Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant - PubMed (original) (raw)

Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant

Yong Zi Tan et al. Nat Commun. 2018.

Abstract

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we exploit several technical advances to improve resolution, including per-particle contrast transfer function (CTF) refinement and correction for Ewald sphere curvature. The latter is demonstrated with several experimental samples and should become more standard as resolutions increase or at lower microscope accelerating voltages. The combined application of the described methods to micrographs recorded on a Titan Krios enables structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle. The resulting structural details provide an improved model for understanding the biology of AAV that will guide future vector development for gene therapy.

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Conflict of interest statement

M.A.M. is a SAB member for Voyager Therapeutics, Inc., and AGTC, has a sponsored research agreement with AGTC and Voyager Therapeutics, and is a consultant for Intima Biosciences, Inc. M.A.M. is a co-founder of StrideBio, Inc. This is a biopharmaceutical company with interest in developing AAV vectors for gene delivery application. R.J.S. is the scientific founder of Bamboo Therapeutics, Asklepios Biopharmaceutics, Chatham Therapeutics, and Merlin. These companies also have interest in the development of AAV for gene delivery applications. The remaining authors declare no competing interests.

Figures

Fig. 1

Procedures and implications for obtaining a sub-2 Å resolution reconstruction of AAV2L336C. a Fourier shell correlation (FSC) curves showing independent contributions of each operation to the final resolution. The resolution cut-off of 0.143 (gray dotted line) is indicated. Each FSC curve was generated by “turning off” one of the respective operations from the final refinement (1A). Correcting for rotational beam-induced movement (1B) improves the FSC at low spatial frequency. Discriminating particles based on their score (3) led to a lower resolution reconstruction (1.89 Å) as compared to equally weighting all particles (1.84 Å). Cumulative loss by “turning off” all the tested operations is indicated by (7). b ResLog plot of the dataset. Five replicates were performed for each data point with random particle distributions and the standard deviation is plotted. Linear regression was fitted and the _b_-factor indicated. c Fourier shell correlation (FSC) curves describing the half-map (blue solid line) and map-to-model (purple solid line) resolutions, as well as a histogram of directional resolutions sampled evenly over the 3DFSC (yellow), and corresponding sphericity value. The resolution cut-offs of 0.143 (blue dotted line) and 0.5 (purple dotted line) were used respectively. d 1.86 Å reconstruction of the AAV2 viral capsid using single-particle cryo-EM, colored by local resolution. Slices through the reconstruction displayed in sub-panels start from the capsid center (left) move progressively towards the front (top right and bottom right). These show few deviations from the global resolution, with the best local resolution at 1.78 Å (primarily located inside the core region of the capsid shell). Both scales correspond to a distance of 100 Å

Fig. 2

Ewald sphere curvature correction improves the resolution of AAV2L336C and rotavirus VP6. ResLog plot of the a AAV2L336C dataset and b rotavirus VP6 dataset from Grant and Grigorieff, with (green) and without (red) Ewald sphere curvature correction in Frealign9. Five replicates were performed for each data point with random particle distributions, and the standard deviation is plotted. Linear regression is plotted for the first linear portion of each set of points. The resolution limits predicted by Spence,, DeRosier, and Downing & Glaeser for a 250 Å and 700 Å macromolecule respectively are indicated. c The theoretical resolution limits caused by curvature of the Ewald sphere at 300 keV plotted against particle diameter. Reconstructions from this study and a previous study that have shown an improvement in final resolution with Ewald sphere curvature correction are plotted (orange with and green without Ewald sphere curvature correction). The minimum resolutions at which noticeable improvements due to Ewald sphere curvature correction occur are also plotted in blue for AAV2L336C and rotavirus VP6, and follow closely with the Spence limit. d The theoretical resolution limits caused by the curvature of the Ewald sphere for particles smaller than 300 Å in diameter at 300 keV. Points on the Spence limit corresponding to 50, 100, 150, and 200 Å particle diameters are indicated and labeled in blue. Rest of the coloring scheme is the same as in c

Fig. 3

High-resolution information within the 1.86 Å reconstruction of AAV2L336C. a Stereo view of a slice through the map and model containing both amino-acid residues and water molecules. b Stereo view of a slice through a beta sheet. c Map densities for each of the 20 types of amino-acid residues. The amino-acid residues are shown as stick representation and colored according to atom type: C = yellow, O = red, N = blue, S = green inside either a translucent solid density (a, b) or black mesh density map (c). H = white atoms are displayed for c. The scale bars correspond to 10 Å

Fig. 4

High-resolution map aids in placement of correct rotamers for AAV2L336C. At 1.86 Å (map in black mesh), uncertainty in rotamer and backbone conformation in a previous model of AAV2 (1LP3, in cyan) determined by X-ray crystallography to 3 Å resolution can now be accurately modeled (yellow)

Fig. 5

Comparison of AAV2WT and AAV2L336C. a The density map with the modeled residues for the DE-loop for AAV2WT and AAV2L336C. The wild-type L336 and substituted C336 residues are shown. The AAV2WT has been the highest resolution density map for AAV2 published to date, at 3 Å. The amino-acid residues are shown as stick representation and colored according to atom type: C = yellow, O = red, N = blue, H = white, S = green inside a black mesh density map. b Superposed pentamer models of AAV2WT (blue) and AAV2L336C (yellow). Lower panel show close-up views of the fivefold region as side view and top view perspectives. Side-chain atoms for individual residues of interest are shown

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Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant - PubMed (original) (raw)