Structure determination

  • Article
    | Open Access

    Here, the authors present a hierarchical disorder model for the analysis of disorder in both crystal and cryo-EM structures. They apply their approach to several structures of three proteins, including SARS-CoV-2 proteins, and discuss mechanistic and dynamical implications.

    • Nicholas M. Pearce
    •  & Piet Gros
  • Article
    | Open Access

    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.

    • Jingjing Zhao
    • , Hongyi Xu
    •  & Xiaodong Zou
  • Article
    | Open Access

    Sequencing methods such as icSHAPE were developed to probe RNA structures transcriptome-wide in cells. To probe intact RNA structures, the authors develop icSHAPE-MaP and apply to Dicer-bound substrates showing that distance measuring is important for Dicer cleavage of pre-miRNAs.

    • Qing-Jun Luo
    • , Jinsong Zhang
    •  & Qiangfeng Cliff Zhang
  • Article
    | Open Access

    Adaptation of current algorithms to 3D SMLM data is currently problematic. Here the authors report a method that increases the signal-to-noise ratio and resolution of 3D single particle analysis in localization microscopy and enables determination of the symmetry groups of macromolecular complexes.

    • Hamidreza Heydarian
    • , Maarten Joosten
    •  & Bernd Rieger
  • Article
    | Open Access

    Tomographic reconstructions of cryopreserved specimens enable in-situ structural studies. Here, the authors present the beam image-shift electron cryo-tomography (BISECT) approach that accelerates data collection speed and improves the map resolution compared to earlier approaches and present the in vitro structure of a 300 kDa protein complex that was solved at 3.6 Å resolution as a test case.

    • Jonathan Bouvette
    • , Hsuan-Fu Liu
    •  & Alberto Bartesaghi
  • Article
    | Open Access

    Time-resolved crystallography (TRX) is used for monitoring only small conformational changes of biomacromolecules within the same lattice. Here, the authors report the interplay between synchronous molecular rearrangements and lattice phase transitions in RNA crystals, providing the basis for the investigation of large conformational changes using TRX.

    • Saminathan Ramakrishnan
    • , Jason R. Stagno
    •  & Yun-Xing Wang
  • Article
    | Open Access

    The viral Protein Kinase-1 (PK-1) phosphorylates the regulatory protein p6.9, which facilitates baculoviral genome release. Here, the authors combine X-ray crystallography with biophysical and biochemical analyses as well as molecular dynamics simulations to characterize Cydia pomenella granulovirus PK-1, which forms a dimer with a parallel side-to-side arrangement of the kinase domains and furthermore, they provide insights into its catalytic mechanism and evolutionary relationships with other kinases.

    • Michael R. Oliver
    • , Christopher R. Horne
    •  & James M. Murphy
  • Article
    | Open Access

    Accurately predicting the secondary structure of non-coding RNAs can help unravel their function. Here the authors propose a method integrating thermodynamic information and deep learning to improve the robustness of RNA secondary structure prediction compared to several existing algorithms.

    • Kengo Sato
    • , Manato Akiyama
    •  & Yasubumi Sakakibara
  • Article
    | Open Access

    Spy is an ATP independent chaperone that can act as both a holdase and a foldase towards topologically simple substrates. Assessing the interaction of Spy and apoflavodoxin, a complex client, the authors show that Spy’s activity is substrate specific. Spy binds partially unfolded states of apoflavodoxin tightly, which limits the possibility of folding and converts Spy to a pure holdase.

    • Rishav Mitra
    • , Varun V. Gadkari
    •  & James C. A. Bardwell
  • Article
    | Open Access

    Sorghum is a source of lignocellulosic biomass for the production of renewable fuels. Here the authors characterise the sorghum secondary cell wall using multi-dimensional magic angle spinning solid-state NMR and present a model dominated by interactions between three-fold screw xylan and amorphous cellulose.

    • Yu Gao
    • , Andrew S. Lipton
    •  & Jenny C. Mortimer
  • Article
    | Open Access

    The structure and dynamics of large proteins and complexes can be studied by methyl-NMR but resonance assignment is still challenging. Here, the authors present a NMR method that leverages optimal control pulse design to unambiguously distinguish between Leu and Val using a simple 2D HMQC experiment and they apply it to several proteins including Cas9, interleukin, and human translation initiation factor eIF4a.

    • Soumya P. Behera
    • , Abhinav Dubey
    •  & Haribabu Arthanari
  • Article
    | Open Access

    Due to the pulsed nature of X-ray free electron laser (XFEL) instruments the majority of protein crystals, which are injected using continuous jet injection techniques are wasted. Here, the authors present a microfluidic device to deliver aqueous protein crystal laden droplets segmented with an immiscible oil and demonstrate that with this device an approx. 60% reduction in sample waste was achieved for data collection of 3-deoxy-D-manno-octulosonate 8-phosphate synthase crystals at the EuXFEL.

