bims-strubi Biomed News
on Advances in structural biology
Issue of 2021–10–24
eight papers selected by
Alessandro Grinzato, European Synchrotron Radiation Facility
  1. Temperature artifacts in protein structures bias ligand-binding predictions.
  2. Lipid Nanodiscs for High-Resolution NMR Studies of Membrane Proteins.
  3. Structural basis of soluble membrane attack complex packaging for clearance.
  4. Deep learning improves macromolecule identification in 3D cellular cryo-electron tomograms.
  5. Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA.
  6. Advances in Xmipp for Cryo-Electron Microscopy: From Xmipp to Scipion.
  7. Structure of the human RNA polymerase I elongation complex.
  8. Crystal structures of the SARS-CoV-2 nucleocapsid protein C-terminal domain and development of nucleocapsid-targeting nanobodies.
  1. Chem Sci. 2021 Sep 01. 12(34): 11275-11293
    Temperature artifacts in protein structures bias ligand-binding predictions.
    Shanshan Y C Bradford, Léa El Khoury, Yunhui Ge, Meghan Osato, David L Mobley, Marcus Fischer.
      X-ray crystallography is the gold standard to resolve conformational ensembles that are significant for protein function, ligand discovery, and computational methods development. However, relevant conformational states may be missed at common cryogenic (cryo) data-collection temperatures but can be populated at room temperature. To assess the impact of temperature on making structural and computational discoveries, we systematically investigated protein conformational changes in response to temperature and ligand binding in a structural and computational workhorse, the T4 lysozyme L99A cavity. Despite decades of work on this protein, shifting to RT reveals new global and local structural changes. These include uncovering an apo helix conformation that is hidden at cryo but relevant for ligand binding, and altered side chain and ligand conformations. To evaluate the impact of temperature-induced protein and ligand changes on the utility of structural information in computation, we evaluated how temperature can mislead computational methods that employ cryo structures for validation. We find that when comparing simulated structures just to experimental cryo structures, hidden successes and failures often go unnoticed. When using structural information in ligand binding predictions, both coarse docking and rigorous binding free energy calculations are influenced by temperature effects. The trend that cryo artifacts limit the utility of structures for computation holds across five distinct protein classes. Our results suggest caution when consulting cryogenic structural data alone, as temperature artifacts can conceal errors and prevent successful computational predictions, which can mislead the development and application of computational methods in discovering bioactive molecules.
    DOI:  https://doi.org/10.1039/d1sc02751d
  2. Chem Rev. 2021 Oct 19.
    Lipid Nanodiscs for High-Resolution NMR Studies of Membrane Proteins.
    Umut Günsel, Franz Hagn.
      Membrane proteins (MPs) play essential roles in numerous cellular processes. Because around 70% of the currently marketed drugs target MPs, a detailed understanding of their structure, binding properties, and functional dynamics in a physiologically relevant environment is crucial for a more detailed understanding of this important protein class. We here summarize the benefits of using lipid nanodiscs for NMR structural investigations and provide a detailed overview of the currently used lipid nanodisc systems as well as their applications in solution-state NMR. Despite the increasing use of other structural methods for the structure determination of MPs in lipid nanodiscs, solution NMR turns out to be a versatile tool to probe a wide range of MP features, ranging from the structure determination of small to medium-sized MPs to probing ligand and partner protein binding as well as functionally relevant dynamical signatures in a lipid nanodisc setting. We will expand on these topics by discussing recent NMR studies with lipid nanodiscs and work out a key workflow for optimizing the nanodisc incorporation of an MP for subsequent NMR investigations. With this, we hope to provide a comprehensive background to enable an informed assessment of the applicability of lipid nanodiscs for NMR studies of a particular MP of interest.
    DOI:  https://doi.org/10.1021/acs.chemrev.1c00702
  3. Nat Commun. 2021 Oct 19. 12(1): 6086
    Structural basis of soluble membrane attack complex packaging for clearance.
    Anaïs Menny, Marie V Lukassen, Emma C Couves, Vojtech Franc, Albert J R Heck, Doryen Bubeck.
      Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane β-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis.
    DOI:  https://doi.org/10.1038/s41467-021-26366-w
  4. Nat Methods. 2021 Oct 21.
    Deep learning improves macromolecule identification in 3D cellular cryo-electron tomograms.
    Emmanuel Moebel, Antonio Martinez-Sanchez, Lorenz Lamm, Ricardo D Righetto, Wojciech Wietrzynski, Sahradha Albert, Damien Larivière, Eric Fourmentin, Stefan Pfeffer, Julio Ortiz, Wolfgang Baumeister, Tingying Peng, Benjamin D Engel, Charles Kervrann.
      Cryogenic electron tomography (cryo-ET) visualizes the 3D spatial distribution of macromolecules at nanometer resolution inside native cells. However, automated identification of macromolecules inside cellular tomograms is challenged by noise and reconstruction artifacts, as well as the presence of many molecular species in the crowded volumes. Here, we present DeepFinder, a computational procedure that uses artificial neural networks to simultaneously localize multiple classes of macromolecules. Once trained, the inference stage of DeepFinder is faster than template matching and performs better than other competitive deep learning methods at identifying macromolecules of various sizes in both synthetic and experimental datasets. On cellular cryo-ET data, DeepFinder localized membrane-bound and cytosolic ribosomes (roughly 3.2 MDa), ribulose 1,5-bisphosphate carboxylase-oxygenase (roughly 560 kDa soluble complex) and photosystem II (roughly 550 kDa membrane complex) with an accuracy comparable to expert-supervised ground truth annotations. DeepFinder is therefore a promising algorithm for the semiautomated analysis of a wide range of molecular targets in cellular tomograms.
