particle analysis
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2022 ◽  
Author(s):  
Guiqing Hu ◽  
Mark A Silveria ◽  
Michael S Chapman ◽  
Scott M Stagg

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE: With 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.


2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Jamie S. Depelteau ◽  
Ludovic Renault ◽  
Nynke Althof ◽  
C. Keith Cassidy ◽  
Luiza M. Mendonça ◽  
...  

AbstractCryo-electron microscopy has become an essential tool to understand structure and function of biological samples. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high-resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 Å structure indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.


2022 ◽  
Author(s):  
Martin Obr ◽  
Wim JH Hagen ◽  
Robert A Dick ◽  
Lingbo Yu ◽  
Abhay Kotecha ◽  
...  

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo- EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Angstrom resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Angstrom. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.


2021 ◽  
Vol 2 (3) ◽  
pp. 242-257
Author(s):  
Abdul Rafeeq ◽  
Syed Azhar Ali ◽  
Asad Khan Tanoli ◽  
Naseem Akhter ◽  
Ghulam Raza

The concentrations of Cd, Cr, Cu, Fe, Ni, Pb, & Zn were determined in the samples of soil, dismantling dust and rubber collected from the electronic waste dumping site of Shershah market and rubber from the Local market of Karachi city Pakistan. The city e-waste dumping and dismantling sites toxicity were not conducted before using modern techniques. The subsequent data of the heavy metals concentration were obtained using Atomic Absorption Spectrometry (AAS), Scanning Electron Microscopy (SEM), and Wavelength Dispersive- X-Ray Fluorescence Spectrometry (WD-XRF). Cu, Pb, and Zn were found as 133.17, 104.53 & 113.26 in soil, whereas in dismantling dust Pb & Zn were remained 10.56 and 12.65 mg/kg similarly. The China toy particle analysis by SEM was resulting metallic trend as Fe > Zn > Pb > Cd > Ni > Cr. The estimated data were compared with the levels allowed by the Pakistan Environmental Protection Agency (Pak-EPA) and the United States Environmental Protection Agency (USEPA). The maximum allowable limit for Cd is 3; Cr, Cd, and Pb are 100 for Ni is 50 and 300 mg/kg for Zn. The presence of these heavy metals from e-waste dumping would become soon a significant reason to cause serious health problems for the nearby residents and as well as city too. It has been concluded that the dumping of e-waste is the major source of contamination of heavy metals in the studied media. It is recommended that the e-waste must be recycled formally to prevent the soil from being polluted. .


Author(s):  
Anjana V Mani ◽  
R Rajam

Rasa sastra is an ancient science dealing with various drugs of mineral and metallic origin Tuttha (copper sulphate) is a mineral useful in various clinical conditions externally as well as internally. According to Rasasastra, through the process of Shodhana (purification) and Marana (incineration) the harmful effects of Tuttha bhasma (incinerated copper sulphate) is nullified resulting in the formation of a newer compound that is therapeutically more potent. To prepare Tuttha Bhasma by adopting standard manufacturing procedure explained in Ayurvedic texts as well as to study it’s physical and chemical characters using traditional and modern analytical tools. The pharmaceutical processing of Tuttha bhasma was carried out in three Kukkuta puta (incineration) with a peak temperature of 6100C according to Rasa Tarangini reference. Physico chemical analysis, Energy-Dispersive X-ray Fluorescence (EDXRF), X-ray Diffraction (XRD), Particle Size Analysis (PSA) were conducted. The final product shows presence of nano particles which was confirmed by particle analysis. XRD results of Tuttha bhasma revealed the presence of copper sulphide (covellite) with hexagonal lattice and sodium sulphate with orthorhombic structure. All of the preparation stages and changes in the properties were documented and validated, and they may now be used as a valuable tool for standardization and quality assurance of Tuttha bhasma.


2021 ◽  
Author(s):  
Qixiang He ◽  
Xiuhua Lin ◽  
Bianca L Chavez ◽  
Benjamin L Lusk ◽  
Ci Ji Lim

Telomere replication and regulation protect mammalian chromosome ends and promote genome stability. An essential step in telomere maintenance is the C-strand fill-in process, which is the de novo synthesis of the complementary strand of the telomere overhang. This step is catalyzed by polymerase-alpha/primase complex (pol-α/primase) and coordinated by an accessory factor, CTC1-STN1-TEN1 (CST). Using cryogenic-electron microscopy single-particle analysis, we report the structure of the human telomere C-strand fill-in preinitiation complex (PIC) at 3.9 Å resolution. The structure reveals a CST and a pol-α/primase co-bound to a single telomere overhang, poised for de novo RNA primer synthesis. Upon PIC assembly, the pol-α/primase undergoes large conformation change from its apo-state; CST partitions the DNA and RNA catalytic centers of pol-α/primase into two separate domains and positions the 3' end of an extended telomere single-stranded DNA template towards the RNA catalytic center (PRIM1 or p49). The telomeric single-stranded DNA template is further positioned by the POLA1 (or p180) catalytically dead exonuclease domain. Together with CST, the exonuclease domain forms a tight-fit molecular tunnel for template direction. Given the structural homology of CST to Replication Protein A (RPA), our structure provides the structural basis for a new model of how pol-α/primase lagging-strand DNA synthesis is coordinated by single-stranded DNA-binding accessory factors.


2021 ◽  
Author(s):  
Roman Koning ◽  
Hildo Vader ◽  
Martijn van Nugteren ◽  
Peter Grocutt ◽  
Wen Yang ◽  
...  

Abstract Speed and efficiency of data collection and image processing in cryo electron microscopy have increased over the last decade. However, cryo specimen preparation techniques have lagged behind and faster, more reproducible specimen preparation devices are needed. Here we present a new vitrification device with highly automated sample handling, requiring only limited user interaction. Moreover, the device allows inspection of thin films using light microscopy, since excess liquid is removed through suction by tubes, not blotting paper. In combination with dew-point control, this enables thin film preparation in a controlled and reproducible manner. The advantage is that quality of the prepared cryo specimen is characterized prior to electron microscopy data acquisition. Practicality and performance of the device are illustrated by experimental results obtained by vitrification of protein suspensions, lipid vesicles, bacterial and human cells, followed by imaged using single particle analysis, cryo electron tomography and cryo correlated light and electron microscopy.


2021 ◽  
Author(s):  
Shimon Harel ◽  
Yarin Altaras ◽  
Dikla Nachmias ◽  
Noa Rotem-Dai ◽  
Inbar Segal ◽  
...  

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Whereas the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (< 100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wildtype (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 30% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.


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