    • Austin Echelmeier
    • , Jorvani Cruz Villarreal
    •  & Alexandra Ros
  • Article
    | Open Access

    Ribosome biogenesis in eukaryotes is a complex process that involves more than 200 protein factors. Here the authors present a structural analysis of a collection of human pre-60S structures sampled through a nuclear export adaptor NMD3, representing structural snapshots of pre-60S particles immediately before and after passing through nuclear pore complex.

    • Xiaomeng Liang
    • , Mei-Qing Zuo
    •  & Ning Gao
  • Article
    | Open Access

    Cryo-electron microscopy is widely employed in structural biology and uses phase contrast imaging. Here, the authors employ electron ptychography, a quantitative phase retrieval method for high-contrast, low-dose phase imaging of cryo-state rotavirus and immature HIV-1 virus-like particles, and show that electron ptychography is more efficient for phase recovery than conventional phase contrast imaging.

    • Liqi Zhou
    • , Jingdong Song
    •  & Peng Wang
  • Article
    | Open Access

    Whole-cell catalyst-based hydroxytyrosol production is low. Here, the authors increase the efficiency of its production in E. coli by de-bottlenecking two enzymatic steps catalyzed by monooxygenase and tyramine oxidase using structure-based enzyme redesign or in vivo-directed evolution with the aid of a newly developed biosensor.

    • Jun Yao
    • , Yang He
    •  & Shuang-Yan Tang
  • Article
    | Open Access

    CKK domain containing CAMSAP/Patronins recognise and regulate microtubule (MT) minus end dynamics. Here the authors compare cryo-EM structures of MT-bound human CKK and Naegleria gruberi CKK which lacks minus-end binding preference, finding NgCKK has a different interaction with, and inability to remodel, its MT binding site, shedding light on the CAMSAP/Patronin end binding mechanism.

    • Joseph Atherton
    • , Yanzhang Luo
    •  & Carolyn A. Moores
  • Article
    | Open Access

    The transient receptor potential channel member 2 (TRPM2) ion channel has a function in redox-dependent signaling. Here the authors present the cryo-EM structures of zebrafish TRPM2 in the ligand-free form, with Ca2+ and both ADP-ribose/Ca2+ and observe two-fold symmetric quaternary structure rearrangements in the ligand-bound structures that likely represent intermediate gating states.

    • Ying Yin
    • , Mengyu Wu
    •  & Seok-Yong Lee
  • Perspective
    | Open Access

    Lack of best practice guidelines currently limits the application of metabolomics in the regulatory sciences. Here, the MEtabolomics standaRds Initiative in Toxicology (MERIT) proposes methods and reporting standards for several important applications of metabolomics in regulatory toxicology.

    • Mark R. Viant
    • , Timothy M. D. Ebbels
    •  & Ralf J. M. Weber
  • Article
    | Open Access

    Human antibodies cross-reactive for several viruses within the Ebolavirus genus have been identified. Here the authors present the crystal structure of such a neutralizing monoclonal antibody (mAb) targeting the stalk of Bundibugyo virus glycoprotein and show that mAb binding may interfere with trimeric bundle assembly and/or the viral membrane.

    • Liam B. King
    • , Brandyn R. West
    •  & Erica Ollmann Saphire
  • Article
    | Open Access

    Adenovirus based (AdV) vectors are promising platforms for therapeutics and vaccines, but receptor usage of serotypes in clinical development remains unclear. Here, based on crystal structures and modeling, Baker et al. show that HAdV-D26/48 fiber knob protein interacts weakly with CAR but not with CD46 or DSG2.

    • Alexander T. Baker
    • , Alexander Greenshields-Watson
    •  & Alan L. Parker
  • Article
    | Open Access

    Quaternary contacts mediated by an extended heavy-chain framework region 3 (FR3) have been shown to improve binding to HIV envelope and virus neutralization for a few antibodies. Here, Liu et al. engraft such an FR3 loop onto several potent broadly neutralizing antibodies, resulting in improved neutralization activity and pharmacokinetics.

    • Qingbo Liu
    • , Yen-Ting Lai
    •  & Paolo Lusso
  • Article
    | Open Access

    The interactions of lignin with polysaccharides in plant secondary cell walls are not well understood. Here the authors employ solid-state NMR measurements to analyse intact stems of maize, Arabidopsis, switchgrass and rice and observe that lignin self-aggregates and forms highly hydrophobic microdomains that make extensive surface contacts to xylan.

    • Xue Kang
    • , Alex Kirui
    •  & Tuo Wang
  • Article
    | Open Access

    Eukaryotic transcription requires passage of RNA polymerase II (Pol II) through chromatin, which is impaired by nucleosomes. Here the authors report the cryo-EM structure of transcribing Pol II engaged with a downstream nucleosome core particle at an overall resolution of 4.4 Å, providing insights into the mechanism of chromatin transcription.

