Biology subjects

McCammon, J. A.

Publications and source records attributed to McCammon, J. A..

At least 19 recordsLinked to original sources

bioRxiv2025

Constitutive activation and allosteric mechanisms underlying Gi/Gq signaling bias in SUCNR1 revealed by AlphaFold-based modeling and enhanced sampling simulations

Succinate Receptor 1 (SUCNR1) is a key metabolite sensing GPCR activated by high local succinate concentrations through Gi and Gq signaling pathways. By combining AlphaFold-based modeling, Gaussian accelerated molecular dynamics simulations (72s in total), receptor mutagenesis, and signaling assays, we here investigate the spontaneous and G-protein binding-associated activation mechanism of SUCNR1. Our molecular simulations predict that, in the absence of G protein or ligand, human SUCNR1 (hSUCNR1), in contrast to murine SUCNR1 (mSUCNR1), adopts canonical active- like conformations in its intracellular domains and undergoes critical conformational changes in its extracellular domain. This finding is consistent with our signaling-assay results showing that hSUCNR1, unlike mSUCNR1, displays a high degree of constitutive signaling. When complexed with either Gi or Gq, both h- and mSUCNR1 adopt highly energetically favorable conformations in the extracellular domain, characterized by the unlocking of extracellular loop 2b (ECL2b) and an expanded ligand entry pathway. Interestingly, the simulations reveal that helix 5 of Gq binds less firmly to the TM3/6 cleft of mSUCNR1, resulting in an unstable extracellular domain of the Gq- mSUCNR1 complex, which however can be stabilized by agonist binding. This result is supported by our BRET assays, which show that, in contrast to hSUCNR1, mSUCNR1 fails to recruit mini-Gq to the cell surface in the absence of an agonist. Furthermore, the signaling effects of key receptor residues at G-protein binding site and within ECL2b, as predicted by our simulations, were confirmed by mutagenesis assays. Overall, our integrative approach demonstrates that hSUCNR1 can spontaneously adopt all key conformations associated with receptor activation, and that G protein binding further primes the extracellular receptor domain for agonist binding, which in turn stabilizes the active complex.

biophysics↗

bioRxiv2025

Markovian State Models uncover Casein Kinase 1 dynamics that govern circadian period

Circadian rhythms in mammals are tightly regulated through phosphorylation of Period (PER) proteins by Casein Kinase 1 (CK1, subtypes {delta} and {varepsilon}). CK1 acts on at least two different regions of PER with opposing effects: phosphorylation of phosphodegron (pD) regions leads to PER degradation, while phosphorylation of the Familial Advanced Sleep Phase (FASP) region leads to PER stabilization. To investigate how substrate selectivity is encoded by the conformational dynamics of CK1, we performed a large set of independent molecular dynamics (MD) simulations of wildtype CK1 and the tau mutant (R178C) that biases kinase activity toward a pD. We used Markovian State Models (MSMs) to integrate the simulations into a single model of the conformational landscape of CK1 and used Gaussian accelerated molecular dynamics (GaMD) to build the first molecular model of CK1 and the unphosphorylated FASP motif. Together, these findings provide a mechanistic view of CK1, establishing how the activation loop acts as a key molecular switch to control substrate selectivity. We show that the tau mutant favors an alternative conformation of the activation loop and significantly accelerates the dynamics of CK1. This reshapes the binding cleft in a way that impairs FASP binding and would ultimately lead to PER destabilization and shorter circadian periods. Finally, we identified an allosteric pocket that could be targeted to bias this molecular switch. Our integrated approach offers a detailed model of CK1s conformational landscape and its relevance to normal, mutant, and druggable circadian timekeeping. Statement of SignificanceDisruption of circadian rhythms alters the temporal orchestration of vital cellular processes and increases the propensity for sleep disorders, metabolic disease, and cancer. Circadian rhythms are generated by a vast gene expression program controlled at the cellular level by a molecular clock comprised of dedicated clock proteins. Amongst the essential protein characters is Casein kinase 1 (CK1), which acts on multiple clock protein substrates. A delicate balance of CK1 activity on these substrates is crucial for proper circadian timekeeping, highlighting CK1 as a promising drug target to tune clock timing. This work aims to identify the conformational landscape of CK1 that underlies its substrate specificity and provide molecular insight for pharmacologic development that could modulate CK1 function for those suffering from clock-related syndromes.