    DOI:  https://doi.org/10.1038/s41592-021-01275-4
  5. Nat Commun. 2021 Oct 19. 12(1): 6095
    Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA.
    Claudia Lancey, Muhammad Tehseen, Souvika Bakshi, Matthew Percival, Masateru Takahashi, Mohamed A Sobhy, Vlad S Raducanu, Kerry Blair, Frederick W Muskett, Timothy J Ragan, Ramon Crehuet, Samir M Hamdan, Alfredo De Biasio.
      Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.
    DOI:  https://doi.org/10.1038/s41467-021-26251-6
  6. Molecules. 2021 Oct 15. pii: 6224. [Epub ahead of print]26(20):
    Advances in Xmipp for Cryo-Electron Microscopy: From Xmipp to Scipion.
    David Strelak, Amaya Jiménez-Moreno, José L Vilas, Erney Ramírez-Aportela, Ruben Sánchez-García, David Maluenda, Javier Vargas, David Herreros, Estrella Fernández-Giménez, Federico P de Isidro-Gómez, Jan Horacek, David Myska, Martin Horacek, Pablo Conesa, Yunior C Fonseca-Reyna, Jorge Jiménez, Marta Martínez, Mohamad Harastani, Slavica Jonić, Jiri Filipovic, Roberto Marabini, José M Carazo, Carlos O S Sorzano.
      Xmipp is an open-source software package consisting of multiple programs for processing data originating from electron microscopy and electron tomography, designed and managed by the Biocomputing Unit of the Spanish National Center for Biotechnology, although with contributions from many other developers over the world. During its 25 years of existence, Xmipp underwent multiple changes and updates. While there were many publications related to new programs and functionality added to Xmipp, there is no single publication on the Xmipp as a package since 2013. In this article, we give an overview of the changes and new work since 2013, describe technologies and techniques used during the development, and take a peek at the future of the package.
    Keywords:  Cryo-EM; Scipion; Xmipp; single-particle analysis
    DOI:  https://doi.org/10.3390/molecules26206224
  7. Cell Discov. 2021 Oct 20. 7(1): 97
    Structure of the human RNA polymerase I elongation complex.
    Dan Zhao, Weida Liu, Ke Chen, Zihan Wu, Huirong Yang, Yanhui Xu.
      Eukaryotic RNA polymerase I (Pol I) transcribes ribosomal DNA and generates RNA for ribosome synthesis. Pol I accounts for the majority of cellular transcription activity and dysregulation of Pol I transcription leads to cancers and ribosomopathies. Despite extensive structural studies of yeast Pol I, structure of human Pol I remains unsolved. Here we determined the structures of the human Pol I in the pre-translocation, post-translocation, and backtracked states at near-atomic resolution. The single-subunit peripheral stalk lacks contacts with the DNA-binding clamp and is more flexible than the two-subunit stalk in yeast Pol I. Compared to yeast Pol I, human Pol I possesses a more closed clamp, which makes more contacts with DNA. The Pol I structure in the post-cleavage backtracked state shows that the C-terminal zinc ribbon of RPA12 inserts into an open funnel and facilitates "dinucleotide cleavage" on mismatched DNA-RNA hybrid. Critical disease-associated mutations are mapped on Pol I regions that are involved in catalysis and complex organization. In summary, the structures provide new sights into human Pol I complex organization and efficient proofreading.
    DOI:  https://doi.org/10.1038/s41421-021-00335-5
  8. FEBS J. 2021 Oct 19.
    Crystal structures of the SARS-CoV-2 nucleocapsid protein C-terminal domain and development of nucleocapsid-targeting nanobodies.
    Zhenghu Jia, Chen Liu, Yuewen Chen, Heng Jiang, Zijing Wang, Jialu Yao, Jie Yang, Jiaxin Zhu, Boqing Zhang, Zhiguang Yuchi.
      The ongoing outbreak of COVID-19 caused by SARS-CoV-2 has resulted in a serious public health threat globally. Nucleocapsid protein is a major structural protein of SARS-CoV-2 that plays important roles in the viral RNA packing, replication, assembly, and infection. Here, we report two crystal structures of nucleocapsid protein C-terminal domain (CTD) at resolutions of 2.0 Å and 3.1 Å, respectively. These two structures, crystallized under different conditions, contain 2 and 12 CTDs in asymmetric unit, respectively. Interestingly, despite different crystal packing, both structures show a similar dimeric form as the smallest unit, consistent with its solution form measured by the size-exclusion chromatography, suggesting an important role of CTD in the dimerization of nucleocapsid proteins. By analyzing the surface charge distribution, we identified a stretch of positively charged residues between Lys257 and Arg262 that are involved in RNA-binding. Through screening a single-domain antibodies (sdAbs) library, we identified four sdAbs targeting different regions of nucleocapsid protein with high affinities that have future potential to be used in viral detection and therapeutic purposes.
    Keywords:  Crystal structure; Nanobodies; Nucleocapsid protein; SARS-CoV-2
    DOI:  https://doi.org/10.1111/febs.16239
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