title: Testing the Validity of Single-Particle Maps at Low and High Resolution.
abstract: Single-particle electron cryomicroscopy may be used to determine the structure of biological assemblies by aligning and averaging low-contrast projection images recorded in the electron microscope. Recent progress in both experimental and computational methods has led to higher resolution three-dimensional maps, including for more challenging low molecular weight proteins, and this has highlighted the problems of model bias and over-fitting during iterative refinement that can potentially lead to incorrect map features at low or high resolution. This chapter discusses the principles and practice of specific validation tests that demonstrate the consistency of a 3D map with projection images. In addition, the chapter describes tests that detect over-fitting during refinement and lead to more robust assessment of both global and local map resolution. Application of several of these tests together demonstrates the reliability of single-particle maps that underpins their correct biological interpretation.
authors: Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom. Electronic address: peter.rosenthal@crick.ac.uk.
authors: P B
authors: PB
authors: Rosenthal
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000648:ultrastructure; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods / Q000706:statistics & numerical data*; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods; D007658:Ketone Oxidoreductases / Q000648:ultrastructure; D008958:Models, Molecular; D011487:Protein Conformation; D012984:Software; D001616:beta-Galactosidase / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+); D001616:beta-Galactosidase
keywords: Absolute hand; CryoEM; Electron microscopy; Model bias; Over-fitting; Resolution; Single-particle reconstruction; Tilt pair; Validation