Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Cryo-electron Microscopy01:28

Cryo-electron Microscopy

4.1K
Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
4.1K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Prothrombinase processivity is conferred by substrate allostery.

The EMBO journal·2026
Same author

Recruitment of bifunctional regulator thermospermine to methylated ribosomes directs xylem fate.

Science (New York, N.Y.)·2026
Same author

The preclinical profile of SerpinPC: a potential new treatment for hemophilia.

Blood advances·2025
Same author

Serious issues with cryo-EM structures of human prothrombinase.

Open biology·2025
Same author

The chemical landscape of the human ribosome at 1.67 Å resolution.

bioRxiv : the preprint server for biology·2023
Same author

Teaching old drugs new tricks.

eLife·2022

Related Experiment Video

Updated: Jan 14, 2026

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
08:27

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Published on: January 7, 2019

9.9K

A 3.3-Å cryo-EM structure of an engineered high-affinity human prothrombinase complex.

Fatma Işık Üstok1, Alexandre Faille1, James A Huntington1

  • 1Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

Blood
|October 27, 2025
PubMed
Summary

Researchers engineered a high-affinity human prothrombinase complex, M17, that functions independently of phospholipid membranes. This breakthrough enabled cryo-electron microscopy (cryo-EM) structural analysis, revealing key interactions within the prothrombinase complex.

More Related Videos

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
12:24

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Published on: June 3, 2014

12.7K
Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit
22:10

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Published on: June 28, 2013

13.7K

Related Experiment Videos

Last Updated: Jan 14, 2026

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
08:27

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Published on: January 7, 2019

9.9K
Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
12:24

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Published on: June 3, 2014

12.7K
Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit
22:10

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Published on: June 28, 2013

13.7K

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Medicine

Background:

  • Thrombin generation from prothrombin is catalyzed by prothrombinase (factor Xa and factor Va).
  • Native prothrombinase requires phospholipid membranes for assembly and function due to low factor Xa-factor Va affinity.
  • Venom factor Xa variants exhibit high affinity for factor Va, enabling prothrombin activation independent of phospholipids.

Purpose of the Study:

  • To engineer a high-affinity, phospholipid-independent human prothrombinase complex.
  • To determine the high-resolution structure of the engineered prothrombinase complex using cryo-electron microscopy (cryo-EM).
  • To elucidate the molecular interactions governing prothrombinase assembly and function.

Main Methods:

  • Site-directed mutagenesis of human factor Xa to create a high-affinity variant (M17).
  • Cryogenic electron microscopy (cryo-EM) for structural determination of the M17-prothrombinase complex at 3.3 Å resolution.
  • High-resolution crystal structure analysis of factor Xa complexed with a synthetic a2-peptide to resolve loop interactions.

Main Results:

  • A novel M17-prothrombinase complex with significantly increased affinity and phospholipid independence was successfully engineered.
  • Cryo-EM structure revealed detailed interactions between factor Xa (serine protease and EGF2 domains) and factor Va (A2 and A3 domains), burying 4,900 Ų of surface area.
  • Structural analysis identified key side-chain interactions, including the interaction of the acidic a2-loop with the basic heparin-binding site of factor Xa.

Conclusions:

  • The engineered M17-prothrombinase provides a structural basis for understanding high-affinity complex formation.
  • The structure is consistent with existing biochemical and mutagenesis data, offering new insights into prothrombinase assembly.
  • This work advances the understanding of blood coagulation factor interactions and potential therapeutic targets.