The structure of the yeast Ctf3 complex

Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

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The structure of the yeast Ctf3 complex

10.1101/628149 ◽

2019 ◽

Author(s):

Stephen M. Hinshaw ◽

Andrew N. Dates ◽

Stephen C. Harrison

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

High Resolution ◽

Atomic Model ◽

Kinetochore Assembly ◽

Cryo Electron Microscopy ◽

Molecular Interpretation ◽

Spindle Microtubules ◽

And Function ◽

Assembly Site

ABSTRACTKinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture. We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

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The structure of the Ctf19c/CCAN from budding yeast

eLife ◽

10.7554/elife.44239 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 36

Author(s):

Stephen M Hinshaw ◽

Stephen C Harrison

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

Budding Yeast ◽

Histone H3 ◽

Kinetochore Assembly ◽

Microtubule Attachment ◽

Cryo Electron Microscopy ◽

Histone H3 Variant

Eukaryotic kinetochores connect spindlemicrotubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.

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Crystal Structures of Fragments D and Double-D from Fibrinogen and Fibrin

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615842 ◽

1999 ◽

Vol 82 (08) ◽

pp. 271-276 ◽

Cited By ~ 8

Author(s):

Glen Spraggon ◽

Stephen Everse ◽

Russell Doolittle

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Crystal Structures ◽

Structure And Function ◽

X Ray ◽

Long Period ◽

And Function

IntroductionAfter a long period of anticipation,1 the last two years have witnessed the first high-resolution x-ray structures of fragments from fibrinogen and fibrin.2-7 The results confirmed many aspects of fibrinogen structure and function that had previously been inferred from electron microscopy and biochemistry and revealed some unexpected features. Several matters have remained stubbornly unsettled, however, and much more work remains to be done. Here, we review several of the most significant findings that have accompanied the new x-ray structures and discuss some of the problems of the fibrinogen-fibrin conversion that remain unresolved. * Abbreviations: GPR—Gly-Pro-Arg-derivatives; GPRPam—Gly-Pro-Arg-Pro-amide; GHRPam—Gly-His-Arg-Pro-amide

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Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s143192761601196x ◽

2016 ◽

Vol 22 (6) ◽

pp. 1316-1328 ◽

Cited By ~ 7

Author(s):

Michael Marko ◽

Chyongere Hsieh ◽

Eric Leith ◽

David Mastronarde ◽

Sohei Motoki

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Practical Experience ◽

Phase Plate ◽

Native State ◽

Automated Data Collection ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

Phase Plates ◽

Set Up

AbstractPhase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

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A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody

Journal of Virology ◽

10.1128/jvi.01597-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 5

Author(s):

Aušra Domanska ◽

Justin W. Flatt ◽

Joonas J. J. Jukonen ◽

James A. Geraets ◽

Sarah J. Butcher

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

High Resolution ◽

Cultured Cells ◽

Human Monoclonal Antibody ◽

Neutralizing Antibody ◽

Molecular Diagnostic ◽

Diagnostic Tools ◽

Human Parechovirus ◽

Cryo Electron Microscopy

ABSTRACTHuman parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCEHuman parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

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Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

10.1101/2021.06.15.448552 ◽

2021 ◽

Author(s):

Nicole Dimos ◽

Carl P.O. Helmer ◽

Andrea M. Chanique ◽

Markus C. Wahl ◽

Robert Kourist ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Structural Biology ◽

Structure Analysis ◽

Cryo Electron Microscopy ◽

High Resolution Structure ◽

Fast Development ◽

Pharmaceutical Industries ◽

Exquisite Specificity ◽

Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

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Hands on Methods for High Resolution Cryo-Electron Microscopy Structures of Heterogeneous Macromolecular Complexes

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2019.00033 ◽

2019 ◽

Vol 6 ◽

Cited By ~ 8

Author(s):

Marina Serna

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Macromolecular Complexes ◽

Cryo Electron Microscopy ◽

Hands On

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High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome

Nature ◽

10.1038/nature11872 ◽

2013 ◽

Vol 494 (7437) ◽

pp. 385-389 ◽

Cited By ~ 94

Author(s):

Yaser Hashem ◽

Amedee des Georges ◽

Jie Fu ◽

Sarah N. Buss ◽

Fabrice Jossinet ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Trypanosoma Brucei ◽

Cryo Electron Microscopy

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How Good Can Single-Particle Cryo-EM Become? What Remains Before It Approaches Its Physical Limits?

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070317-032828 ◽

2019 ◽

Vol 48 (1) ◽

pp. 45-61 ◽

Cited By ~ 26

Author(s):

Robert M. Glaeser

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Single Particle ◽

Energy Use ◽

Optimal Choice ◽

Structural Basis ◽

Detective Quantum Efficiency ◽

Quantum Metrology ◽

Cryo Electron Microscopy ◽

Induced Motion

Impressive though the achievements of single-particle cryo–electron microscopy are today, a substantial gap still remains between what is currently accomplished and what is theoretically possible. As is reviewed here, twofold or more improvements are possible as regards ( a) the detective quantum efficiency of cameras at high resolution, ( b) converting phase modulations to intensity modulations in the image, and ( c) recovering the full amount of high-resolution signal in the presence of beam-induced motion of the specimen. In addition, potential for improvement is reviewed for other topics such as optimal choice of electron energy, use of aberration correctors, and quantum metrology. With the help of such improvements, it does not seem to be too much to imagine that determining the structural basis for every aspect of catalytic control, signaling, and regulation, in any type of cell of interest, could easily be accelerated fivefold or more.

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