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The autosomal dominant monogenetic disease neurofibromatosis type 1 (NF1) affects approximately one in 3,000 individuals and is caused by mutations in the NF1 tumour suppressor gene, leading to dysfunction in the protein neurofibromin (Nf1)1,2. As a GTPase-activating protein, a key function of Nf1 is repression of the Ras oncogene signalling cascade. We determined the human Nf1 dimer structure at an overall resolution of 3.3 Å. The cryo-electron microscopy structure reveals domain organization and structural details of the Nf1 exon 23a splicing3 isoform 2 in a closed, self-inhibited, Zn-stabilized state and an open state. In the closed conformation, HEAT/ARM core domains shield the GTPase-activating protein-related domain (GRD) so that Ras binding is sterically inhibited. In a distinctly different, open conformation of one protomer, a large-scale movement of the GRD occurs, which is necessary to access Ras, whereas Sec14-PH reorients to allow interaction with the cellular membrane4. Zn incubation of Nf1 leads to reduced Ras-GAP activity with both protomers in the self-inhibited, closed conformation stabilized by a Zn binding site between the N-HEAT/ARM domain and the GRD–Sec14-PH linker. The transition between closed, self-inhibited states of Nf1 and open states provides guidance for targeted studies deciphering the complex molecular mechanism behind the widespread neurofibromatosis syndrome and Nf1 dysfunction in carcinogenesis., Cryo-EM structure of Nf1 protein is reported, revealing closed and open conformations that regulate interaction with Ras oncogene, setting the stage for understanding the mechanistic action of Nf1 and how disease mutations lead to dysfunction.

NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochromec-dependent nitric oxide reductase (cNOR).cNOR catalyzes the NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeBinsertion, and the general mechanism of the MoxR/VWA interacting protein families. We show that NorQ-catalyzed ATP hydrolysis, an intact VWA-domain in NorD and specific surface carboxylates oncNOR are all features required forcNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that ‘plugs’ the NorQ ring pore to the VWA domain of NorD with a protruding ‘finger’ inserting through the pore, and suggest this binding mode to be general for MoxR/VWA couples. We present a tentative model for the mechanism of NorQD-catalyzedcNOR remodelling and suggest many of its features to be applicable to the whole MoxR/VWA family.

An overview of the Scipion plugin for the RELION software package is presented and various image-processing capabilities within the Scipion framework are discussed., Scipion is a modular image-processing framework that integrates several software packages under a unified interface while taking care of file formats and conversions. Here, new developments and capabilities of the Scipion plugin for the widely used RELION software package are presented and illustrated with an image-processing pipeline for published data. The user interfaces of Scipion and RELION are compared and the key differences are highlighted, allowing this manuscript to be used as a guide for both new and experienced users of this software. Different on-the-fly image-processing options are also discussed, demonstrating the flexibility of the Scipion framework.

Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here Single Particle Automated Raman Trapping Analysis (SPARTA) is combined with small-angle X-ray and neutron scattering (SAXS/SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP-phospholipase D (PLD) interactions. This analysis demonstrates that PLD, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate is an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated.

Glycoprotein 2 (GP2) and uromodulin (UMOD) filaments protect against gastrointestinal and urinary tract infections by acting as decoys for bacterial fimbrial lectin FimH. By combining AlphaFold2 predictions with X-ray crystallography and cryo-EM, we show that these proteins contain a bipartite decoy module whose new fold presents the high-mannose glycan recognized by FimH. The structure rationalizes UMOD mutations associated with kidney diseases and visualizes a key epitope implicated in cast nephropathy. Ministry of Health (MOH) National Medical Research Council (NMRC) Published version This work was supported by the Swedish Research Council (project grants 2016-03999 and 2020-04936 to L.J.), the Karolinska Institutet Research Foundation (grant 2016fobi50035 to L.J.), the Knut and Alice Wallenberg Foundation (project grant 2018.0042 to L.J.) and the Ministry of Health, Singapore, NMRC grant (MOH-000382-00 to B.W.). Open access funding provided by Karolinska Institute.

The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1–ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity., Journal of Biological Chemistry, 298 (8), ISSN:0021-9258, ISSN:1083-351X

Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate carbon hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal concentrations. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED., Micro-crystal electron diffraction (MicroED) has shown great potential for the structure determination of crystals that are too small for X-ray diffraction but MicroED sample preparation remains challenging. Here, the authors present Preassis, a pressure-assisted method for the preparation of MicroED specimens and demonstrate that Preassis can be applied to a wide range of protein crystal suspensions with low and high viscosities, as well as those with low crystal concentrations.

