Comparing Cryo-EM Reconstructions and Validating Atomic Model Fit Using Difference Maps.

Agnel Praveen Joseph,Maya Topf,Tom Burnley,Martyn Winn,Arjen Jakobi,Ardan Patwardhan,Ingvar Lagerstedt

doi:10.1021/acs.jcim.9b01103

Abstract

Cryogenic electron microscopy (cryo-EM) is a powerful technique for determining structures of multiple conformational or compositional states of macromolecular assemblies involved in cellular processes. Recent technological developments have led to a leap in the resolution of many cryo-EM data sets, making atomic model building more common for data interpretation. We present a method for calculating differences between two cryo-EM maps or a map and a fitted atomic model. The proposed approach works by scaling the maps using amplitude matching in resolution shells. To account for variability in local resolution of cryo-EM data, we include a procedure for local amplitude scaling that enables appropriate scaling of local map contrast. The approach is implemented as a user-friendly tool in the CCP-EM software package. To obtain clean and interpretable differences, we propose a protocol involving steps to process the input maps and output differences. We demonstrate the utility of the method for identifying conformational and compositional differences including ligands. We also highlight the use of difference maps for evaluating atomic model fit in cryo-EM maps.

Highlights

Over the past few years, cryogenic electron microscopy has had an enormous impact on the structure determination of large and dynamic molecular machines
There has been a large influx of structures solved using Cryogenic electron microscopy (cryo-EM) in the central repository the Electron
Difference maps are calculated for such comparisons, and the maps are scaled to an equivalent density range prior to such calculations

Summary

Introduction

Over the past few years, cryogenic electron microscopy (cryo-EM) has had an enormous impact on the structure determination of large and dynamic molecular machines. Over the past few years, cryogenic electron microscopy Better detectors and algorithms for three-dimensional structure reconstruction from images have helped in achieving near atomic resolutions. There has been a large influx of structures solved using cryo-EM in the central repository the Electron. Microscopy Data Bank (EMDB, https://www.ebi.ac.uk/pdbe/ emdb/statistics_main.html/) and this is expected to rise dramatically in the coming years. Cryo-EM enables structure determination of different functional forms of biological macromolecules in the near-native state.[4] Comparison of individual forms gives insights into the biological pathway of the molecule. Approaches for global density scaling exist; e.g., Relion[5] (relion_image_handler), EMAN26 (e2proc3d), diffmap (http://grigoriefflab.janelia.org/ diffmap), and BSoft[7] (bscale) work by scaling amplitudes in each resolution shell of a map to that of a reference power spectrum (usually based on an atomic model)

Methods

Discussion

Conclusion