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An atomic model of brome mosaic virus using direct electron detection and real-space optimization.

Wang Z, Hryc CF, Bammes B, Afonine PV, Jakana J, Chen DH, Liu X, Baker ML, Kao C, Ludtke SJ, Schmid MF, Adams PD, Chiu W - Nat Commun (2014)

Bottom Line: We used the map to derive an all-atom model with a newly implemented real-space optimization protocol.The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps.This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.

View Article: PubMed Central - PubMed

Affiliation: 1] National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA [2].

ABSTRACT
Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.

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Related in: MedlinePlus

Cryo-EM density maps of two independent data sets and 3D reconstructions after 38 refinement iterations.With maps generated from data sets 1 and 2 (a) and their combined maps (b). The initial model for each data set/reconstruction was generated using EMAN1. Subsequent refinement was computed using MPSA. The final five iterations were completed in EMAN1, resulting in the final 3D density maps.
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f2: Cryo-EM density maps of two independent data sets and 3D reconstructions after 38 refinement iterations.With maps generated from data sets 1 and 2 (a) and their combined maps (b). The initial model for each data set/reconstruction was generated using EMAN1. Subsequent refinement was computed using MPSA. The final five iterations were completed in EMAN1, resulting in the final 3D density maps.

Mentions: Our goal is to produce the structure with the best quality and resolution from direct detection movie-mode data. To this end, we performed multiple reconstructions using various sums of frames and damage compensation. To properly assess the resolution of our final density maps and to further validate our reconstructions, we followed the gold-standard procedure, that is, dividing the entire set of particle data into two separate data subsets prior to further image processing and 3D reconstruction2223. Each data subset used an independent initial model generated by starticos of EMAN1 (ref. 24)24 (Fig. 2), which builds a rough reconstruction from particles close to the five, three and twofold symmetry axes. Multi-path simulated annealing (MPSA)25 was used to quickly refine the map to 5 Å. MPSA utilizes cross common lines in Fourier space to determine both centre and orientation of a particle image simultaneously. When the independent reconstructions reached a resolution of ~5 Å, we switched to EMAN1 and used a finer particle orientation search (Supplementary Fig. 2). Resolution was estimated by the 0.143 Fourier shell correlation (FSC) criterion between the two independent reconstructions, with an inner mask to remove the RNA, which lacks overall icosahedral symmetry. The mask had a Gaussian profile with a width of 5 Å to avoid the sharp mask-edge effects, which may cause resolution exaggeration. A combined map was then generated from all of the data, and filtered. The size scale of the map was refined during the model optimization process (Methods). All reported resolutions are based on the final adjusted map scale of 0.99 Å pixel−1.


An atomic model of brome mosaic virus using direct electron detection and real-space optimization.

Wang Z, Hryc CF, Bammes B, Afonine PV, Jakana J, Chen DH, Liu X, Baker ML, Kao C, Ludtke SJ, Schmid MF, Adams PD, Chiu W - Nat Commun (2014)

Cryo-EM density maps of two independent data sets and 3D reconstructions after 38 refinement iterations.With maps generated from data sets 1 and 2 (a) and their combined maps (b). The initial model for each data set/reconstruction was generated using EMAN1. Subsequent refinement was computed using MPSA. The final five iterations were completed in EMAN1, resulting in the final 3D density maps.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4155512&req=5

f2: Cryo-EM density maps of two independent data sets and 3D reconstructions after 38 refinement iterations.With maps generated from data sets 1 and 2 (a) and their combined maps (b). The initial model for each data set/reconstruction was generated using EMAN1. Subsequent refinement was computed using MPSA. The final five iterations were completed in EMAN1, resulting in the final 3D density maps.
Mentions: Our goal is to produce the structure with the best quality and resolution from direct detection movie-mode data. To this end, we performed multiple reconstructions using various sums of frames and damage compensation. To properly assess the resolution of our final density maps and to further validate our reconstructions, we followed the gold-standard procedure, that is, dividing the entire set of particle data into two separate data subsets prior to further image processing and 3D reconstruction2223. Each data subset used an independent initial model generated by starticos of EMAN1 (ref. 24)24 (Fig. 2), which builds a rough reconstruction from particles close to the five, three and twofold symmetry axes. Multi-path simulated annealing (MPSA)25 was used to quickly refine the map to 5 Å. MPSA utilizes cross common lines in Fourier space to determine both centre and orientation of a particle image simultaneously. When the independent reconstructions reached a resolution of ~5 Å, we switched to EMAN1 and used a finer particle orientation search (Supplementary Fig. 2). Resolution was estimated by the 0.143 Fourier shell correlation (FSC) criterion between the two independent reconstructions, with an inner mask to remove the RNA, which lacks overall icosahedral symmetry. The mask had a Gaussian profile with a width of 5 Å to avoid the sharp mask-edge effects, which may cause resolution exaggeration. A combined map was then generated from all of the data, and filtered. The size scale of the map was refined during the model optimization process (Methods). All reported resolutions are based on the final adjusted map scale of 0.99 Å pixel−1.

Bottom Line: We used the map to derive an all-atom model with a newly implemented real-space optimization protocol.The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps.This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.

View Article: PubMed Central - PubMed

Affiliation: 1] National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA [2].

ABSTRACT
Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.

Show MeSH
Related in: MedlinePlus