Electron Cryo‐microscopy as a Tool for Structure‐Based Drug Development

Abstract

For decades, X-ray crystallography and NMR have been the most important techniques for studying the atomic structure of macromolecules. However, as a result of size, instability, low yield, and other factors, many macromolecules are difficult to crystallize or unsuitable for NMR studies. Electron cryo-microscopy (cryo-EM) does not depend on crystals and has therefore been the method of choice for many macromolecular complexes that cannot be crystallized, but atomic resolution has mostly been beyond its reach. A new generation of detectors that are capable of sensing directly the incident electrons has recently revolutionized the field, with structures of macromolecules now routinely being solved to near-atomic resolution. In this review, we summarize some of the most recent examples of high-resolution cryo-EM structures. We put particular emphasis on proteins with pharmacological relevance that have traditionally been inaccessible to crystallography. Furthermore, we discuss examples where interactions with small molecules have been fully characterized at atomic resolution. Finally, we stress the current limits of cryo-EM, and methodological issues related to its usage as a tool for drug development.

Presence and structure‐activity relationship of intrinsically disordered regions across mucins

“…For many proteins implicated in cancer, structural biology information has proven invaluable for understanding their functional implications as well as discovering novel therapeutic modalities. [49][50][51][52] Unfortunately, it is difficult to study mucins structurally by traditional methods. Crystallographic methods falter because of the sheer size of mucins (up to 14 000 kDa), extensive variation in the number of tandem repeats within the VNTR (up to 120), sequence variation, and inability to clone, express, and purify fully folded and glycosylated forms.…”

“…For many proteins implicated in cancer, structural biology information has proven invaluable for understanding their functional implications as well as discovering novel therapeutic modalities . Unfortunately, it is difficult to study mucins structurally by traditional methods.…”

Electron cryomicroscopy as a powerful tool in biomedical research

“…The Ca 2+ channel was also resolved in complex with the spider peptide toxin DkTx and a small vanilloid agonist [61]. The small molecules were clearly resolved in the structures, demonstrating that cryo-EM is indeed able to visualize ligands [62]. …”

Kryo‐Elektronenmikroskopie revolutioniert die Strukturbestimmung von Biomolekülen

“…[9] Dennoch kann die Kryo-EM bislang nicht als Hochdurchsatz-Verfahren eingesetzt werden. Sie ermçglichte in den letzten vier Jahren die Bestimmung hochaufgelçster Strukturen vieler Proteinkomplexe,die durch Rçntgenkristallographie nicht hätten gelçst werden kçnnen, darunter Spliceosomenkomplexe,R ibosomen, Kationenkanäle,r espiratorische Superkomplexe und Aktomyosinkomplexe.D urch die mit Einzelpartikel-Kryo-EM erreichten Auflçsungen kçnnen nicht nur einzelne Aminosäureseitenketten präzise platziert werden, in vielen Fällen ist sogar das exakte Positionieren kleiner Moleküle mçglich (Abbildung 2).…”

“…Den entscheidenden Impuls für diesen Ansatz lieferte Joachim Frank und trieb über viele Jahre die Entwicklung dieser computerbasierten Bildverarbeitung voran. [8] Aufgrund physikalischer Grenzen der klassischen Bildaufnahme konnten jedoch keine optimalen Signal-zu- Rausch [9] Dennoch kann die Kryo-EM bislang nicht als Hochdurchsatz-Verfahren eingesetzt werden. Viele Schritte bei der Probenvorbereitung sowie der Datenaufnahme und -verarbeitung erfordern nach wie vor erfahrene Nutzer.D ie Bedingungen müssen fürj ede Probe individuell angepasst werden.…”