bims-strubi Biomed News
on Advances in structural biology
Issue of 2021–11–28
nineteen papers selected by
Alessandro Grinzato, European Synchrotron Radiation Facility
  1. Molecular structure of an open human KATP channel.
  2. Combining Cryo-EM Density Map and Residue Contact for Protein Secondary Structure Topologies.
  3. Structure of Mycobacterium tuberculosis cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates.
  4. High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism.
  5. Structure of the translating Neurospora ribosome arrested by cycloheximide.
  6. High-resolution single-particle cryo-EM of samples vitrified in boiling nitro-gen.
  7. Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy.
  8. Crystal structure of the potato leafroll virus coat protein and implications for viral assembly.
  9. High-resolution structure of native toxin A from Clostridioides difficile.
  10. Cryo-EM structures of intermediates suggest an alternative catalytic reaction cycle for cytochrome c oxidase.
  11. Structure of the human SAGA coactivator complex.
  12. Structure of the class C orphan GPCR GPR158 in complex with RGS7-Gβ5.
  13. Cryo-TEM simulations of amorphous radiation-sensitive samples using multislice wave propagation.
  14. Michaelis-like complex of SARS-CoV-2 main protease visualized by room-temperature X-ray crystallography.
  15. Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization.
  16. Approximating deformation fields for the analysis of continuous heterogeneity of biological macromolecules by 3D Zernike polynomials.
  17. Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.
  18. Experimental evidence for the benefits of higher X-ray energies for macromolecular crystallography.
  19. Structural and functional analysis of the promiscuous AcrB and AdeB efflux pumps suggests different drug binding mechanisms.
  1. Proc Natl Acad Sci U S A. 2021 Nov 30. pii: e2112267118. [Epub ahead of print]118(48):
    Molecular structure of an open human KATP channel.
    Chen Zhao, Roderick MacKinnon.
      KATP channels are metabolic sensors that translate intracellular ATP/ADP balance into membrane excitability. The molecular composition of KATP includes an inward-rectifier potassium channel (Kir) and an ABC transporter-like sulfonylurea receptor (SUR). Although structures of KATP have been determined in many conformations, in all cases, the pore in Kir is closed. Here, we describe human pancreatic KATP (hKATP) structures with an open pore at 3.1- to 4.0-Å resolution using single-particle cryo-electron microscopy (cryo-EM). Pore opening is associated with coordinated structural changes within the ATP-binding site and the channel gate in Kir. Conformational changes in SUR are also observed, resulting in an area reduction of contact surfaces between SUR and Kir. We also observe that pancreatic hKATP exhibits the unique (among inward-rectifier channels) property of PIP2-independent opening, which appears to be correlated with a docked cytoplasmic domain in the absence of PIP2.
    Keywords:  ATP-sensitive potassium channel; KATP; ion channel
    DOI:  https://doi.org/10.1073/pnas.2112267118
  2. Molecules. 2021 Nov 22. pii: 7049. [Epub ahead of print]26(22):
    Combining Cryo-EM Density Map and Residue Contact for Protein Secondary Structure Topologies.
    Maytha Alshammari, Jing He.
      Although atomic structures have been determined directly from cryo-EM density maps with high resolutions, current structure determination methods for medium resolution (5 to 10 Å) cryo-EM maps are limited by the availability of structure templates. Secondary structure traces are lines detected from a cryo-EM density map for α-helices and β-strands of a protein. A topology of secondary structures defines the mapping between a set of sequence segments and a set of traces of secondary structures in three-dimensional space. In order to enhance accuracy in ranking secondary structure topologies, we explored a method that combines three sources of information: a set of sequence segments in 1D, a set of amino acid contact pairs in 2D, and a set of traces in 3D at the secondary structure level. A test of fourteen cases shows that the accuracy of predicted secondary structures is critical for deriving topologies. The use of significant long-range contact pairs is most effective at enriching the rank of the maximum-match topology for proteins with a large number of secondary structures, if the secondary structure prediction is fairly accurate. It was observed that the enrichment depends on the quality of initial topology candidates in this approach. We provide detailed analysis in various cases to show the potential and challenge when combining three sources of information.
