Implications of intracrystalline OC17 on the protection of lattice incorporated proteins.

Acknowledgements: H. B. C. would like to thank the Cambridge Commonwealth, European & International Trust, the Department of Engineering, the Nanoscience Centre, and Prof Jim Huntington from the Cambridge Institute for Medical Research, Department of Haematology, Cambridge, UK.Biogenic CaCO3 formation is regulated by crystallization proteins during crystal growth. Interactions of proteins with nascent mineral surfaces trigger proteins to be incorporated into the crystal lattice. As a result of incorporation, these intracrystalline proteins are protected in the lattice, an example of which is ancient eggshell proteins that have persisted in CaCO3 for thousands of years even under harsh environmental conditions. OC17 is an eggshell protein known to interact with CaCO3 during eggshell formation during which OC17 becomes incorporated into the lattice. Understanding protein incorporation into CaCO3 could offer insights into protein stability inside crystals. Here, we study the protection of OC17 in the CaCO3 lattice. Using thermogravimetric analysis we show that the effect of temperature on intracrystalline proteins of eggshells is negligible below 250 °C. Next, we show that lattice incorporation protects the OC17 structure despite a heat-treatment step that is shown to denature the protein. Because incorporated proteins need to be released from crystals, we verify metal chelation as a safe crystal dissolution method to avoid protein denaturation during reconstitution. Finally, we optimize the recombinant expression of OC17 which could allow engineering OC17 for engineered intracrystalline entrapment studies

Serious Issues with Cryo-EM Structures of Human Prothrombinase

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. The strict order of cleavage is first at Arg320 to produce the active intermediate meizothrombin, followed by cleavage at Arg271 to release thrombin from its membrane-anchoring pro-domains. Since thrombin is the effector enzyme in blood coagulation, a structural understanding of the assembly and function of prothrombinase is of critical importance. The affinity of fXa for fVa is low, with assembly dependent on a phospholipid membrane surface, which makes structural studies challenging. In a recent paper published in the journal Blood, Ruben and colleagues from the group of Enrico Di Cera reported a major breakthrough in the field (DOI: 10.1182/blood.2022015807): the cryo-EM structures of human prothrombinase on nanodiscs at 5.5Å resolution (7TPQ) and of a catalytically inert human prothrombinase with its substrate prothrombin in the absence of any membrane at 4.1Å resolution (7TPP). The breakthrough warranted a commentary in Blood and featured as a State of the Art lecture at the ISTH conference in 2022, including an accompanying article in the Journal of Thrombosis and Haemostasis (DOI:https://doi.org/10.1002/rth2.12830). As is the norm in structural biology, the original paper was reviewed without access to the deposited 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. We initially hypothesized that the low information content of the map EMD26060 and its unusual features were evidence of extreme reference bias, sometimes called ‘Einstein from noise’. However, the authors acceded to our request to deposit their data on the EMPIAR server (11615) and it is now clear that this is not the case (https://pubpeer.com/publications/1A47110B82A77E2902BFC5BE8E97B0). Rather, the pathology of the map is due to extreme orientation bias, resulting in poor quality, striation and discontinuity in the map, coupled with an over-estimation of resolution. We conclude that the map EMD26060 and coordinates 7TPP are deeply flawed and do not contain any useful information regarding the assembly or function of the prothrombinase complex