scholarly journals Two new polymorphic structures of alpha-synuclein solved by cryo-electron microscopy

2019 ◽  
Author(s):  
Ricardo Guerrero-Ferreira ◽  
Nicholas M.I. Taylor ◽  
Ana-Andrea Arteni ◽  
Pratibha Kumari ◽  
Daniel Mona ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Alpha Synuclein ◽  
Fibril Formation ◽  
Salt Bridges ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
N Terminus ◽  
Hydrophobic Cleft ◽  
Fibril Diameter

AbstractIntracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson’s disease (PD). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.Impact statementTwo new polymorphic structures of recombinant human alpha-synuclein fibrils show striking differences to previous structures, while familial PD mutation sites remain crucial for protofilament interaction and fibril stability.

scholarly journals Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ricardo Guerrero-Ferreira ◽  
Nicholas MI Taylor ◽  
Ana-Andreea Arteni ◽  
Pratibha Kumari ◽  
Daniel Mona ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Alpha Synuclein ◽  
Fibril Formation ◽  
Salt Bridges ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
N Terminus ◽  
Hydrophobic Cleft ◽  
Fibril Diameter

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson’s disease (PD). Previously, we reported the structure of alpha-synuclein fibrils (residues 1–121), composed of two protofibrils that are connected via a densely-packed interface formed by residues 50–57 (Guerrero-Ferreira, eLife 218;7:e36402). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.

scholarly journals Cryo-EM structure of alpha-synuclein fibrils

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Ricardo Guerrero-Ferreira ◽  
Nicholas MI Taylor ◽  
Daniel Mona ◽  
Philippe Ringler ◽  
Matthias E Lauer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Lewy Bodies ◽  
Alpha Synuclein ◽  
Diagnosis And Treatment ◽  
Lewy Neurites ◽  
Cryo Electron Microscopy ◽  
High Concentrations ◽  
Hydrophobic Cleft

Parkinson’s disease is a progressive neuropathological disorder that belongs to the class of synucleinopathies, in which the protein alpha-synuclein is found at abnormally high concentrations in affected neurons. Its hallmark are intracellular inclusions called Lewy bodies and Lewy neurites. We here report the structure of cytotoxic alpha-synuclein fibrils (residues 1–121), determined by cryo-electron microscopy at a resolution of 3.4 Å. Two protofilaments form a polar fibril composed of staggered β-strands. The backbone of residues 38 to 95, including the fibril core and the non-amyloid component region, are well resolved in the EM map. Residues 50–57, containing three of the mutation sites associated with familial synucleinopathies, form the interface between the two protofilaments and contribute to fibril stability. A hydrophobic cleft at one end of the fibril may have implications for fibril elongation, and invites for the design of molecules for diagnosis and treatment of synucleinopathies.

scholarly journals Cryo-EM structure of alpha-synuclein fibrils

2018 ◽  
Author(s):  
Ricardo Guerrero-Ferreira ◽  
Nicholas M. I. Taylor ◽  
Daniel Mona ◽  
Philippe Ringler ◽  
Matthias E. Lauer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rational Design ◽  
Alpha Synuclein ◽  
Diagnosis And Treatment ◽  
Cryo Electron Microscopy ◽  
Cell Propagation ◽  
Hydrophobic Cleft

AbstractIntracellular inclusions of alpha-synuclein are the neuropathological hallmark of progressive disorders called synucleinopathies. Alpha-synuclein fibrils are associated with transmissive cell-to-cell propagation of pathology. We report the structure of an alpha-synuclein fibril (residues 1-121) determined by cryo-electron microscopy at 3.4Å resolution. Two protofilaments form a polar fibril composed of staggered β-strands. The backbone of residues 38 to 95, including the fibril core and the non-amyloid component region, are well resolved in the EM map. Residues 50-57, containing three mutation sites associated with familial synucleinopathies, form the interface between the two protofilaments and contribute to fibril stability. A hydrophobic cleft may have implications for fibril elongation, and inform the rational design of molecules for diagnosis and treatment of synucleinopathies.

Cryo-EM Is a Powerful Tool, But Helical Applications Can Have Pitfalls

Soft Matter ◽  
2021 ◽  
Author(s):  
Edward Egelman ◽  
Fengbin Wang
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Macromolecular Complexes ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Main Technique

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct...

scholarly journals Antibody-Based Affinity Cryo-Electron Microscopy at 2.6 Å Resolution

2016 ◽  
Author(s):  
Guimei Yu ◽  
Kunpeng Li ◽  
Pengwei Huang ◽  
Xi Jiang ◽  
Wen Jiang
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
High Concentration ◽  
Low Concentration ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Grid Approach ◽  
Reconstruction Quality ◽  
Tulane Virus ◽  
Quantitative Analyses

AbstractThe affinity cryo-electron microscopy (cryo-EM) approach has been explored in recent years to simplify and improve the sample preparation for cryo-EM. Despite the demonstrated successes for low-concentration and unpurified specimens, the lack of near-atomic structures using this approach has led to a common perception of affinity cryo-EM as a niche technique incapable of reaching high resolutions. Here, we report a ~2.6 Å structure solved using the antibody-based affinity grid approach with a Tulane virus sample of low concentration. This is the first near-atomic structure solved using the affinity cryo-EM approach. Quantitative analyses of the structure indicate data and reconstruction quality comparable to conventional grid preparation method using samples at high concentration. With the shifting of bottlenecks of cryo-EM structural studies to sample grid preparation, our demonstration of the sub-3 Å capability of affinity cryo-EM approach indicates its potential in revolutionizing cryo-EM sample preparation for a broader spectrum of specimens.

scholarly journals Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xiaowei Hou ◽  
Ian R Outhwaite ◽  
Leanne Pedi ◽  
Stephen Barstow Long
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Calcium Channel ◽  
Calcium Release ◽  
Structural Information ◽  
Excitable Cells ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Open Conformation ◽  
Hydrophobic Amino Acids

The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca2+ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou et al., 2018). Here we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-electron microscopy. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel’s gate. Structured M1–M2 turrets, not evident previously, form the channel’s extracellular entrance.

scholarly journals New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy

2020 ◽  
Vol 61 ◽  
pp. 89-95 ◽  
Author(s):  
Ricardo Guerrero-Ferreira ◽  
Lubomir Kovacik ◽  
Dongchun Ni ◽  
Henning Stahlberg
Keyword(s):  
Electron Microscopy ◽  
Alpha Synuclein ◽  
Cryo Electron Microscopy

scholarly journals Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a

2021 ◽  
Author(s):  
Yuqing Hou ◽  
Lei Zhao ◽  
Tomohiro Kubo ◽  
Xi Cheng ◽  
Nathan McNeill ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Chlamydomonas Reinhardtii ◽  
Model Organism ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Cryo Electron Microscopy ◽  
N Terminus ◽  
Central Apparatus

Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a “9+2” axoneme composed of 9 outer doublet microtubules plus 2 central microtubules; the central microtubules together with their projections is termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70, which also has been reported to be associated with the doublet microtubules. Here we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the CA's C2a projection. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 and FAP147. Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65, and FAP147 as the first defining components of the C2a projection.

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