    Keywords:  amino acid; constraints; contact; cryo-electron microscopy; image; protein structure; secondary structure; topology
    DOI:  https://doi.org/10.3390/molecules26227049
  3. Elife. 2021 Nov 25. pii: e69418. [Epub ahead of print]10
    Structure of Mycobacterium tuberculosis cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates.
    Shan Zhou, Weiwei Wang, Xiaoting Zhou, Yuying Zhang, Yuezheng Lai, Yanting Tang, Jinxu Xu, Dongmei Li, Jianping Lin, Xiaolin Yang, Ting Ran, Hongming Chen, Luke W Guddat, Quan Wang, Yan Gao, Zihe Rao, Hongri Gong.
      Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of Mycobacterium tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol-binding pockets. We observe Q203 and TB47 bound at the quinol-binding site and stabilized by hydrogen bonds with the side chains of QcrBThr313 and QcrBGlu314, residues that are conserved across pathogenic mycobacteria. These high-resolution images provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad-spectrum drugs to treat mycobacterial infections.
    Keywords:  Mycobacterium tuberculosis; Q203; TB47; cryo-electron microscopy; cytochrome bcc complex; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.69418
  4. Elife. 2021 Nov 23. pii: e73724. [Epub ahead of print]10
    High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism.
    Sabrina Pospich, H Lee Sweeney, Anne Houdusse, Stefan Raunser.
      The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin.
    Keywords:  human; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.73724
  5. Proc Natl Acad Sci U S A. 2021 Nov 30. pii: e2111862118. [Epub ahead of print]118(48):
    Structure of the translating Neurospora ribosome arrested by cycloheximide.
    Lunda Shen, Zhaoming Su, Kailu Yang, Cheng Wu, Thomas Becker, Deborah Bell-Pedersen, Junjie Zhang, Matthew S Sachs.
      Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX's mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.
    Keywords:  cryo-electron microscopy; protein synthesis; translation
    DOI:  https://doi.org/10.1073/pnas.2111862118
  6. IUCrJ. 2021 Nov 01. 8(Pt 6): 867-877
    High-resolution single-particle cryo-EM of samples vitrified in boiling nitro-gen.
    Tyler Engstrom, Jonathan A Clinger, Katherine A Spoth, Oliver B Clarke, David S Closs, Richard Jayne, Benjamin A Apker, Robert E Thorne.
      Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures. These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. However, ethane and propane are flammable, they must be liquified in liquid nitro-gen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitro-gen for storage, complicating workflows and increasing the chance of sample damage during handling. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only ∼250 000 K s-1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitro-gen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect. Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitro-gen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitro-gen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.
    Keywords:  cryocooling; cryoelectron microscopy; vitrification
    DOI:  https://doi.org/10.1107/S2052252521008095
  7. J Vis Exp. 2021 Nov 06.
    Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy.
    David P Klebl, Frank Sobott, Howard D White, Stephen P Muench.
      The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 µL sample at concentrations of ≥ 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.
    DOI:  https://doi.org/10.3791/62199
  8. J Struct Biol. 2021 Nov 20. pii: S1047-8477(21)00116-7. [Epub ahead of print] 107811
    Crystal structure of the potato leafroll virus coat protein and implications for viral assembly.
    Myfanwy C Adams, Carl J Schiltz, Michelle L Heck, Joshua S Chappie.
      Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of staple food crops. Previous cryo-electron microscopy studies of virus-like particles indicate that luteovirid viral capsids are built from a structural coat protein that organizes with T=3 icosahedral symmetry. Here we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.80-Å resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation and suggest that the CP N-terminus and its interactions with RNA play an important role in generating capsid curvature.
