RELION: implementation of a Bayesian approach to cryo-EM structure determination.

Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 A compared to previous RELION versions.

/pdf/new-tools-for-automated-high-resolution-cryo-em-structure-d3sztfqg71.pdf

New tools for automated high-resolution cryo-EM structure determination in RELION-3.

Gctf: Real-time CTF determination and correction

The N-terminal domains of gasdermin proteins cause pyroptotic cell death by oligomerizing to form membrane pores.

Pore-forming activity and structural autoinhibition of the gasdermin family

Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

/pdf/structures-of-cas9-endonucleases-reveal-rna-mediated-e5eo54au8k.pdf

Structures of Cas9 Endonucleases Reveal RNA- Mediated Conformational Activation

EMAN2: an extensible image processing suite for electron microscopy.

As high-resolution biological transmission electron microscopy (TEM) has increased in popularity over recent years, the volume of data and number of projects underway has risen dramatically. A robust tool for effective data management is essential to efficiently process large data sets and extract maximum information from the available data. We present the Electron Microscopy Electronic Notebook (EMEN), a portable, object-oriented, web-based tool for TEM data archival and project management. EMEN has several unique features. First, the database is logically organized and annotated so multiple collaborators at different geographical locations can easily access and interpret the data without assistance. Second, the database was designed to provide flexibility to the user, so it can be used much as a lab notebook would be, while maintaining a structure suitable for data mining and direct interaction with data-processing software. Finally, as an object-oriented database, the database structure is dynamic and can be easily extended to incorporate information not defined in the original database specification.

Object oriented database and electronic notebook for transmission electron microscopy.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

/pdf/eman2-software-for-image-analysis-and-single-particle-4xtf4fkdry.pdf

Liwei Peng

Papers

EMAN2: an extensible image processing suite for electron microscopy.

Object oriented database and electronic notebook for transmission electron microscopy.

EMAN2: Software for Image Analysis and Single Particle Reconstruction