Cryo-EM structures and molecular simulations of a pentameric ligand-gated ion channel, GLIC, in resting and activating conditions implicate protonation sites and altered dynamics in sequential gating., Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1–β2 and M2–M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

Scipion is a modular image processing framework integrating several software packages under a unified interface while taking care of file formats and conversions. Here new developments and capabilities of the Scipion plugin for the Relion software are presented and illustrated with the image processing pipeline of published data. The user interfaces of Scipion and Relion are compared and the key differences highlighted, allowing this manuscript to be used as a guide for both new and experienced users of these software. Different streaming image processing options are also discussed demonstrating the flexibility of the Scipion framework.SynopsisAn overview of the Scipion plugin for the Relion software is presented and various capabilities of image processing within Scipion framework are discussed.

Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo‐electron microscopy study of uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant protein in human urine and an archetypal ZP module‐containing molecule, in its mature homopolymeric state. UMOD forms a one‐start helix with an unprecedented 180‐degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD‐based models of heteromeric vertebrate egg coat filaments identify a common sperm‐binding region at the interface between subunits., Insights into the architecture of uromodulin filaments involved in the capture of uropathogenic bacteria, and structurally‐related vertebrate egg coat material, suggest how a widespread extracellular polymerization module can support multiple functions.

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological development in this family relies on high-quality structural data in multiple, subtly distinct functional states. However, structural data remain limited, particularly for the unliganded or resting state. Here we report cryo-electron microscopy structures of the Gloeobacter violaceus ligand-gated ion channel (GLIC) under resting and activating conditions (neutral and low pH). Parallel models were built either manually or using recently developed density-guided molecular simulations. The moderate resolution of resting-state reconstructions, particularly in the extracellular domain, was improved under activating conditions, enabling the visualization of residues at key subunit interfaces including loops B, C, F, and M2–M3. Combined with molecular dynamics simulations, the cryo-electron microscopy structures at different pH describe a heterogeneous population of closed channels, with activating conditions condensing the closed-channel energy landscape on a pathway towards gating.

Density guided simulations files .mrc files - CryoEM density half-maps for pH 7, pH 5, pH 3 .mdp - Option files for GROMACS simulations. One density guided mdp file and one annealing mdp file per pH data set .gro - Equilibrated starting model of manually built pH 7,the same was used for all three pH data sets .itp - topology files .top - topology master file Example command for density guided simulations gmx grompp --f mdrun_ip_ph3_tc40_sp02_tau0_3.mdp --p topology.top --o mdrun_ip_ph3_tc40_sp02_tau0_3.tpr --c minimized.gro gmx mdrun --s mdrun_ip_ph3_tc40_sp02_tau0_3_test.tpr --v --x mdrun.xtc --g md.log --e ener.edr --c final_ip_ph3_tc40_sp02_tau0_3_test.gro Example command for custom GROMACS FSC tool, available at (https://github.com/blauc/gromacs/tree/fscavg) gromacs-fsc/bin/gmx fsc -f whole.xtc -s mdrun_ip_ph3_tc40_sp02_tau0_3_test.tpr -refmap pH3_half1_class001_unfil.mrc-dt 100 -xvg none -shells 186 -o fsc_ip_ph3_tc40_sp02_tau0_3.xvg -similarity similarity_ip_ph3_tc40_sp02_tau0_3.xvg -avg fscavg_ip_ph3_tc40_sp02_tau0_3.xvg -fscmove fscmove_ip_ph3_tc40_sp02_tau0_3.xvg -ordinate-axis fscaxis_ip_ph3_tc40_sp02_tau0_3.xvg -fscmoveavg fscmoveavg_ip_ph3_tc40_sp02_tau0_3.xvg Molecular dynamics simulations .prep_file -topology files and forcefield files for preparing the simulations. .run_tpr -tpr files for production runs .prodution_traj -concatenated trajectories and starting snapshots (after equilibration). analysis_results - csv: analaysis data - ipynb: jupyter notebook for generating the figures in the paper.

SUMMARYAssembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to human, no information is available on the filamentous conformation of any ZP module protein. Here we report the cryo-electron microscopy structure of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of mammalian and avian heteromeric egg coat filaments identify a common sperm-binding region at the interface between subunits.

Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD–PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach — combining cryo-EM, X-ray crystallography, protein–protein interaction measurements, and activity assays — we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication., Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. Here, the authors present a cryo-EM structure of the DNA-bound PolD–PCNA complex from Pyrococcus abyssi to reveal the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA.

SummaryReplicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex fromPyrococcus abyssiat 3.77Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography and protein-protein interaction measurements - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA with an unprecedented level of detail. PolD recruits PCNAviaa complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation phase and a processive phase during replication.

Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal contents. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED.

Bacteriophage PR772, a member of theTectiviridaefamily, has a 70-nm diameter icosahedral protein capsid that encapsulates a lipid membrane, dsDNA, and various internal proteins. An icosahedrally averaged CryoEM reconstruction of the wild-type virion and a localized reconstruction of the vertex region reveals the composition and the structure of the vertex complex along with new protein conformations that play a vital role in maintaining the capsid architecture of the virion. The overall resolution of the virion is 2.75 Å, while the resolution of the protein capsid is 2.3 Å. The conventional penta-symmetron formed by the capsomeres is replaced by a large vertex complex in the pseudo T=25 capsid. All the vertices contain the host-recognition protein, P5; two of these vertices show the presence of the receptor-binding protein, P2. The 3D structure of the vertex complex shows interactions with the viral membrane, indicating a possible mechanism for viral infection.

The E.coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Δloop ribosomes, while two ~100-residue protein normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Δloop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

Respiratory chain complexes execute energy conversion by connecting electron transport with proton translocation over the inner mitochondrial membrane to fuel ATP synthesis. Notably, these complexes form multi-enzyme assemblies known as respiratory supercomplexes. Here we used single-particle cryo-EM to determine the structures of the yeast mitochondrial respiratory supercomplexes III

Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevín et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world., Spanish Ministry of Economy and Competitiveness through Grants BIO2016-76400R(AEI/FEDER, UE) and AEI/FEDER BFU 2016 74868P, the Comunidad Autónoma de Madrid through Grant: S2017/BMD-3817, European Union (EU) and Horizon 2020 through grant Corbel (INFRADEV-1-2014-1, Proposal: 654248). The “Knut & Alice Wallenberg Foundation”, and “A Pilot Facility development grant from Science for Life Laboratory”. European Union (EU) and Horizon 2020 through grant EOSCpilot (INFRADEV-04-2016, Proposal: 739563).This work used the EGI Infrastructure and is co-funded by the EGIEngage project (Horizon 2020) under Grant No. 654142. European Union (EU) and Horizon 2020 through grant West-Life (EINFRA-2015-1, Proposal: 675858) European Union (EU) and Horizon 2020 through grant Elixir - EXCELERATE (INFRADEV-3-2015, Proposal: 676559) European Union (EU) and Horizon 2020 through grant iNEXT (INFRAIA-1-2014-2015, Proposal: 653706).

Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson’s disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.

GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM. This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble. Reconstruction of the double-tetrameric form, combined with small-angle X-ray scattering data, allows the localisation of the B domain of the Psf1 subunit in the free GINS complex, which was not visible in previous studies and is essential for the formation of a functional replication fork.

ATP-driven bacterial AAA+ proteases have been recognized as drug targets. They possess an AAA+ protein (e.g., ClpC), which threads substrate proteins into an associated peptidase (e.g., ClpP). ATPase activity and substrate selection of AAA+ proteins are regulated by adapter proteins that bind to regulatory domains, such as the N-terminal domain (NTD). The antibacterial peptide Cyclomarin A (CymA) kills Mycobacterium tuberculosis cells by binding to the NTD of ClpC. How CymA affects ClpC function is unknown. Here, we reveal the mechanism of CymA-induced toxicity. We engineered a CymA-sensitized ClpC chimera and show that CymA activates ATPase and proteolytic activities. CymA mimics adapter binding and enables autonomous protein degradation by ClpC/ClpP with relaxed substrate selectivity. We reconstitute CymA toxicity in E. coli cells expressing engineered ClpC and ClpP, demonstrating that gain of uncontrolled proteolytic activity causes cell death. This validates drug-induced overriding of AAA+ protease activity control as effective antibacterial strategy.

Highlights • Structural biology. • Cryo electron microscopy. • Macromolecular complexes. • Single particle analysis., Cryo-electron microscopy (cryo-EM) is a structural molecular and cellular biology technique that has experienced major advances in recent years. Technological developments in image recording as well as in processing software make it possible to obtain three-dimensional reconstructions of macromolecular assemblies at near-atomic resolution that were formerly obtained only by X-ray crystallography or NMR spectroscopy. In parallel, cryo-electron tomography has also benefitted from these technological advances, so that visualization of irregular complexes, organelles or whole cells with their molecular machines in situ has reached subnanometre resolution. Cryo-EM can therefore address a broad range of biological questions. The aim of this review is to provide a brief overview of the principles and current state of the cryo-EM field.