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Electron Microscopy Solutions
Detailed reconstructions are now available with modern cryo-EM.
5N8Y. KaiCBA circadian clock backbone model based on a Cryo-EM density
DOI: 10.1126/science.aag3218

2017 Nobel Prize in Chemistry

A New Era in Cryo-EM

 

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.

Read the press release

What is Cryo-EM?

Cryo-Electron Microscopy (Cryo-EM) begins with vitrification, in which the protein solution is cooled so rapidly that water molecules do not have time to crystallize, forming an amorphous solid that does little or no damage to the sample structure (a process known as vitrification). The sample is then screened for particle concentration, distribution and orientation. Next, a series of images is acquired, and two-dimensional classes are computationally extracted. In the final step, the data is processed by reconstruction software, yielding accurate, detailed, 3D models of intricate biological structures at the sub-cellular and molecular scales. These models can reveal interactions that were impossible to visualize previously, a key to scientific results.

History of Cryo-EM

Cryo-EM possesses a long history that includes many groups of researchers who contributed to the evolution and revolution of cryo-EM in equally important ways. It has taken 40+ years of dedicated work for the structural biology community to develop the myriad technological advancements—detectors, automation, software and more—that have made modern cryo-EM possible. The Nobel Prize in Chemistry 2017 was awarded to three researchers who had spent decades advancing cryo-EM:

  • In 1975, Joachim Frank began work on the algorithms that would analyze fuzzy 2D images and reconstruct them into sharp 3D structures. 
  • In the early 1980s, Jacques Dubochet succeeded in vitrifying water, which allowed the biomolecules to retain their shape in a vacuum.
  • In 1990, Richard Henderson was the first to use an electron microscope to generate a 3D image of a protein at atomic resolution.

Courtesy of nobelprize.org

Cryo-EM structures of tau filaments from Alzheimer's disease.
DOI: 10.1038/nature23002

The Rise of Cryo-EM

One of the greatest advantages of cryo-EM relative to conventional structural biology techniques is its ability to analyze large, complex and flexible structures. Oftentimes these cannot be crystallized for X-ray crystallography (XRD) or are too large and complex for nuclear magnetic resonance (NMR) spectroscopy. These include many biologically important proteins, especially those with variable or flexible structures like membrane proteins. The established methods for structure determination, XRD and NMR, are now routinely integrated with cryo-EM density maps to achieve atomic-resolution models of complex, dynamic molecular assemblies.

Since the first model based on cryo-EM reconstruction was deposited with the PDB (Protein Data Bank) in 1997, the number of deposited structures has grown exponentially. Cryo-EM was responsible for entries to the EMDB surpassing 1,000 in 2016, with a quarter of all entries deposited that year alone. Many of these most recent depositions include critical macromolecular assemblies previously thought impervious to structure determination.

Resolution Revolution

Cryo-EM now regularly breaks the 3Å barrier, allowing for high-resolution solving of structures that are impossible (or nearly so) with the traditional techniques of XRD and NMR. Thermo Fisher Scientific pioneered this modern “Resolution Revolution” era of cryo-EM with its introduction of the Titan Krios™ transmission electron microscope in 2008. Recent technological advancements in the microscope design and imaging hardware, along with enhanced image processing and automation, have helped to catapult the technique’s success. Many leading scientists have recently adopted the technique as one of the most critical tools in their laboratory. This increase in adoption, along with an unprecedented sharp rise in associated publications, led to Nature Methods naming cryo-EM as its Method of the Year for 2015.

Data courtesy of National University of Singapore.

Scientific Results

Cryo-EM has enabled researchers to define the world’s first protein structures for human ataxia telangiectasia mutated (ATM). ATM is a key trigger protein in the DNA damage response and a prime therapeutic target in cancer research. The research could prove invaluable in developing new cancer drugs. Science Advances, May 10, 2017.

Researchers have used cryo-EM to determine the ternary complex structure of the calcitonin receptor for a class B G protein-coupled receptor (GPCR), an important membrane protein complex that has been difficult to analyze by other means. Nature, June 1, 2017.

The Thermo Scientific™ Krios Cryo-TEM and SPA Workflow have enabled researchers to more clearly resolve the structure of several viruses, including HIV, Zika, Ebola and rhinovirus C. PNA, August 9, 2016

Thermo Fisher Scientific's Solutions for Cryo-EM

Single Particle Analysis Workflow

The Challenges of Protein Complex Analysis

Drug development at the pace requested by today's society requires the study of molecular mechanisms as close as possible to in vivo at high resolution. Until recently, the main technique available to achieve high resolution structures of biological molecules was crystallography.

Cryo-EM, the Nature 2015 Method of the Year, is now able to solve near-atomic-resolution structures with volume and clarity. Using Cryo-EM to understand molecular structures such as proteins, protein complexes, and protein-ribonucleic acid associations-the fundamental building blocks of life-will lead to wide scale pharmaceutical solutions for treating diseases and disorders.

