scholarly journals Structure and transport mechanism of P5B-ATPases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ping Li ◽  
Kaituo Wang ◽  
Nina Salustros ◽  
Christina Grønberg ◽  
Pontus Gourdon
Keyword(s):  
Electron Microscopy ◽  
Parkinson’S Disease ◽  
Transport Mechanism ◽  
Human Cells ◽  
Transmembrane Segments ◽  
Cryo Electron Microscopy ◽  
N Terminus ◽  
Transport Cycle ◽  
Yeast Homolog ◽  
Uptake And Transport

AbstractIn human cells, P5B-ATPases execute the active export of physiologically important polyamines such as spermine from lysosomes to the cytosol, a function linked to a palette of disorders. Yet, the overall shape of P5B-ATPases and the mechanisms of polyamine recognition, uptake and transport remain elusive. Here we describe a series of cryo-electron microscopy structures of a yeast homolog of human ATP13A2-5, Ypk9, determined at resolutions reaching 3.4 Å, and depicting three separate transport cycle intermediates, including spermine-bound conformations. Surprisingly, in the absence of cargo, Ypk9 rests in a phosphorylated conformation auto-inhibited by the N-terminus. Spermine uptake is accomplished through an electronegative cleft lined by transmembrane segments 2, 4 and 6. Despite the dramatically different nature of the transported cargo, these findings pinpoint shared principles of transport and regulation among the evolutionary related P4-, P5A- and P5B-ATPases. The data also provide a framework for analysis of associated maladies, such as Parkinson’s disease.

scholarly journals Cryo-EM structures and transport mechanism of human P5B type ATPase ATP13A2

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Xudong Chen ◽  
Mingze Zhou ◽  
Sensen Zhang ◽  
Jian Yin ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Neurodegenerative Disorders ◽  
Mammalian Cells ◽  
Transport Mechanism ◽  
Transmembrane Domain ◽  
Normal Function ◽  
Cryo Electron Microscopy ◽  
Polyamine Transport ◽  
Adenosine Triphosphatases ◽  
Transport Cycle

AbstractPolyamines are important polycations that play critical roles in mammalian cells. ATP13A2 belongs to the orphan P5B adenosine triphosphatases (ATPase) family and has been established as a lysosomal polyamine exporter to maintain the normal function of lysosomes and mitochondria. Previous studies have reported that several human neurodegenerative disorders are related to mutations in the ATP13A2 gene. However, the transport mechanism of ATP13A2 in the lysosome remains unclear. Here, we report the cryo-electron microscopy (cryo-EM) structures of three distinct intermediates of the human ATP13A2, revealing key insights into the spermine (SPM) transport cycle in the lysosome. The transmembrane domain serves as a substrate binding site and the C-terminal domain is essential for protein stability and may play a regulatory role. These findings advance our understanding of the polyamine transport mechanism, the lipid-associated regulation, and the disease-associated mutants of ATP13A2.

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 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.

scholarly journals Molecular Tectonic Model of Virus Structural Transitions: the Putative Cell Entry States of Poliovirus

2000 ◽  
Vol 74 (3) ◽  
pp. 1342-1354 ◽  
Author(s):  
David M. Belnap ◽  
David J. Filman ◽  
Benes L. Trus ◽  
Naiqian Cheng ◽  
Frank P. Booy ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Infected Cell ◽  
Conformational Changes ◽  
Viral Protein ◽  
Cell Entry ◽  
Tectonic Plates ◽  
Tectonic Model ◽  
Beta Barrel ◽  
Cryo Electron Microscopy ◽  
N Terminus

ABSTRACT Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ∼22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ∼4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

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 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 Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

2021 ◽  
Author(s):  
Man Pan ◽  
Qingyun Zheng ◽  
Tian Wang ◽  
Lujun Liang ◽  
Junxiong Mao ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
E3 Ligase ◽  
Degradation Pathways ◽  
Single Chain ◽  
Cryo Electron Microscopy ◽  
N Terminus ◽  
Specific Manner ◽  
Ubiquitin Conjugating Enzyme ◽  
Structural Insights ◽  
Conjugating Enzyme

The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches. In these two structures, Ubr1 adopts different conformations to facilitate the transfer of Ub from Ubc2 to either an N-degron peptide or a monoubiquitinated degron. These structures not only reveal the architecture of the Ubr1 complex but also provide mechanistic insights into the initiation and elongation steps of ubiquitination catalyzed by Ubr1.

Use of SEM, X-ray analysis and TEM to localize Fe3+ reduction in roots

1988 ◽  
Vol 46 ◽  
pp. 392-393 ◽  
Author(s):  
William P. Wergin ◽  
P. F. Bell ◽  
Rufus L. Chaney
Keyword(s):  
Electron Microscopy ◽  
Root Hairs ◽  
Rapid Reduction ◽  
X Ray ◽  
Physical Evidence ◽  
Transmission Electron ◽  
Young Root ◽  
Insoluble Precipitate ◽  
Uptake And Transport ◽  
Scanning Electron

In dicotyledons, Fe3+ must be reduced to Fe2+ before uptake and transport of this essential macronutrient can occur. Ambler et al demonstrated that reduction along the root could be observed by the formation of a stain, Prussian blue (PB), Fe4 [Fe(CN)6]3 n H2O (where n = 14-16). This stain, which is an insoluble precipitate, forms at the reduction site when the nutrient solution contains Fe3+ and ferricyanide. In 1972, Chaney et al proposed a model which suggested that the Fe3+ reduction site occurred outside the cell membrane; however, no physical evidence to support the model was presented at that time. A more recent study using the PB stain indicates that rapid reduction of Fe3+ occurs in a region of the root containing young root hairs. Furthermore the most pronounced activity occurs in plants that are deficient in Fe. To more precisely localize the site of Fe3+ reduction, scanning electron microscopy (SEM), x-ray analysis, and transmission electron microscopy (TEM) were utilized to examine the distribution of the PB precipitate that was induced to form in roots.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

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