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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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Side-chain details from regions in subunit B shown with map and model.Comparable regions from the other two capsid subunits are shown in Supplementary Fig. 5.
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f5: Side-chain details from regions in subunit B shown with map and model.Comparable regions from the other two capsid subunits are shown in Supplementary Fig. 5.

Mentions: Following the real-space model optimization of individual capsid subunits and adjusting map pixel scale as described above, a complete asymmetric unit was assembled (Supplementary Fig. 4). From these models, an additional round of real-space optimization was performed to improve interfaces and eliminate clashes. The asymmetric unit was iteratively modelled using the real-space optimization routine with minor manual adjustments made in COOT. After five rounds of optimization the asymmetric unit model converged to a final asymmetric unit model with MolProbity and clash score statistics in the top 90% for structures at equivalent resolution. At the next level of interactions, the asymmetric unit interfaces, seven surrounding asymmetric units were added to the original asymmetric unit and real-space optimization was performed on this complex (Supplementary Movie 2). After real-space optimization, our model revealed good fit-to-density and ranked high in terms of protein geometry and clash score (Table 1) when compared with models in the Protein Data Bank (PDB)42 at equivalent resolution. Figure 5 and Supplementary Fig. 6 show examples of regions of each subunit for their match between density and the model with unambiguous side-chain resolvability.


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)

Side-chain details from regions in subunit B shown with map and model.Comparable regions from the other two capsid subunits are shown in Supplementary Fig. 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Side-chain details from regions in subunit B shown with map and model.Comparable regions from the other two capsid subunits are shown in Supplementary Fig. 5.
Mentions: Following the real-space model optimization of individual capsid subunits and adjusting map pixel scale as described above, a complete asymmetric unit was assembled (Supplementary Fig. 4). From these models, an additional round of real-space optimization was performed to improve interfaces and eliminate clashes. The asymmetric unit was iteratively modelled using the real-space optimization routine with minor manual adjustments made in COOT. After five rounds of optimization the asymmetric unit model converged to a final asymmetric unit model with MolProbity and clash score statistics in the top 90% for structures at equivalent resolution. At the next level of interactions, the asymmetric unit interfaces, seven surrounding asymmetric units were added to the original asymmetric unit and real-space optimization was performed on this complex (Supplementary Movie 2). After real-space optimization, our model revealed good fit-to-density and ranked high in terms of protein geometry and clash score (Table 1) when compared with models in the Protein Data Bank (PDB)42 at equivalent resolution. Figure 5 and Supplementary Fig. 6 show examples of regions of each subunit for their match between density and the model with unambiguous side-chain resolvability.

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