biophysics↗

bioRxiv2024

Targeting Tuberculosis: Novel Scaffolds for Inhibiting Cytochrome bd Oxidase

Discovered in the 1920s, cytochrome bd is a terminal oxidase that has received renewed attention as a drug target since its atomic structure was first solved in 2016. Only found in prokaryotes, we study it here as a drug target for Mycobacterium tuberculosis (Mtb). Most previous drug discovery efforts towards cytochrome bd have involved analogs of the canonical substrate quinone, known as Aurachin D. Here we report six new cytochrome bd inhibitor scaffolds determined from a computational screen totaling over one million molecules and confirmed on target activity through in vitro testing. These scaffolds provide new avenues for lead optimization towards Mtb therapeutics.

biochemistry↗

bioRxiv2023

Dilated cardiomyopathy mutation in beta-cardiac myosin enhances actin activation of the power stroke and phosphate release

Inherited mutations in human beta-cardiac myosin (M2{beta}) can lead to severe forms of heart failure. The E525K mutation in M2{beta} is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2{beta} subfragment 1 (S1) we found that E525K enhances (3-fold) the maximum steady-state actin-activated ATPase activity (kcat) and decreases (6-fold) the actin concentration at which ATPase is one-half maximal (KATPase). We also found a 3 to 4-fold increase in the actin-activated power stroke and phosphate release rate constants at 30 M actin, which overall enhanced the duty ratio 3-fold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2{beta} S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt-bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt-bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis. Significance StatementHeart disease can be caused by inherited mutations in beta-cardiac myosin, the molecular motor that powers systolic contraction in the ventricles of the heart. However, it remains unclear how these mutations lead to contractile dysfunction and pathogenic remodeling of the heart. We investigated a unique dilated cardiomyopathy mutation (E525K) that dramatically stabilizes the autoinhibited state while enhancing intrinsic motor function. Thus, we examined how this mutation impacts transient kinetic steps of the ATPase cycle, motile properties, and structural changes associated with the power stroke and phosphate release. Our results provide a kinetic and structural basis for how beta-cardiac myosin mutations may disrupt molecular-level contractile function in complex ways, which may inform the development of targeted therapeutics.

biophysics↗

bioRxiv2023

Forward Genetic Stomatal CO2 Response Screen in Grass Brachypodium distachyon Reveals Central Function of a MAP Kinase in Early Stomatal CO2 Signal Transduction

Plants respond to increased CO2 concentrations through rapid stomatal closure which can contribute to increased water use efficiency. Grasses display faster stomatal responses than eudicots due to dumbbell-shaped guard cells flanked by subsidiary cells working in opposition. However, forward genetic screening for stomatal CO2 signal transduction mutants in grasses has not been reported. The grass model Brachypodium distachyon is closely related to agronomically important cereal crops, sharing largely collinear genomes. To gain insights into CO2 control mechanisms of stomatal movements in grasses, we developed a forward genetics screen with an EMS-mutagenized Brachypodium distachyon M5 generation population using infrared imaging to identify plants with altered canopy leaf temperature at elevated CO2. Among isolated mutants, a "chill1" mutant exhibited cooler leaf temperatures than wildtype Bd21-3 parent control plants after exposure to increased [CO2]. chill1 plants showed strongly impaired high CO2-induced stomatal closure, despite retaining a robust abscisic acid-induced stomatal closing response. Through bulked segregant whole-genome-sequencing analyses followed by analyses of further backcrossed F4 generation plants and generation and characterization of CRISPR-cas9 mutants, chill1 was mapped to a protein kinase, BdMPK5. The chill1 mutation impaired BdMPK5 protein-mediated CO2/HCO3- sensing in vitro. Furthermore, AlphaFold2-directed structural modeling suggests that the identified BdMPK5-D90N chill1 mutant residue is located at the interface with the HT1 Raf-like kinase. BdMPK5 is a key signaling component involved in CO2-induced stomatal movements, potentially functioning as a component of the CO2 sensor in grasses.

plant biology↗

bioRxiv2023

Glycosylation and Crowded Membrane Effects on Influenza Neuraminidase Stability and Dynamics