Learn more about Single Particle Analysis Workflow

Krios G3i Cryo-TEM for Life Sciences

Unravelling ‘Life’ at the Molecular Level

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.

Learn more about Krios G3i Cryo-TEM

Publication list

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

Read abstract

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

Read abstract

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.

Protein Data Bank Structures

(Click on images for more details)
  • Life in the extremes: atomic structure of Sulfolobus Turreted Icosahedral Virus
    Author: Veesler, D. et al.
    Taken on a FEI TITAN KRIOS at 4.5 Å resolution
  • Cryo-EM structure of Dengue virus serotype 3 at 28 degrees C
    Author: Fibriansah, G. et al.
    Taken on a FEI TITAN KRIOS at 6.0 Å resolution
  • Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy
    Author: Bartesaghi, A. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • CryoEM single particle reconstruction of anthrax toxin protective antigen pore at 2.9 Angstrom resolution
    Author: Jiang, J. et al.
    Taken on a FEI TITAN KRIOS at 2.9 Å resolution
  • Atomic structure of a non-enveloped virus reveals pH sensors for a coordinated process of cell entry
    Author: Zhang, X. et al.
    Taken on a FEI TITAN KRIOS at 3.3 Å resolution
  • Structure of alpha-1 glycine receptor by single particle electron cryo-microscopy, strychnine-bound state
    Author: Du, J. et al.
    Taken on a FEI TITAN KRIOS at 3.9 Å resolution
  • Electron cryo-microscopy of the IST1-CHMP1B ESCRT-III copolymer
    Author: McCullough, J. et al.
    Taken on a FEI TITAN KRIOS at 4.0 Å resolution
  • Structure of Escherichia coli EF4 in posttranslocational ribosomes (Post EF4)
    Author: Zhang, D. et al.
    Taken on a FEI TITAN KRIOS at 3.700 Å resolution
  • Cryo-EM structure of the magnesium channel CorA in the closed symmetric magnesium-bound state
    Author: Matthies, D. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Structure of the yeast 60S ribosomal subunit in complex with Arx1, Alb1 and C-terminally tagged Rei1
    Author: Greber, B.J. et al.
    Taken on a FEI TITAN KRIOS at 3.410 Å resolution
  • Structure of a Chaperone-Usher pilus reveals the molecular basis of rod uncoilin
    Author: Hospenthal, M.K. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Cryo-EM structure of the flagellar hook of Campylobacter jejuni
    Author: Matsunami, H. et al.
    Taken on a FEI TITAN KRIOS at 3.5 Å resolution
  • Structure of F-ATPase from Pichia angusta, state1
    Author: Vinothkumar, K.R. et al.
    Taken on a FEI TITAN KRIOS at 7 Å resolution
  • Cryo-EM structure of a BG505 Env-sCD4-17b-8ANC195 complex
    Author: Wang, H. et al.
    Taken on a FEI TITAN KRIOS at 8.9 Å resolution
  • Ca2+ bound aplysia Slo1
    Author: MacKinnon, R. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Dephosphorylated, ATP-free cystic fibrosis transmembrane conductance regulator (CFTR) from zebrafish
    Author: Zhang, Z. et al.
    Taken on a FEI TITAN KRIOS at 3.73 Å resolution
  • The 2.8 A Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparanoid Pentasaccharide
    Author: Xie, Q. et al.
    Taken on a FEI TITAN KRIOS at 2.8 Å resolution
  • Volta phase plate cryo-electron microscopy structure of a calcitonin receptor-heterotrimeric Gs protein complex
    Author: Liang, Y.L. et al.
    Taken on a FEI TITAN KRIOS at 4.1 Å resolution
  • Cryo-EM structure of the human ether-a-go-go related K+ channel
    Author: Wang, W.W. et al.
    Taken on a FEI TITAN KRIOS at 3.7 Å resolution
  • CryoEM structure of type II secretion system secretin GspD in E.coli K12
    Author: Yan, Z. et al.
    Taken on a FEI TITAN KRIOS at 3.04 Å resolution
  • Structure of a Pancreatic ATP-sensitive Potassium Channel
    Author: Li, N. et al.
    Taken on a FEI TITAN KRIOS at 5.6 Å resolution
  • The resting state of yeast proteasome
    Author: Ding, Z. et al.
    Taken on a FEI TITAN KRIOS at 6.3 Å resolution
  • Structure of a eukaryotic voltage-gated sodium channel at near atomic resolution
    Author: Shen, H. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Prefusion structure of MERS-CoV spike glycoprotein, three-fold symmetry
    Author: Yuan, Y. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • Prefusion structure of MERS-CoV spike glycoprotein, conformation 2
    Author: Yuan, Y. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • Structure of SARS-CoV spike glycoprotein
    Author: Gui, M. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution