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Transmission Electron Microscope

Krios G3i Cryo-TEM for Life Sciences

Unravelling ‘Life’ at the Molecular Level – Easier, Faster, and more Reliably 

The new Thermo Scientific™ Krios™ G3i Cryo Transmission Electron Microscope (Cryo-TEM) enables life science researchers to unravel life at the molecular level—easier, faster, and more reliably than ever before. Its highly stable 300 kV TEM platform and industry-leading Autoloader (cryogenic sample manipulation robot) are designed for automated applications, such as single particle analysis (SPA) and cryo-tomography. Designed-in connectivity ensures a robust and risk-free pathway throughout the entire workflow, from sample preparation and optimization to image acquisition and data processing.

Setting up data acquisition has been made easier and quicker by enhanced automation and systematic user guidance. This allows every user to achieve the ultimate performance for every experiment. Simultaneously, the high-resolution performance and throughput of the Krios G3i Cryo-TEM have been further improved by cleverly combining hardware improvements with advanced software capabilities.





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Key Benefits

Enhanced ease of use through automation

The Krios G3i Cryo-TEM reduces the number of complex alignments that a user has to perform. For recurring alignments that need to be done regularly, automated alignment routines have been implemented into the EPU software for SPA. Furthermore the EPU user interface has been further streamlined to provide comprehensive user guidance.

Reproducible, optimal tool performance always guarantee

The optimal thermal and mechanical stability of the Krios G3i Cryo-TEM ensure perfect optical performance. The instrument features a self-assessment function that automatically evaluates the optical status of the microscope, providing feedback for any steps that require optimization. Additionally, automated alignment routines allow the instrument to be tuned to its optimal starting point for SPA or tomography experiments.

Maximum Throughput

EPU is the native software package for SPA automated screening and acquisition of large data sets on the Thermo Fisher cryo-TEMs. With full control of the Autoloader from within EPU, all grids in a cassette can be batch-screened: after the creation of a grid atlas, ice quality (presence, thickness) of the vitrified grids is automatically categorized to support guided selection of grid squares.

High resolution performance

The Krios Cryo-TEM has a proven track record of high resolution imaging of a wide variety of particles: the vast majority of published structures at or below 4 Å have been determined using Thermo Fisher cryo-TEMs . Constant power lenses reduce thermal drift, and contribute to the excellent system stability during long automated acquisition sessions. To allow imaging of increasingly smaller particles at increasingly higher resolution, the Krios G3i comes with Volta Phase Plate integration and a guaranteed <1% anisotropic magnification distortion.

Workflow connectivity

For successful cryo-EM data acquisition, optimization of both biochemistry and vitrification requires an efficient screening process. The Krios G3i Cryo-TEM can be integrated in a SPA workflow to where samples will be evaluated and optimized using the Talos Arctica or Glacios Cryo-TEMs, before imaging them at high-resolution in the Krios G3i Cryo-TEM

The designed-in connectivity of the Autoloader capsule and cassette systems ensure a robust and contamination-free transfer of samples between Glacios, Arctica and Krios Cryo-TEMs, without the need for manipulation of individual small grids.

Featured Document

Krios G3i Cryo-TEM Datasheet

Our newest Cryo-TEM enables life science researchers to unravel life at the molecular level. Its highly stable 300 kV TEM platform and industry-leading Autoloader (cryogenic sample manipulation robot) are designed for automated applications, such as single particle analysis (SPA) and cryo-tomography. Designed-in connectivity ensures a robust and risk-free pathway throughout the entire workflow, from sample preparation and optimization to image acquisition and data processing.

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Featured Document

Titan Krios TEM Publications

List of published articles and research results with the Titan Krios

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Featured News

Congratulations to the winners of the 2017 Nobel Prize in Chemistry.

Three scientists; Dr. Jacques Dubochet Dr. Joachim Frank, and Dr. Richard Henderson, were awarded the prize for their developments within Cryo-Electron Microscopy.

We are extremely proud of what these researchers and the structural biology community have achieved.

Publication list for Titan Krios for Life Sciences

1.
C. Engel, T. Gubbey, S. Neyer, S. Sainsbury, C. Oberthuer, C. Baejen, C. Bernecky, P. Cramer   (2017)   Structural Basis of RNA Polymerase I Transcription Initiation.   Cell   169

DOI:  10.1016/j.cell.2017.03.003

References PDB protein(s):  5N5Y, 5N5Z, 5N60, 5N61

Read abstract

Structural Basis of RNA Polymerase I Transcription Initiation.

C. Engel, T. Gubbey, S. Neyer, S. Sainsbury, C. Oberthuer, C. Baejen, C. Bernecky, P. Cramer

Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis. Comparison with the Pol II system suggests that promoter specificity relies on a distinct "bendability" and "meltability" of the promoter sequence that enables contacts between initiation factors, DNA, and polymerase.