    Keywords:  capsid; icosahedral virus; plant virus; potato leafroll virus; quasi-equivalence; viral assembly
    DOI:  https://doi.org/10.1016/j.jsb.2021.107811
  9. EMBO Rep. 2021 Nov 24. e53597
    High-resolution structure of native toxin A from Clostridioides difficile.
    Aria Aminzadeh, Christian Engelbrecht Larsen, Thomas Boesen, René Jørgensen.
      Clostridioides difficile infections have emerged as the leading cause of healthcare-associated infectious diarrhea. Disease symptoms are mainly caused by the virulence factors, TcdA and TcdB, which are large homologous multidomain proteins. Here, we report a 2.8 Å resolution cryo-EM structure of native TcdA, unveiling its conformation at neutral pH. The structure uncovers the dynamic movement of the CROPs domain which is induced in response to environmental acidification. Furthermore, the structure reveals detailed information about the interaction area between the CROPs domain and the tip of the delivery and receptor-binding domain, which likely serves to shield the C-terminal part of the hydrophobic pore-forming region from solvent exposure. Similarly, extensive interactions between the globular subdomain and the N-terminal part of the pore-forming region suggest that the globular subdomain shields the upper part of the pore-forming region from exposure to the surrounding solvent. Hence, the TcdA structure provides insights into the mechanism of preventing premature unfolding of the pore-forming region at neutral pH, as well as the pH-induced inter-domain dynamics.
    Keywords:   Clostridioides difficile ; TcdA; Toxin A; cryo-EM; native structure
    DOI:  https://doi.org/10.15252/embr.202153597
  10. Nat Commun. 2021 Nov 25. 12(1): 6903
    Cryo-EM structures of intermediates suggest an alternative catalytic reaction cycle for cytochrome c oxidase.
    F Kolbe, S Safarian, Ż Piórek, S Welsch, H Müller, H Michel.
      Cytochrome c oxidases are among the most important and fundamental enzymes of life. Integrated into membranes they use four electrons from cytochrome c molecules to reduce molecular oxygen (dioxygen) to water. Their catalytic cycle has been considered to start with the oxidized form. Subsequent electron transfers lead to the E-state, the R-state (which binds oxygen), the P-state (with an already split dioxygen bond), the F-state and the O-state again. Here, we determined structures of up to 1.9 Å resolution of these intermediates by single particle cryo-EM. Our results suggest that in the O-state the active site contains a peroxide dianion and in the P-state possibly an intact dioxygen molecule, the F-state may contain a superoxide anion. Thus, the enzyme's catalytic cycle may have to be turned by 180 degrees.
    DOI:  https://doi.org/10.1038/s41467-021-27174-y
  11. Nat Struct Mol Biol. 2021 Nov 22.
    Structure of the human SAGA coactivator complex.
    Dominik A Herbst, Meagan N Esbin, Robert K Louder, Claire Dugast-Darzacq, Gina M Dailey, Qianglin Fang, Xavier Darzacq, Robert Tjian, Eva Nogales.
      The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.
    DOI:  https://doi.org/10.1038/s41594-021-00682-7
  12. Nat Commun. 2021 Nov 23. 12(1): 6805
    Structure of the class C orphan GPCR GPR158 in complex with RGS7-Gβ5.
    Eunyoung Jeong, Yoojoong Kim, Jihong Jeong, Yunje Cho.
      GPR158, a class C orphan GPCR, functions in cognition, stress-induced mood control, and synaptic development. Among class C GPCRs, GPR158 is unique as it lacks a Venus flytrap-fold ligand-binding domain and terminates Gαi/o protein signaling through the RGS7-Gβ5 heterodimer. Here, we report the cryo-EM structures of GPR158 alone and in complex with one or two RGS7-Gβ5 heterodimers. GPR158 dimerizes through Per-Arnt-Sim-fold extracellular and transmembrane (TM) domains connected by an epidermal growth factor-like linker. The TM domain (TMD) reflects both inactive and active states of other class C GPCRs: a compact intracellular TMD, conformations of the two intracellular loops (ICLs) and the TMD interface formed by TM4/5. The ICL2, ICL3, TM3, and first helix of the cytoplasmic coiled-coil provide a platform for the DHEX domain of one RGS7 and the second helix recruits another RGS7. The unique features of the RGS7-binding site underlie the selectivity of GPR158 for RGS7.