All protein simulations are conducted with varying degrees of simplifications, oftentimes with unknown ramifications on how these simplifications affect the interpretability of the results. In this work we investigated how protein glycosylation and lateral crowding effects modulate an array of properties characterizing the stability and dynamics of influenza neuraminidase. We constructed three systems: 1) Glycosylated neuraminidase in a whole virion (i.e. crowded membrane) environment 2) Glycosylated neuraminidase in its own lipid bilayer 3) Unglycosylated neuraminidase in its own lipid bilayer. We saw that glycans tend to stabilize the protein structure and reduce its conformational flexibility while restricting solvent movement. Conversely, a crowded membrane environment encouraged exploration of the free energy landscape and a large scale conformational change while making the protein structure more compact. Understanding these effects informs what factors one must consider while attempting to recapture the desired level of physical accuracy. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/556910v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@ec7a9org.highwire.dtl.DTLVardef@563495org.highwire.dtl.DTLVardef@1965cd7org.highwire.dtl.DTLVardef@a9659d_HPS_FORMAT_FIGEXP M_FIG TOC. Schematic of the three systems used here. (A) 2009-H1N1-ungly is the unglycosylated, single protein NA system. (B) 2009-H1N1-gly is the glycosylated, single protein NA system. (C) 2009-H1N1-vir is the NA in the virion context. C_FIG

biophysics↗

bioRxiv2022

Capturing differences in the regulation of LRRK2 dynamics and conformational states by small molecule kinase inhibitors

Mutations in the human leucine rich repeat protein kinase-2 (LRRK2) create risk factors for Parkinsons Disease, and pathological functions of LRRK2 are often correlated with aberrant kinase activity. Past research has focused on developing selective LRRK2 kinase inhibitors. We showed previously that in addition to influencing intrinsic kinase activity, the global conformation of the LRRK2 protein plays a vital role in regulating LRRK2 signaling pathways. Deciphering the allosteric regulation in LRRK2 provides novel strategies for drug discovery. In this study, we combined enhanced sampling simulations with HDX-MS to analyze the inhibitor-induced dynamic changes and the allosteric communications in the C-terminal half of LRRK2, LRRK2RCKW. We find that a type I inhibitor (MLi-2) locks the kinase into a closed, active-like configuration, whereas a type II inhibitor (Rebastinib) shifts the kinase to an open, inactive configuration. While both type I and type II inhibitors reduce the kinase activity effectively, they have distinct effects on the LRRK2 conformational dynamics. Specifically, binding of MLi-2 stabilizes the kinase domain in a closed conformation and reduces the global dynamics of LRRK2RCKW, leading to a more compact LRRK2RCKW structure. In contrast, binding of Rebastinib stabilizes an open conformation where communication between the N- and C-lobe is severed, which promotes a more extended LRRK2RCKW structure. Rebastinib, based on HDX-MS, creates a more dynamic kinase domain especially at domain interfaces associated with the C-lobe. Our results also reveal the importance of the Dk-helix, which plays a crucial role in propagating communication between the kinase domain and the GTPase domain.

biochemistry↗

bioRxiv2022

Integrating comparative modeling and accelerated simulations reveals conformational and energetic basis of actomyosin force generation

Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin-actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structurefunction relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin-actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the pre-powerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions. Significance StatementInteractions between myosin and actin are essential in producing various cellular forces. Targeting cardiac myosin, several small molecules have been developed to treat cardiomyopathy. A clear mechanistic picture for the allosteric control in the actomyosin complex can potentially facilitate drug design by uncovering functionally important intermediate states. Here, integrating Rosetta comparative modeling and accelerated molecular dynamics, we reveal how ATP-hydrolysis product release correlates with powerstroke and myosin tight binding to actin. The predicted metastable states and corresponding energetics complement available experimental data and provide insights into the timing of elementary mechanochemical events. Our method establishes a framework to characterize at an atomistic level how a molecular motor translocates along a filament.