2.
H. Zhao, K. Li, A. Lynn, K. Aron, G. Yu, W. Jiang, L. Tang   (2017)   Structure of a headful DNA-packaging bacterial virus at 2.9 Å resolution by electron cryo-microscopy.   Proceedings of the National Academy of Sciences of the United States of America   114

DOI:  10.1073/pnas.1615025114

References PDB protein(s):  5L35

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Structure of a headful DNA-packaging bacterial virus at 2.9 Å resolution by electron cryo-microscopy.

H. Zhao, K. Li, A. Lynn, K. Aron, G. Yu, W. Jiang, L. Tang

The enormous prevalence of tailed DNA bacteriophages on this planet is enabled by highly efficient self-assembly of hundreds of protein subunits into highly stable capsids. These capsids can stand with an internal pressure as high as ∼50 atmospheres as a result of the phage DNA-packaging process. Here we report the complete atomic model of the headful DNA-packaging bacteriophage Sf6 at 2.9 Å resolution determined by electron cryo-microscopy. The structure reveals the DNA-inflated, tensed state of a robust protein shell assembled via noncovalent interactions. Remarkable global conformational polymorphism of capsid proteins, a network formed by extended N arms, mortise-and-tenon-like intercapsomer joints, and abundant β-sheet-like mainchain:mainchain intermolecular interactions, confers significant strength yet also flexibility required for capsid assembly and DNA packaging. Differential formations of the hexon and penton are mediated by a drastic α-helix-to-β-strand structural transition. The assembly scheme revealed here may be common among tailed DNA phages and herpesviruses.

3.
X. Wang, P. Cimermancic, C. Yu, A. Schweitzer, N. Chopra, J. Engel, C. Greenberg, A. Huszagh, F. Beck, E. Sakata, Y. Yang, E. Novitsky, A. Leitner, P. Nanni, A. Kahraman, X. Guo, J. Dixon, S. Rychnovsky, R. Aebersold, W. Baumeister, A. Sali, L. Huang   (2017)   Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome.   Molecular & cellular proteomics : MCP   

DOI:  10.1074/mcp.M116.065326

References PDB protein(s):  5LN3

Read abstract

Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome.

X. Wang, P. Cimermancic, C. Yu, A. Schweitzer, N. Chopra, J. Engel, C. Greenberg, A. Huszagh, F. Beck, E. Sakata, Y. Yang, E. Novitsky, A. Leitner, P. Nanni, A. Kahraman, X. Guo, J. Dixon, S. Rychnovsky, R. Aebersold, W. Baumeister, A. Sali, L. Huang

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based in vivo and in vitro XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 inter-protein and 26 intra-protein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6 and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino-acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes. .

4.
G. Demo, E. Svidritskiy, R. Madireddy, R. Diaz-Avalos, T. Grant, N. Grigorieff, D. Sousa, A. Korostelev   (2017)   Mechanism of ribosome rescue by ArfA and RF2.   eLife   6

DOI:  10.7554/eLife.23687

References PDB protein(s):  5U9F, 5U9G

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Mechanism of ribosome rescue by ArfA and RF2.

G. Demo, E. Svidritskiy, R. Madireddy, R. Diaz-Avalos, T. Grant, N. Grigorieff, D. Sousa, A. Korostelev

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Å resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA 'senses' the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

5.
J. Gu, M. Wu, R. Guo, K. Yan, J. Lei, N. Gao, M. Yang   (2016)   The architecture of the mammalian respirasome.   Nature   537

DOI:  10.1038/nature19359

References PDB protein(s):  5GPN

Read abstract

The architecture of the mammalian respirasome.

J. Gu, M. Wu, R. Guo, K. Yan, J. Lei, N. Gao, M. Yang

The respiratory chain complexes I, III and IV (CI, CIII and CIV) are present in the bacterial membrane or the inner mitochondrial membrane and have a role of transferring electrons and establishing the proton gradient for ATP synthesis by complex V. The respiratory chain complexes can assemble into supercomplexes (SCs), but their precise arrangement is unknown. Here we report a 5.4 Å cryo-electron microscopy structure of the major 1.7 megadalton SCI1III2IV1 respirasome purified from porcine heart. The CIII dimer and CIV bind at the same side of the L-shaped CI, with their transmembrane domains essentially aligned to form a transmembrane disk. Compared to free CI, the CI in the respirasome is more compact because of interactions with CIII and CIV. The NDUFA11 and NDUFB9 supernumerary subunits of CI contribute to the oligomerization of CI and CIII. The structure of the respirasome provides information on the precise arrangements of the respiratory chain complexes in mitochondria.

 
 
 

2017 Nobel Prize in Chemistry

Congratulations to the winners of the 2017 Nobel Prize in Chemistry. Three scientists; Dr. Jacques Dubochet, Dr. Joachim Frank, and Dr. Richard Henderson, were awarded the prize for their developments within Cryo-Electron Microscopy.

We are extremely proud of what these researchers and the structural biology community have achieved.