bims-strubi Biomed News
on Advances in structural biology
Issue of 2021‒10‒10
nine papers selected by
Alessandro Grinzato
European Synchrotron Radiation Facility


  1. J Struct Biol. 2021 Oct 01. pii: S1047-8477(21)00107-6. [Epub ahead of print] 107802
      While cryo-electron microscopy (cryo-EM) has revolutionized the structure determination of supramolecular protein complexes that are refractory to structure determination by X-ray crystallography, structure determination by cryo-EM can nonetheless be complicated by excessive conformational flexibility or structural heterogeneity resulting from weak or transient protein-protein association. Since such transient complexes are often critical for function, specialized approaches must be employed for the determination of meaningful structure-function relationships. Here, we outline examples in which transient protein-protein interactions have been visualized successfully by cryo-EM in the biosynthesis of fatty acids, polyketides, and terpenes. These studies demonstrate the utility of chemical crosslinking to stabilize transient protein-protein complexes for cryo-EM structural analysis, as well as the use of partial signal subtraction and localized reconstruction to extract useful structural information out of cryo-EM data collected from inherently dynamic systems. While these approaches do not always yield atomic resolution insights on protein-protein interactions, they nonetheless enable direct experimental observation of complexes in assembly-line biosynthesis that would otherwise be too fleeting for structural analysis.
    DOI:  https://doi.org/10.1016/j.jsb.2021.107802
  2. Proc Natl Acad Sci U S A. 2021 Oct 12. pii: e2115001118. [Epub ahead of print]118(41):
      We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.
    Keywords:  Legobody; cryo-EM; nanobody; scaffold; small protein
    DOI:  https://doi.org/10.1073/pnas.2115001118
  3. Acta Crystallogr D Struct Biol. 2021 Oct 01. 77(Pt 10): 1251-1269
      Metalloproteins catalyze a range of reactions, with enhanced chemical functionality due to their metal cofactor. The reaction mechanisms of metalloproteins have been experimentally characterized by spectroscopy, macromolecular crystallography and cryo-electron microscopy. An important caveat in structural studies of metalloproteins remains the artefacts that can be introduced by radiation damage. Photoreduction, radiolysis and ionization deriving from the electromagnetic beam used to probe the structure complicate structural and mechanistic interpretation. Neutron protein diffraction remains the only structural probe that leaves protein samples devoid of radiation damage, even when data are collected at room temperature. Additionally, neutron protein crystallography provides information on the positions of light atoms such as hydrogen and deuterium, allowing the characterization of protonation states and hydrogen-bonding networks. Neutron protein crystallography has further been used in conjunction with experimental and computational techniques to gain insight into the structures and reaction mechanisms of several transition-state metal oxidoreductases with iron, copper and manganese cofactors. Here, the contribution of neutron protein crystallography towards elucidating the reaction mechanism of metalloproteins is reviewed.
    Keywords:  X-ray diffraction; enzymatic mechanisms; metalloproteins; neutron protein crystallography; protonation; radiation damage
    DOI:  https://doi.org/10.1107/S2059798321009025
  4. Acta Crystallogr D Struct Biol. 2021 Oct 01. 77(Pt 10): 1282-1291
      In 2020, cryo-EM single-particle analysis achieved true atomic resolution thanks to technological developments in hardware and software. The number of high-resolution reconstructions continues to grow, increasing the importance of the accurate determination of atomic coordinates. Here, a new Python package and program called Servalcat is presented that is designed to facilitate atomic model refinement. Servalcat implements a refinement pipeline using the program REFMAC5 from the CCP4 package. After the refinement, Servalcat calculates a weighted Fo - Fc difference map, which is derived from Bayesian statistics. This map helps manual and automatic model building in real space, as is common practice in crystallography. The Fo - Fc map helps in the visualization of weak features including hydrogen densities. Although hydrogen densities are weak, they are stronger than in the electron-density maps produced by X-ray crystallography, and some H atoms are even visible at ∼1.8 Å resolution. Servalcat also facilitates atomic model refinement under symmetry constraints. If point-group symmetry has been applied to the map during reconstruction, the asymmetric unit model is refined with the appropriate symmetry constraints.