biophysics↗

bioRxiv2022

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

PERIOD (PER) and Casein Kinase 1{delta} regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1{delta} phosphorylation of the Familial Advanced Sleep Phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1{delta}. Co-crystal structures in conjunction with accelerated molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1{delta}. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortens circadian period in human cells. We found that Drosophila PER also regulates CK1{delta} via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

biochemistry↗

bioRxiv2022

Architecture and self-assembly of the jumbo bacteriophage nuclear shell

Bacteria encode myriad defenses that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR/Cas systems. In response, one family of large bacteriophage employs a nucleus-like compartment to protect their replicating genomes by excluding host defense factors. However, the principle composition and structure of this compartment remain unknown. Here, we find that the bacteriophage nuclear shell assembles primarily from one protein, termed chimallin. Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin cooperatively self-assembles as a flexible sheet into closed micron-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

biophysics↗

bioRxiv2021

A glycan gate controls opening of the SARS-CoV-2 spike protein

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded "down" to an exposed "up" state in order to bind the human ACE2 receptor and infect cells. While snapshots of the "up" and "down" states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 s of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with ManifoldEM analysis of cryo-EM data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

biophysics↗

bioRxiv2020

Multiscale simulations examining glycan shield effects on drug binding to influenza neuraminidase

Influenza neuraminidase is an important drug target. Glycans are present on neuraminidase, and are generally considered to inhibit antibody binding via their glycan shield. In this work we studied the effect of glycans on the binding kinetics of antiviral drugs to the influenza neuraminidase. We created all-atom in silico systems of influenza neuraminidase with experimentally-derived glycoprofiles consisting of four systems with different glycan conformations and one system without glycans. Using Brownian dynamics simulations, we observe a two- to eight-fold decrease in the rate of ligand binding to the primary binding site of neuraminidase due to the presence of glycans. These glycans are capable of covering much of the surface area of neuraminidase, and the ligand binding inhibition is derived from glycans sterically occluding the primary binding site on a neighboring monomer. Our work also indicates that drugs preferentially bind to the primary binding site (i.e. the active site) over the secondary binding site, and we propose a binding mechanism illustrating this. These results help illuminate the complex interplay between glycans and ligand binding on the influenza membrane protein neuraminidase. Statement of SignificanceThe influenza glycoprotein neuraminidase is the target for three FDA-approved influenza drugs in the US. However, drug resistance and low drug effectiveness merits further drug development towards neuraminidase, which is hindered by our limited understanding of glycan effects on ligand binding. Generally, drug developers do not include glycans in their development pipelines. Here, we show that even though glycans can reduce drug binding towards neuraminidase, we recommend future drug development work to focus on strong binders with a long lifetime. Furthermore, we examine the binding competition between the primary and secondary binding sites on neuraminidase, leading us to propose a new, to the best of our knowledge, multivalent binding mechanism.

biophysics↗

bioRxiv2020

Elucidation of cryptic and allosteric pockets within the SARS-CoV-2 protease

The SARS-CoV-2 pandemic has rapidly spread across the globe, posing an urgent health concern. Many quests to computationally identify treatments against the virus rely on in silico small molecule docking to experimentally determined structures of viral proteins. One limit to these approaches is that protein dynamics are often unaccounted for, leading to overlooking transient, druggable conformational states. Using Gaussian accelerated molecular dynamics to enhance sampling of conformational space, we identified cryptic pockets within the SARS-CoV-2 main protease, including some within regions far from the active site and assed their druggability. These pockets can aid in virtual screening efforts to identify a protease inhibitor for the treatment of COVID-19. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2020

Structural and Dynamic Basis of Molecular Recognition between Acyltransferase and Carrier Protein in E. coli Fatty Acid Synthesis

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the first x-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity. Significance StatementThe essential role of acyltransferases (ATs) in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways, namely the selection and loading of starter and extender units onto acyl carrier proteins (ACPs), relies on catalytically productive ACP-AT interactions. Here, we describe and interrogate the first structure of a type II FAS malonyl-CoA:ACP-transacylase (MAT) in covalent complex with its cognate ACP. We combine structural, mutational, crosslinking and kinetic data with molecular dynamics simulations to describe a highly flexible and robust protein-protein interface, substrate-induced active site reorganization, and key subdomain motions that likely govern FAS function. These findings strengthen a mechanistic understanding of molecular recognitions between ACPs and partner enzymes and provide new insights for engineering AT-dependent biosynthetic pathways.