    • Lucas Farnung
    • , Seychelle M. Vos
    •  & Patrick Cramer
  • Article
    | Open Access

    Mutations within immunological epitope containing regions of influenza A virus can impair the established immune response between influenza strains and could impact rational vaccine design. Here Grant et al. examine the presence, structural impact and cross reactivity of two human immunodominant influenza epitope variants.

    • Emma J. Grant
    • , Tracy M. Josephs
    •  & Katherine Kedzierska
  • Article
    | Open Access

    TssA is an important component of the bacterial type VI secretion system (T6SS). Here, Dix et al. integrate structural, phylogenetic and functional analysis of the TssA subunits, providing new insights into their role in T6SS assembly and function.

    • Samuel R. Dix
    • , Hayley J. Owen
    •  & Mark S. Thomas
  • Article
    | Open Access

    Current Förster resonance energy transfer (FRET)-based fluorescence spectroscopy methods are suffering from some limitations in the field of structural biology. Here, the authors present an optical approach, distance-encoding photoinduced electron transfer (DEPET), capable of the simultaneous study of protein structure and function.

    • Antonios Pantazis
    • , Karin Westerberg
    •  & Riccardo Olcese
  • Article
    | Open Access

    The determination of high resolution structures of G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins is challenging. Here authors develop an antibody fragment, mAB16, which stabilizes GPCR/G-protein complexes and facilitates the application of high resolution cryo-EM.

    • Shoji Maeda
    • , Antoine Koehl
    •  & Brian K. Kobilka
  • Article
    | Open Access

    Conventional reconstruction methods used in cryo-EM single particle analysis do not take the depth of field effect into account. Here the authors present a block-based reconstruction method to deal with the depth of field effect and show that this approach can improve the resolution of cryo-EM virus structures.

    • Dongjie Zhu
    • , Xiangxi Wang
    •  & Xinzheng Zhang
  • Article
    | Open Access

    BMI1, a core element of the polycomb repressive complex 1, is suggested to have oncogenic activity in a variety of cancers. Here, the authors report the structure of BMI1 bound to the protein PHC2, identify BMI1 homo-oligomerization interfaces, and analyse the role of BMI1 protein-protein interactions in PRC1 function.

    • Felicia Gray
    • , Hyo Je Cho
    •  & Tomasz Cierpicki
  • Article
    | Open Access

    Tetrameric cationic channels specificity is determined by the sequence and structural conformation of their selectivity filter. Here, the authors show that a cationic channel from Tsukamurella paurometabola is non-selective due to a Ca2+-binding motif within its unusual proline-rich filter.

    • Balasundaresan Dhakshnamoorthy
    • , Ahmed Rohaim
    •  & Benoît Roux
  • Article
    | Open Access

    Large-scale dense reconstruction of neuronal circuits (or connectomics) requires methods for large-volume dense en-blocelectron microscopy (EM) staining. Here the authors develop a protocol for staining tissue blocks from mouse neocortex sized at least 1 mm in diameter, enabling correlated functional and structural circuit analyses.

    • Yunfeng Hua
    • , Philip Laserstein
    •  & Moritz Helmstaedter
  • Article
    | Open Access

    The peptidoglycan (PG) layer of the Gram-positive bacteria cell wall resists turgor pressure, but the architecture of this layer is largely unknown. Here the authors use high resolution atomic force microscopy to image the PG layer from live Streptococcusto reveal a net-like arrangement that resists osmotic challenge by stretching and stiffening.

    • Ron Saar Dover
    • , Arkady Bitler
    •  & Yechiel Shai
  • Article
    | Open Access

    The determination of RNA structures within high-molecular weight protein-RNA complexes in non-crystalline state is technically challenging. Here, the authors describe a solid-state NMR protocol for the determination of RNA structures at high resolution.

    • Alexander Marchanka
    • , Bernd Simon
    •  & Teresa Carlomagno
  • Article |

    High-resolution structures of large RNAs and protein–RNA complexes are difficult to solve due to inherent structural flexibility and a high risk of crystallization artefacts. Here, Duss et al. present a novel EPR-based approach to aid structure determination of large RNAs and protein–RNA complexes in solution.

    • Olivier Duss
    • , Maxim Yulikov
    •  & Frédéric H.-T. Allain
  • Article |

    RNA is central to many cellular functions, but in vivo structures of most RNAs are unknown. Here Kwok et al. present a universally applicable method, DMS/SHAPE-LMPCR, to identify structures of low-abundance transcripts in living cells, which reveals important features that are uniquely present in vivo.

    • Chun Kit Kwok
    • , Yiliang Ding
    •  & Philip C Bevilacqua