    Keywords:  REFMAC5; Servalcat; cryo-EM; structure refinement
    DOI:  https://doi.org/10.1107/S2059798321009475
  5. J Phys Chem B. 2021 Oct 07.
      Antibodies are crucial biomolecules that bring high therapeutic efficacy in medicine and accurate molecular detection in diagnosis. Many studies have been devoted to analyzing the antigen-antibody interaction from the importance of understanding the antibody recognition mechanism. However, most of the previous studies examined the characteristic of the antibody for interaction. It is also informative to clarify the significant antigen residues contributing to the binding. To characterize the molecular interaction of antigens, we computationally analyzed 350 antigen-antibody complex structures by molecular mechanics (MM) calculations and molecular dynamics (MD) simulations. Based on the MM calculations, the antigen residues contributing to the binding were extracted from all the 350 complexes. The extracted residues are located at the antigen-antibody interface and are responsible for making contact with the antibody. The appearances of the charged polar residues, Asp, Glu, Arg, and Lys, were noticeably large. In contrast, the populations of the hydrophobic residues, Leu, Val, and Ala, were relatively low. The appearance frequencies of the other amino acid residues were almost close to the abundance of general proteins of eukaryotes. The binding score indicated that the hydrophilic interaction was dominant at the antigen-antibody contact instead of the hydrophobic one. The positively charged residues, Arg and Lys, remarkably contributed to the binding compared to the negatively charged ones, Asp and Glu. Considerable contributions were also observed for the noncharged polar residues, Asn and Gln. The analysis of the secondary structures of the extracted antigen residues suggested that there was no marked difference in recognition by antibodies among helix, sheet, turn, and coil. A long helix of the antigen sometimes made contact with antibody complementarity-determining regions, and a large sheet also frequently covered the antibody heavy and light chains. The turn structure was the most popularly observed at the contact with antibody among 350 complexes. Three typical complexes were picked up for each of the four secondary structures. MD simulations were performed to examine the stability of the interfacial structures of the antigens for these 12 complex models. The alterations of secondary structures were monitored through the simulations. The structural fluctuations of the contact residues were low compared with the other domains of antigen molecules. No drastic conversion was observed for every model during the 100 ns simulation. The motions of the interfacial antigen residues were small compared to the other residues on the protein surface. Therefore, diverse molecular conformations are possible for antibody recognition as long as the target areas are polar, nonflexible, and protruding on the protein surface.
    DOI:  https://doi.org/10.1021/acs.jpcb.1c05997
  6. Nat Commun. 2021 Oct 08. 12(1): 5902
      ABCA4 is an ATP-binding cassette (ABC) transporter that flips N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to the cytoplasmic leaflet of photoreceptor membranes. Loss-of-function mutations cause Stargardt disease (STGD1), a macular dystrophy associated with severe vision loss. To define the mechanisms underlying substrate binding and STGD1, we determine the cryo-EM structure of ABCA4 in its substrate-free and bound states. The two structures are similar and delineate an elongated protein with the two transmembrane domains (TMD) forming an outward facing conformation, extended and twisted exocytoplasmic domains (ECD), and closely opposed nucleotide binding domains. N-Ret-PE is wedged between the two TMDs and a loop from ECD1 within the lumen leaflet consistent with a lateral access mechanism and is stabilized through hydrophobic and ionic interactions with residues from the TMDs and ECDs. Our studies provide a framework for further elucidating the molecular mechanism associated with lipid transport and disease and developing promising disease interventions.
    DOI:  https://doi.org/10.1038/s41467-021-26161-7
  7. Acta Crystallogr D Struct Biol. 2021 Oct 01. 77(Pt 10): 1233-1240
      Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (∼5-10 ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7-700 ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air-water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.
    Keywords:  electron microscopy; myosin; protein dynamics; structural biology; time-resolved
    DOI:  https://doi.org/10.1107/S2059798321008810
  8. Nat Struct Mol Biol. 2021 Oct;28(10): 858-868
      Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN's cellular functions.
    DOI:  https://doi.org/10.1038/s41594-021-00668-5
  9. Front Mol Biosci. 2021 ;8 716466
      In silico assessment of protein receptor interactions with small ligands is now part of the standard pipeline for drug discovery, and numerous tools and protocols have been developed for this purpose. With the SeamDock web server, we propose a new approach to facilitate access to small molecule docking for nonspecialists, including students. The SeamDock online service integrates different docking tools in a common framework that allows ligand global and/or local docking and a hierarchical approach combining the two for easy interaction site identification. This service does not require advanced computer knowledge, and it works without the installation of any programs with the exception of a common web browser. The use of the Seamless framework linking the RPBS calculation server to the user's browser allows the user to navigate smoothly and interactively on the SeamDock web page. A major effort has been put into the 3D visualization of ligand, receptor, and docking poses and their interactions with the receptor. The advanced visualization features combined with the seamless library allow a user to share with an unlimited number of collaborators, a docking session, and its full visualization states. As a result, SeamDock can be seen as a free, simple, didactic, evolving online docking resource best suited for education and training.
    Keywords:  collaborative sessions; drug discovery; molecular docking; virtual screening; web-server
    DOI:  https://doi.org/10.3389/fmolb.2021.716466