    DOI:  https://doi.org/10.1038/s41467-021-27147-1
  13. IUCrJ. 2021 Nov 01. 8(Pt 6): 943-953
    Cryo-TEM simulations of amorphous radiation-sensitive samples using multislice wave propagation.
    Benjamin Himes, Nikolaus Grigorieff.
      Image simulation plays a central role in the development and practice of high-resolution electron microscopy, including transmission electron microscopy of frozen-hydrated specimens (cryo-EM). Simulating images with contrast that matches the contrast observed in experimental images remains challenging, especially for amorphous samples. Current state-of-the-art simulators apply post hoc scaling to approximate empirical solvent contrast, attenuated image intensity due to specimen thickness and amplitude contrast. This practice fails for images that require spatially variable scaling, e.g. simulations of a crowded or cellular environment. Modeling both the signal and the noise accurately is necessary to simulate images of biological specimens with contrast that is correct on an absolute scale. The 'frozen plasmon' method is introduced to explicitly model spatially variable inelastic scattering processes in cryo-EM specimens. This approach produces amplitude contrast that depends on the atomic composition of the specimen, reproduces the total inelastic mean free path as observed experimentally and allows for the incorporation of radiation damage in the simulation. These improvements are quantified using the matched filter concept to compare simulation and experiment. The frozen plasmon method, in combination with a new mathematical formulation for accurately sampling the tabulated atomic scattering potentials onto a Cartesian grid, is implemented in the open-source software package cisTEM.
    Keywords:  cisTEM; cryo-TEM; frozen plasmon method; multislice wave propagation
    DOI:  https://doi.org/10.1107/S2052252521008538
  14. IUCrJ. 2021 Nov 01. 8(Pt 6): 973-979
    Michaelis-like complex of SARS-CoV-2 main protease visualized by room-temperature X-ray crystallography.
    Daniel W Kneller, Qiu Zhang, Leighton Coates, John M Louis, Andrey Kovalevsky.
      SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145-His41 dyad, can help in guiding drug design. Here, a 2.0 Å resolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3' by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.
    Keywords:  3CL protease; C145A mutant; SARS-CoV-2; catalytic mechanism; enzyme–substrate complex; main protease; room-temperature X-ray crystallography
    DOI:  https://doi.org/10.1107/S2052252521010113
  15. Nat Commun. 2021 Nov 25. 12(1): 6913
    Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization.
    Baobin Li, Christopher M Hoel, Stephen G Brohawn.
      Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.
    DOI:  https://doi.org/10.1038/s41467-021-27283-8
  16. IUCrJ. 2021 Nov 01. 8(Pt 6): 992-1005
    Approximating deformation fields for the analysis of continuous heterogeneity of biological macromolecules by 3D Zernike polynomials.
    David Herreros, Roy R Lederman, James Krieger, Amaya Jiménez-Moreno, Marta Martínez, David Myška, David Strelak, Jiri Filipovic, Ivet Bahar, Jose Maria Carazo, Carlos Oscar S Sanchez.
