Serious issues with cryo-EM structures of human prothrombinase.

Abstract

Thrombin is generated from prothrombin through sequential cleavage at two sites by the enzyme complex prothrombinase, composed of a serine protease, factor (f) Xa and a cofactor, fVa, on phospholipid membranes. In a recent paper published in Blood, Ruben et al. (Ruben et al. 2022 Blood 139, 3463-3473 (doi:10.1182/blood.2022015807)) reported a major breakthrough in the field: the cryogenic electron microscopy structures of human prothrombinase on nanodiscs at 5.5 Å resolution (7TPQ) and of a catalytically inert human prothrombinase with its substrate prothrombin in the absence of any membrane at 4.1 Å resolution (7TPP). As is the norm in structural biology, the original paper was reviewed without access to the coordinates and maps, and it was therefore not possible for referees to assess the validity of the structures or their interpretations. In this article, we provide a post hoc analysis of the quality of the reported coordinates and maps, and look closely at the claimed intermolecular contacts on which the supposed breakthrough depends. We demonstrate that the work is deeply flawed, with not a single claimed intermolecular contact supported by the map, and conclude that the two reported structures do not contain any useful information regarding the assembly or function of the prothrombinase complex.