biochemistry↗

bioRxiv2019

An Open Source Mesh Generation Platform for Biophysical Modeling Using Realistic Cellular Geometries

Advances in imaging methods such as electron microscopy, tomography, and other modalities are enabling high-resolution reconstructions of cellular and organelle geometries. Such advances pave the way for using these geometries for biophysical and mathematical modeling once these data can be represented as a geometric mesh, which, when carefully conditioned, enables the discretization and solution of partial differential equations. In this study, we outline the steps for a naive user to approach GAMer 2, a mesh generation code written in C++ designed to convert structural datasets to realistic geometric meshes, while preserving the underlying shapes. We present two example cases, 1) mesh generation at the subcellular scale as informed by electron tomography, and 2) meshing a protein with structure from x-ray crystallography. We further demonstrate that the meshes generated by GAMer are suitable for use with numerical methods. Together, this collection of libraries and tools simplifies the process of constructing realistic geometric meshes from structural biology data. SIGNIFICANCEAs biophysical structure determination methods improve, the rate of new structural data is increasing. New methods that allow the interpretation, analysis, and reuse of such structural information will thus take on commensurate importance. In particular, geometric meshes, such as those commonly used in graphics and mathematics, can enable a myriad of mathematical analysis. In this work, we describe GAMer 2, a mesh generation library designed for biological datasets. Using GAMer 2 and associated tools PyGAMer and BlendGAMer, biologists can robustly generate computer and algorithm friendly geometric mesh representations informed by structural biology data. We expect that GAMer 2 will be a valuable tool to bring realistic geometries to biophysical models.

biophysics↗

bioRxiv2019

Gating Mechanism of β-Ketoacyl-ACP Synthases

Formation of carbon-carbon bonds via {beta}-ketoacyl-acyl carrier protein (ACP) synthases (KS), are key reactions during de novo fatty acid and polyketide biosynthesis. KSs recognize multiple ACPs and choreograph ping-pong mechanisms often in an iterative fashion. Therefore, KSs must limit non-productive protein-protein interactions (PPIs) to achieve high degrees of reaction fidelity. To better understand the stereochemical features governing substrate discrimination during ACP*KS PPIs, we determined x-ray crystal structures complemented by molecular dynamic simulations of E. coli AcpP in complex with the elongating KSs, FabF and FabB. Covalently trapped substrate analogs were used to interrogate critical catalytic events accompanying carbon-carbon bond formation revealing a previously unknown gating mechanism during the binding and delivery of acyl-AcpPs. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features generally in elongating KSs.

biochemistry↗

bioRxiv2019

GAMer 2: A System for 3D Mesh Processing of Cellular Electron Micrographs

ObjectiveRecent advances in electron microscopy have, for the first time, enabled imaging of single cells in 3D at a nanometer length scale resolution. An uncharted frontier for in silico biology is the ability to simulate cellular processes using these observed geometries. However, this will require a system for going from EM images to 3D volume meshes which can be used in finite element simulations. MethodsIn this paper, we develop an end-to-end pipeline for this task by adapting and extending computer graphics mesh processing and smoothing algorithms. Our workflow makes use of our recently rewritten mesh processing software, GAMer 2, which implements several mesh conditioning algorithms and serves as a platform to connect different pipeline steps. ResultsWe apply this pipeline to a series of electron micrographs of dendrite morphology explored at three different length scales and show that the resultant meshes are suitable for finite element simulations. ConclusionOur pipeline, which consists of free and open-source community driven tools, is a step towards routine physical simulations of biological processes in realistic geometries. SignificanceWe posit that a new frontier at the intersection of computational technologies and single cell biology is now open. Innovations in algorithms to reconstruct and simulate cellular length scale phenomena based on emerging structural data will enable realistic physical models and advance discovery.

systems biology↗

bioRxiv2018

Molecular mechanism of off-target effects in CRISPR-Cas9

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations - using Gaussian accelerated MD (GaMD) - are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a \"lock\" effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to \"lock\" the HNH domain, thereby identifying the ability to \"lock\" HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to \"trap\" somehow the HNH domain in an inactive \"conformational checkpoint\" state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the \"locking\" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

biophysics↗