scholarly journals Sub-2 Å Ewald Curvature Corrected Single-Particle Cryo-EM

2018 ◽  
Author(s):  
Yong Zi Tan ◽  
Sriram Aiyer ◽  
Mario Mietzsch ◽  
Joshua A. Hull ◽  
Robert McKenna ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gene Delivery ◽  
Structure Determination ◽  
Single Particle ◽  
Delivery Vehicle ◽  
Adeno Associated Virus ◽  
Ewald Sphere ◽  
Virus Serotype ◽  
Gene Delivery Vehicle ◽  
Technical Solutions

AbstractSingle-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we have exploited several technical solutions to improve resolution, including sub-Angstrom pixelation, per-particle CTF refinement, and most notably a correction for Ewald sphere curvature. The application of these methods on micrographs recorded on a base model Titan Krios enabled structure determination at ∼1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle.

scholarly journals In situ structure determination using single particle cryo-electron microscopy images

2020 ◽  
Author(s):  
Jing Cheng ◽  
Bufan Li ◽  
Long Si ◽  
Xinzheng Zhang
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Structure Refinement ◽  
Target Function ◽  
Target Protein ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Tilt Series

AbstractCryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

scholarly journals Lactoferrin as a gene delivery vehicle to hepatocytes

1997 ◽  
Vol 29 (2) ◽  
pp. 111-116 ◽  
Author(s):  
Sang Taek Oh ◽  
Jeong Keun Rih ◽  
Heung Sun Kwon ◽  
Deog Su Hwang ◽  
Sun Young Kim ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Delivery Vehicle ◽  
Gene Delivery Vehicle

SPION loaded poly(L-lysine)/hyaluronic acid micelles as MR contrast agent and gene delivery vehicle for cancer theranostics

2017 ◽  
Vol 25 (5) ◽  
pp. 446-451 ◽  
Author(s):  
Reju George Thomas ◽  
Muthunarayanan Muthiah ◽  
MyeongJu Moon ◽  
In-Kyu Park ◽  
Yong Yeon Jeong
Keyword(s):  
Hyaluronic Acid ◽  
Gene Delivery ◽  
Contrast Agent ◽  
Delivery Vehicle ◽  
Gene Delivery Vehicle ◽  
Cancer Theranostics

scholarly journals In situ structure determination using single particle cryo-electron microscopy images

2020 ◽  
Author(s):  
Jing Cheng ◽  
Bufan Li ◽  
Long Si ◽  
Xinzheng Zhang
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Structure Refinement ◽  
Target Function ◽  
Target Protein ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Tilt Series

Abstract Cryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

scholarly journals Cardiomyocyte Progenitor Cells as a Functional Gene Delivery Vehicle for Long-Term Biological Pacing

Molecules ◽  
2019 ◽  
Vol 24 (1) ◽  
pp. 181 ◽  
Author(s):  
Anna M. D. Végh ◽  
A. Dénise Den Haan ◽  
Lucía Cócera Ortega ◽  
Arie O. Verkerk ◽  
Joost P. G. Sluijter ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Progenitor Cells ◽  
Magnetic Beads ◽  
Fluorescent Protein ◽  
Functional Gene ◽  
Neonatal Rat ◽  
Pacemaker Activity ◽  
Delivery Vehicle ◽  
Pacemaker Function ◽  
Gene Delivery Vehicle

Sustained pacemaker function is a challenge in biological pacemaker engineering. Human cardiomyocyte progenitor cells (CMPCs) have exhibited extended survival in the heart after transplantation. We studied whether lentivirally transduced CMPCs that express the pacemaker current If (encoded by HCN4) can be used as functional gene delivery vehicle in biological pacing. Human CMPCs were isolated from fetal hearts using magnetic beads coated with Sca-1 antibody, cultured in nondifferentiating conditions, and transduced with a green fluorescent protein (GFP)- or HCN4-GFP-expressing lentivirus. A patch-clamp analysis showed a large hyperpolarization-activated, time-dependent inward current (−20 pA/pF at −140 mV, n = 14) with properties typical of If in HCN4-GFP-expressing CMPCs. Gap-junctional coupling between CMPCs and neonatal rat ventricular myocytes (NRVMs) was demonstrated by efficient dye transfer and changes in spontaneous beating activity. In organ explant cultures, the number of preparations showing spontaneous beating activity increased from 6.3% in CMPC/GFP-injected preparations to 68.2% in CMPC/HCN4-GFP-injected preparations (P < 0.05). Furthermore, in CMPC/HCN4-GFP-injected preparations, isoproterenol induced a significant reduction in cycle lengths from 648 ± 169 to 392 ± 71 ms (P < 0.05). In sum, CMPCs expressing HCN4-GFP functionally couple to NRVMs and induce physiologically controlled pacemaker activity and may therefore provide an attractive delivery platform for sustained pacemaker function.

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