent on their conformational mobility. However, the relationship between receptor function and static structures determined via crystallography or cryo-electron microscopy is not always clear. This study examines the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide-1 receptor (GLP-1R), an important clinical target. We employ variants of the peptides GLP-1 and exendin-4 to explore the interplay between helical propensity near the agonist N-terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an exendin-4 analogue, the GLP-1R and Gs protein revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.

pharmacology and toxicology↗

bioRxiv2021

Structure and dynamics of semaglutide and taspoglutide bound GLP-1R-Gs complexes

The glucagon-like peptide-1 receptor (GLP-1R) regulates insulin secretion, carbohydrate metabolism and appetite, and is an important target for treatment of type II diabetes and obesity. Multiple GLP-1R agonists have entered into clinical trials, such as semaglutide, progressing to approval. Others, including taspoglutide, failed through high incidence of side-effects or insufficient efficacy. GLP-1R agonists have a broad spectrum of signalling profiles. However, molecular understanding is limited by a lack of structural information on how different GLP-1R agonists engage with the GLP-1R. In this study, we determined cryo-electron microscopy (cryo-EM) structures of GLP-1R-Gs protein complexes bound with semaglutide and taspoglutide. These revealed similar peptide binding modes to that previously observed for GLP-1. However, 3D variability analysis of the cryo-EM micrographs revealed different motions within the bound peptides and the receptor relative to when GLP-1 is bound. This work provides novel insights into the molecular determinants of peptide engagement with the GLP-1R.

biochemistry↗

bioRxiv2021

Evolving cryo-EM structural approaches for GPCR drug discovery

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in biochemical approaches for the stabilisation of GPCR:transducer complexes together with improvements in the technology and application of cryo-EM has recently opened up new possibilities for structure-assisted drug design of GPCR agonists. Nonetheless, limitations in the commercial application of some of these approaches, including the use of nanobody 35 (Nb35) for stabilisation of GPCR:Gs complexes, and the high cost of 300kV imaging have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated formation of stable complexes with a modified Gs protein in the absence of Nb35 that had equivalent resolution in the drug binding pocket to complexes solved in the presence of Nb35, while the G protein displayed increased conformational dynamics. In parallel, we assessed the performance of 200kV versus 300kV image acquisition using a Falcon 4 or K3 direct electron detector. We show that with 300kV Krios, both bottom mounted Falcon 4 and energy filtered (25eV slit) Bio-Quantum K3 produced similar resolution. Moreover, the 200kV Glacios with bottom mounted Falcon 4 yielded a 3.2 [A] map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.

biochemistry↗

bioRxiv2020

Routine sub-2.5 A cryo-EM structure determination of B-family G protein-coupled receptors

Cryo-electron microscopy (cryo-EM) experienced game-changing hardware and software advances about a decade ago. Since then, there have been gradual and steady improvements in experimental and data analysis methods. Nonetheless, structural analysis of nonsymmetric membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Their relatively low molecular weight and obstruction by the micelle/nanodisc result in marginal signal levels, which combined with the intrinsic flexibility of such complexes creates difficult structural study scenarios. Pushing the performance limits of cryo-EM requires careful optimization of all experimental aspects. To this end, it is necessary to build quantitative knowledge of the effect each parameter has on the outcome. Here, we present in-depth analysis of the influence of the main cryo-EM experimental factors on the performance for GPCR structure determination. We used a tandem experiment approach that combined real-world structural studies with parameter testing. We quantified the effects of using a Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. Through such systematic optimization of the experimental conditions, it has been possible to routinely determine class B1 GPCR structures at resolutions better than 2.5 [A]. The improved fidelity of such maps helps to build higher confidence atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines drafted here are not limited to GPCRs and can be applied directly for the study of other challenging membrane protein targets.

biophysics↗

bioRxiv2020

Differential GLP-1R binding and activation by peptide and non-peptide agonists

Peptide drugs targeting class B1 GPCRs can treat multiple diseases, however there remains substantial interest in the development of orally delivered non-peptide drugs. Here we reveal unexpected overlap between signalling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist, PF 06882961, and GLP-1 that was not observed for another compound, OWL-833. Both compounds are currently in clinical trials for treatment of type 2 diabetes. High resolution cryo-EM structures reveal the binding sites for PF-06882961 and GLP-1 substantially overlap, whereas OWL-833 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not OWL-833, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.

pharmacology and toxicology↗

bioRxiv2020

Spectral DQE of the Volta Phase Plate

The Volta Phase Plate (VPP) consists of a heated, thin film that is placed in the same plane as the focused diffraction pattern of an electron microscope. A change in surface potential develops at the point irradiated by the intense, unscattered electron beam, and this altered surface potential produces, in turn, a phase shift between the unscattered and scattered parts of the electron wave. While the VPP thus increases the image contrast for weak-phase objects at low spatial frequencies, we report here that it also leads to the loss of an increasing fraction of the signal at higher resolution. The approximately linear dependence (with increasing resolution) of this loss has been quantified at 200 kV and 300 kV, using evaporated-carbon films of different thicknesses as Volta phase plates. In all cases, the loss of signal remains almost independent of variation of the conditions and parameters that were tested. In spite of having done a number or additional, discovery-based experiments, the cause of this loss of signal remains unexplained at this point.

bioengineering↗

bioRxiv2019

Fast and accurate defocus for improved tunability of cryo-EM experiments

Current data collection strategies in electron cryo-microscopy (cryo-EM) record multiframe movies with static optical settings. This limits the number of adjustable parameters that can be used to optimize the experiment. Here, we propose a method for fast and accurate defocus (FADE) modulation during movie acquisition. It uses the objective lens aperture as an electrostatic pole that locally modifies the electron beam potential. The beam potential variation is converted to defocus modulation by the typically undesired chromatic aberration of the objective lens. The simplicity, electrostatic principle and low electrical impedance of the device will allow fast switching speeds that will enable per-frame defocus values in cryo-EM movies. Researchers will be able to define custom defocus "recipes" and tailor the experiment for optimal information extraction from their sample. The FADE method could help to convert the microscope into a more dynamic and flexible optical platform that delivers better performance in cryo-EM single particle analysis and electron cryo-tomography.

biophysics↗

bioRxiv2016

Cryo-EM structure of haemoglobin at 3.2 A determined with the Volta phase plate

With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way1. These cameras are superior to previous detectors in coping with the intrinsically low contrast of radiation-sensitive organic materials embedded in amorphous ice, and so they have enabled the structure determination of several macromolecular assemblies to atomic or near-atomic resolution. According to one theoretical estimation, a few thousand images should suffice for calculating the structure of proteins as small as 17 kDa at 3 [A] resolution2. In practice, however, we are still far away from this theoretical ideal. Thus far, protein complexes that have been successfully reconstructed to high-resolution by single particle analysis (SPA) have molecular weights of ~100 kDa or larger3. Here, we report the use of Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 [A]. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination.

biophysics↗

bioRxiv2016

Using the Volta phase plate with defocus for cryo-EM single particle analysis

Previously, we reported an in-focus data acquisition method for cryo-EM single particle analysis with the Volta phase plate (VPP) (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also enables 3D reconstructions with resolutions in the 2 [A] range, demonstrating that, in practice, there are no resolution limitations imposed by the VPP.

biophysics↗