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1.
Sci Rep ; 5: 14290, 2015 Sep 22.
Article in English | MEDLINE | ID: mdl-26390853

ABSTRACT

Cryo-Electron Microscopy (cryo-EM) of macromolecular complexes is a fundamental structural biology technique which is expanding at a very fast pace. Key to its success in elucidating the three-dimensional structure of a macromolecular complex, especially of small and non-symmetric ones, is the ability to start from a low resolution map, which is subsequently refined with the actual images collected at the microscope. There are several methods to produce this first structure. Among them, Random Conical Tilt (RCT) plays a prominent role due to its unbiased nature (it can create an initial model based on experimental measurements). In this article, we revise the fundamental mathematical expressions supporting RCT, providing new expressions handling all key geometrical parameters without the need of intermediate operations, leading to improved automation and overall reliability, essential for the success of cryo-EM when analyzing new complexes. We show that the here proposed RCT workflow based on the new formulation performs very well in practical cases, requiring very few image pairs (as low as 13 image pairs in one of our examples) to obtain relevant 3D maps.


Subject(s)
Cryoelectron Microscopy/methods , Macromolecular Substances/ultrastructure , Complement C3b/ultrastructure , Image Processing, Computer-Assisted , Models, Theoretical
2.
J Struct Biol ; 171(2): 197-206, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20362059

ABSTRACT

Two-dimensional analysis of projections of single-particles acquired by an electron microscope is a useful tool to help identifying the different kinds of projections present in a dataset and their different projection directions. Such analysis is also useful to distinguish between different kinds of particles or different particle conformations. In this paper we introduce a new algorithm for performing two-dimensional multireference alignment and classification that is based on a Hierarchical clustering approach using correntropy (instead of the more traditional correlation) and a modified criterion for the definition of the clusters specially suited for cases in which the Signal-to-Noise Ratio of the differences between classes is low. We show that our algorithm offers an improved sensitivity over current methods in use for distinguishing between different projection orientations and different particle conformations. This algorithm is publicly available through the software package Xmipp.


Subject(s)
Microscopy, Electron/methods , Algorithms , Bacteriorhodopsins/ultrastructure , Ribosomes/ultrastructure , Software
3.
J Struct Biol ; 167(3): 252-60, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19555764

ABSTRACT

The 3D reconstruction of biological specimens using Electron Microscopy is currently capable of achieving subnanometer resolution. Unfortunately, this goal requires gathering tens of thousands of projection images that are frequently selected manually from micrographs. In this paper we introduce a new automatic particle selection that learns from the user which particles are of interest. The training phase is semi-supervised so that the user can correct the algorithm during picking and specifically identify incorrectly picked particles. By treating such errors specially, the algorithm attempts to minimize the number of false positives. We show that our algorithm is able to produce datasets with fewer wrongly selected particles than previously reported methods. Another advantage is that we avoid the need for an initial reference volume from which to generate picking projections by instead learning which particles to pick from the user. This package has been made publicly available in the open-source package Xmipp.


Subject(s)
Algorithms , Artificial Intelligence , Imaging, Three-Dimensional/methods , Adenoviridae/chemistry , Antigens, Viral, Tumor/chemistry , Microscopy, Electron , Particle Size , Replication Protein A/chemistry
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