      Structural biology has evolved greatly due to the advances introduced in fields like electron microscopy. This image-capturing technique, combined with improved algorithms and current data processing software, allows the recovery of different conformational states of a macromolecule, opening new possibilities for the study of its flexibility and dynamic events. However, the ensemble analysis of these different conformations, and in particular their placement into a common variable space in which the differences and similarities can be easily recognized, is not an easy matter. To simplify the analysis of continuous heterogeneity data, this work proposes a new automatic algorithm that relies on a mathematical basis defined over the sphere to estimate the deformation fields describing conformational transitions among different structures. Thanks to the approximation of these deformation fields, it is possible to describe the forces acting on the molecules due to the presence of different motions. It is also possible to represent and compare several structures in a low-dimensional mapping, which summarizes the structural characteristics of different states. All these analyses are integrated into a common framework, providing the user with the ability to combine them seamlessly. In addition, this new approach is a significant step forward compared with principal component analysis and normal mode analysis of cryo-electron microscopy maps, avoiding the need to select components or modes and producing localized analysis.
    Keywords:  3D reconstruction and image processing; Zernike polynomials; conformations; multi-dimensional scaling (MDS); single-particle cryo-EM; spherical harmonics
    DOI:  https://doi.org/10.1107/S2052252521008903
  17. Nat Commun. 2021 Nov 26. 12(1): 6933
    Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.
    Christian Tüting, Fotis L Kyrilis, Johannes Müller, Marija Sorokina, Ioannis Skalidis, Farzad Hamdi, Yashar Sadian, Panagiotis L Kastritis.
      Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.
    DOI:  https://doi.org/10.1038/s41467-021-27287-4
  18. IUCrJ. 2021 Nov 01. 8(Pt 6): 896-904
    Experimental evidence for the benefits of higher X-ray energies for macromolecular crystallography.
    Selina L S Storm, Danny Axford, Robin L Owen.
      X-ray-induced radiation damage is a limiting factor for the macromolecular crystallographer and data must often be merged from many crystals to yield complete data sets for the structure solution of challenging samples. Increasing the X-ray energy beyond the typical 10-15 keV range promises to provide an extension of crystal lifetime via an increase in diffraction efficiency. To date, however, hardware limitations have negated any possible gains. Through the first use of a cadmium telluride EIGER2 detector and a beamline optimized for high-energy data collection, it is shown that at higher energies fewer crystals will be required to obtain complete data, as the diffracted intensity per unit dose increases by a factor of more than two between 12.4 and 25 keV. Additionally, these higher energy data can provide more information, as shown by a systematic increase in the high-resolution cutoff of the data collected. Taken together, these gains point to a high-energy future for synchrotron-based macromolecular crystallography.
    Keywords:  X-ray radiation damage; high-energy X-rays; macromolecular crystallography
    DOI:  https://doi.org/10.1107/S2052252521008423
  19. Nat Commun. 2021 Nov 25. 12(1): 6919
    Structural and functional analysis of the promiscuous AcrB and AdeB efflux pumps suggests different drug binding mechanisms.
    Alina Ornik-Cha, Julia Wilhelm, Jessica Kobylka, Hanno Sjuts, Attilio V Vargiu, Giuliano Malloci, Julian Reitz, Anja Seybert, Achilleas S Frangakis, Klaas M Pos.
      Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H+/drug antiporter module that recognizes structurally diverse substances, including antibiotics. Here, we show the 3.5 Å structure of subunit AdeB from the Acinetobacter baumannii AdeABC efflux pump solved by single-particle cryo-electron microscopy. The AdeB trimer adopts mainly a resting state with all protomers in a conformation devoid of transport channels or antibiotic binding sites. However, 10% of the protomers adopt a state where three transport channels lead to the closed substrate (deep) binding pocket. A comparison between drug binding of AdeB and Escherichia coli AcrB is made via activity analysis of 20 AdeB variants, selected on basis of side chain interactions with antibiotics observed in the AcrB periplasmic domain X-ray co-structures with fusidic acid (2.3 Å), doxycycline (2.1 Å) and levofloxacin (2.7 Å). AdeABC, compared to AcrAB-TolC, confers higher resistance to E. coli towards polyaromatic compounds and lower resistance towards antibiotic compounds.
    DOI:  https://doi.org/10.1038/s41467-021-27146-2
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