Combining X-ray crystallography and electron microscopy.

Michael G Rossmann; Marc C Morais; Petr G Leiman; Wei Zhang

doi:10.1016/j.str.2005.01.005

Home/Archive/PubMed

PubMed · 15766536

Combining X-ray crystallography and electron microscopy.

Michael G Rossmann·Marc C Morais·Petr G Leiman·Wei Zhang

View original source ↗Export metadata ↓

Abstract

The combination of cryo-electron microscopy to study large biological assemblies at low resolution with crystallography to determine near atomic structures of assembly fragments is quickly expanding the horizon of structural biology. This technique can be used to advantage in the study of large structures that cannot be crystallized, to follow dynamic processes, and to "purify" samples by visual selection of particles. Factors affecting the quality of cryo-electron microscopy maps and limits of accuracy in fitting known structural fragments are discussed.

Explore related subjects

Cryoelectron Microscopy Crystallography, X-Ray Molecular Structure Multiprotein Complexes Viral Proteins Viruses Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.Review

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Michael G Rossmann, Marc C Morais, Petr G Leiman, Wei Zhang. 2005. Combining X-ray crystallography and electron microscopy.. https://doi.org/10.1016/j.str.2005.01.005

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2025

Human endogenous retrovirus K (HERV-K) envelope structures in pre- and postfusion by cryo-EM.

Human endogenous retroviruses (HERVs) are remnants of ancient infections that comprise ~8% of the human genome. The HERV-K envelope glycoprotein (Env) is aberrantly expressed in cancers, autoimmune disorders, and neurodegenerative diseases, and is targeted by patients' own antibodies. However, a lack of structural information has limited molecular and immunological studies of the roles of HERVs in disease. Here, we present cryo-electron microscopy structures of stabilized HERV-K Env in the prefusion conformation, revealing a distinct fold and architecture compared to HIV and simian immunodeficiency virus. We also generated and characterized a panel of monoclonal antibodies with subunit and conformational specificity, serving as valuable research tools. These antibodies enabled structure determination of the postfusion conformation of HERV-K Env, including its unique "tether" helix, and antibody-bound prefusion Env. Together, these results provide a structural framework that opens the door to mechanistic studies of HERV-K Env and tools for its evaluation as a potential therapeutic target.

Cryoelectron Microscopy↗

PubMed2025

Disassembly activates Retron-Septu for antiphage defense.

Retrons are antiphage defense systems that produce multicopy single-stranded DNA (msDNA) and hold promise for genome engineering. However, the mechanisms of defense remain unclear. The Retron-Septu system integrates retron and Septu antiphage defenses. Cryo-electron microscopy structures reveal asymmetric nucleoprotein complexes comprising a reverse transcriptase, msDNA (a hybrid of msdDNA and msrRNA), and two PtuAB copies. msdDNA and msrRNA are essential for assembling this complex, with msrRNA adopting a conserved lariat-like structure that regulates reverse transcription. Notably, the assembled Retron-Septu complex is inactive, with msdDNA occupying the PtuA DNA binding site. Activation occurs upon disassembly, releasing PtuAB, which degrades single-stranded DNA to restrict phage replication. This "arrest-and-release" mechanism underscores the dynamic regulatory roles of msDNA, advancing our understanding of antiphage defense strategies.

Cryoelectron Microscopy↗

PubMed2025

Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies.

CRISPR-Cas9 has transformed genome editing through its programmability and versatility. Its DNA cleavage activity involves dynamic conformational changes during gRNA binding, DNA recognition, R-loop formation, and endonuclease activation. Understanding these molecular transitions is critical for improving the specificity and efficiency of Cas9, but this remains challenging precisely due to these rapid structural rearrangements. Early structural studies provided foundational insights but were limited to static states under catalytically inactive conditions. Cryo-EM has since enabled visualization of the dynamic nature of active Cas9, by enriching for specific conformations. This chapter introduces a kinetics-informed cryo-EM approach to capture the stepwise activation of Cas9 in real time. With thorough kinetic analyses, such as stopped-flow measurements of R-loop formation, we describe how to identify optimal timepoints to visualize key conformational states with cryo-EM. Integration of kinetic and structural data enables precise mapping of the conformational landscape of Cas9 and other dynamic enzymes, advancing our understanding of their molecular mechanisms and providing a framework for engineering enhanced variants.

Cryoelectron Microscopy↗