scholarly journals Isolation of Disease-relevant p53 for Cryo-Electron Microscopy Analysis

2021 ◽  
Author(s):  
Maria J. Solares ◽  
GM Jonaid ◽  
William Y. Luqiu ◽  
Yanping Liang ◽  
Madison C. Evans ◽  
...  
Keyword(s):  
P53 Protein ◽  
P53 Gene ◽  
Tp53 Gene ◽  
Tumor Suppressor Protein ◽  
Protein Assemblies ◽  
Cryo Electron Microscopy ◽  
Naturally Occurring ◽  
Imaging Protocols ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis

Abstract Tumor suppressor protein TP53 (p53) plays a multi-faceted role in all cells of thehuman body. Sadly, mutations in the TP53 gene are involved in nearly ~50% of tumors,spurring erratic cell growth and disease progression. Until recently, structural informationfor p53 remained incomplete and there are limited studies on native p53 produced inhuman tumors. Here, we present a highly reproducible and effective protocol to extract,enrich, and purify native p53 protein assemblies from cancer cells for downstreamstructural studies. This method does not introduce purification tags into the p53 gene andmaintains naturally occurring modifications. In conjunction with cryo-Electron Microscopytechniques, we determined new structures for p53 monomers (~50 kDa) and tetramers(~200 kDa) at spatial resolutions of ~4.8 Å and ~7 Å, respectively.1 These modelsrevealed new insights for flexible regions of p53 along with biologically-relevantubiquitination sites. Combining biochemical and structural imaging protocols, we aim tobuild a better understanding of native p53’s impact in cancer formation.

scholarly journals Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein

2020 ◽  
Author(s):  
Dongchun Ni ◽  
Kelvin Lau ◽  
Frank Lehmann ◽  
Andri Fränkl ◽  
David Hacker ◽  
...  
Keyword(s):  
Structural Rearrangement ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Angiotensin Converting Enzyme 2 ◽  
Cryo Electron Microscopy ◽  
Monomeric Complex ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Viral Surface

AbstractThe human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19.

scholarly journals Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex

Science ◽  
2020 ◽  
Vol 368 (6489) ◽  
pp. eaaz2449 ◽  
Author(s):  
Xudong Wu ◽  
Marc Siggel ◽  
Sergey Ovchinnikov ◽  
Wei Mi ◽  
Vladimir Svetlov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ubiquitin Ligase ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Ubiquitin Ligase Complex ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Simulation Results ◽  
Membrane Region

Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo–electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two “half-channels” with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.

90S pre-ribosome transformation into the primordial 40S subunit

Science ◽  
2020 ◽  
Vol 369 (6510) ◽  
pp. 1470-1476 ◽  
Author(s):  
Jingdong Cheng ◽  
Benjamin Lau ◽  
Giuseppe La Venuta ◽  
Michael Ameismeier ◽  
Otto Berninghausen ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Rna Helicase ◽  
Rna Cleavage ◽  
En Bloc ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
External Transcribed Spacer ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Shed Light

Production of small ribosomal subunits initially requires the formation of a 90S precursor followed by an enigmatic process of restructuring into the primordial pre-40S subunit. We elucidate this process by biochemical and cryo–electron microscopy analysis of intermediates along this pathway in yeast. First, the remodeling RNA helicase Dhr1 engages the 90S pre-ribosome, followed by Utp24 endonuclease–driven RNA cleavage at site A1, thereby separating the 5′-external transcribed spacer (ETS) from 18S ribosomal RNA. Next, the 5′-ETS and 90S assembly factors become dislodged, but this occurs sequentially, not en bloc. Eventually, the primordial pre-40S emerges, still retaining some 90S factors including Dhr1, now ready to unwind the final small nucleolar U3–18S RNA hybrid. Our data shed light on the elusive 90S to pre-40S transition and clarify the principles of assembly and remodeling of large ribonucleoproteins.

scholarly journals Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shihui Sun ◽  
Hongjing Gu ◽  
Lei Cao ◽  
Qi Chen ◽  
Qing Ye ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
Structural Basis ◽  
Rapid Adaptation ◽  
Cryo Electron Microscopy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
And Gender ◽  
Related Mortality

AbstractThere is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.

scholarly journals Cryo-Electron Microscopy Analysis of a 105 kDa Protein Particle: The Xylulose Kinase from E. Coli

2006 ◽  
Vol 12 (S02) ◽  
pp. 408-409 ◽  
Author(s):  
H-T Chou ◽  
E di Luccio ◽  
D Wilson ◽  
H Stahlberg
Keyword(s):  
Electron Microscopy ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Protein Particle ◽  
Xylulose Kinase

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Material analysis techniques using the ion mill

1996 ◽  
Vol 54 ◽  
pp. 1034-1035
Author(s):  
J. P. Benedict ◽  
R. M. Anderson ◽  
S. J. Klepeis
Keyword(s):  
Polished Surface ◽  
Surface Material ◽  
Analysis Techniques ◽  
Material Analysis ◽  
Optical Inspection ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Argon Ions ◽  
The Impact ◽  
Scanning Electron

Ion mills equipped with flood guns can perform two important functions in material analysis; they can either remove material or deposit material. The ion mill holder shown in Fig. 1 is used to remove material from the polished surface of a sample for further optical inspection or SEM ( Scanning Electron Microscopy ) analysis. The sample is attached to a pohshing stud type SEM mount and placed in the ion mill holder with the polished surface of the sample pointing straight up, as shown in Fig 2. As the holder is rotating in the ion mill, Argon ions from the flood gun are directed down at the top of the sample. The impact of Argon ions against the surface of the sample causes some of the surface material to leave the sample at a material dependent, nonuniform rate. As a result, the polished surface will begin to develop topography during milling as fast sputtering materials leave behind depressions in the polished surface.

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