Structure Article EM-Fold: De Novo Atomic-Detail Protein Structure Determination from Medium-Resolution Density Maps Steffen Lindert,1 Nathan Alexander,1 Nils Wo¨tzel,1 Mert Karakasx,1 Phoebe L. Stewart,2 and Jens Meiler1,* 1Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN 37212, USA 2Department of Pharmacology and Cleveland Center for Membrane . Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR. of roughly 90% X-ray, 8% NMR, and 2% EM The first near atomic resolution structure by cryoEM lay In addition to recent developments in direct detector technology, the determination of the first near-atomic resolution structure of a coronavirus spike glycoprotein trimer was made possible by (i) engineering a pre-fusion stabilized ectodomain construct, (ii) using extensive computational classification of particle images to sort out sample . Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR W. Trent Franks, Benjamin J. Wylie, Heather L. Frericks Schmidt, Andrew J. Nieuwkoop, Rebecca-Maria Mayrhofer, To leverage the unique potential of SSNMR to solve membrane protein and fibril structures (5-8) at atomic resolution therefore requires further advances in SSNMR structure methods. Subsequent developments . Protein crystallography may provide atomic or near atomic resolution, when small details of the protein structure can be resolved with very high accuracy. It has emerged as a powerful technique for the study of the structure and dynamics of proteins, providing detailed insights into biomolecular function. Structure determination is a process to obtain the three dimension coordinates of a molecule or biomolecule by a selected method. Here we demonstrate de novo protein structure determination to a level with accurate atomic detail using medium-resolution density maps to restrain the simulation. At resolutions better than 4 Å, atomic model building starts to become possible, but the direct visualization of true atomic positions in protein structure determination requires much higher (better than 1.5 Å) resolution, which so far has not been attained by cryo-EM. i.e. 1. Termini restraining of small membrane proteins enables structure determination at . Resolution in terms of electron density is a measure of the resolvability in the electron density map of a molecule. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (30 kDa) proteins.We introduce an integrated structure determination approach that . Obtaining de novo atomic-resolution structures of such proteins is extremely challenging, if not impossible; however, results from various biophysical methods, which on their own are incapable of determining a de novo structure, can be integrated to determine a structural model that satisfies the maximum number of experimental restraints. The structure determination process is iterative and involves frequent 1. Although NMR can determine protein structures at atomic resolution, its . The first structure solved was that of lysozyme at 2.9 Å resolution by still diffraction 2 and then at 2.5 Å resolution by continuous rotation. Whereas most atomic-resolution structures are solved using X-ray crystallography, single-particle cryo-EM has emerged as a. The influence ofdynamical scattering onthemeasuredintensities wasthoughtto limit accurate determination of structure factors (23-26). In MicroED an extremely low elec-tron dose is used to collect multiple electron diffraction patterns from each crystal at varying angles (Figures 2A and 3). We have obtained a total of 15 crystal structures of six different proteins (42, 43), three of which are of human origin and two from other vertebrates (Table 1 and Fig. Proteins have too many protons to be resolved by one-dimensional NMR. At resolutions better than 4 Å, atomic model building starts to become possible, but the direct visualization of true atomic positions in protein structure determination requires much higher (better than 1.5 Å) resolution, which so far has not been attained by cryo-EM. Model building is a key step in macromolecular structure determination. Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR Proceedings of The National Academy of Sciences, 2008 H. Frericks Schmidt In X-ray crystallography, resolution is the highest resolvable peak in the diffraction pattern, while resolution in cryo-electron microscopy is a frequency space comparison of two halves of the data, which strives to correlate with the X-ray definition. Furthermore, and perhaps more importantly, the retention of high-resolution diffraction demonstrates that cryo-FIB milling preserves atomic-level structure within the lamellae, suggesting that this method might provide a vehicle for the determination of molecular and atomic structures via electron imaging and diffraction from not only protein . Start studying Methods for Protein Structure Determination: X-Ray Crystallography. Atomic-resolution structure determination is crucial for understanding protein function. At resolutions better than 4 Å, atomic model building starts becoming possible but the direct visualization of true atomic positions in protein structure determination requires significantly higher resolution, which so far could not be attained by cryo-EM. Protein structure determination methods using magic-angle spinning solid-state nuclear magnetic resonance (MAS SSNMR) have experienced a remarkable development in the past decade. ; Protein folding The most powerful tool for determining the residual structures of unfolded . ; Molecular dynamics The most powerful technique for quantifying motional properties of biomacromolecules. An aqueous biological sample is frozen rapidly and irradiated with a beam of electrons from a transmission electron microscope. The key to success was to identify NMR-accessible parameters that unambiguously determine the spatial arrangement of polypeptide chains. 2012 Mar 7;20(3):464-78. doi: 10.1016/j.str.2012.01.023. This has been changed in the last . 51 While many known protein structures were solved using high-resolution techniques, such as X-ray crystallography, for many interesting and biomedically relevant proteins, we can only collect . The [14]' possible, structure determination of biological assemblies, dominated peptides, and chemical compounds has been in X-ray crystallography. Crystal- This technique has been largely limited to protein nanocrystals which grow either as needles or plates measuring only a few hundred nanometers in thickness. At resolutions better than 4 Å, atomic model building starts to become possible, but the direct visualization of true atomic positions in protein structure determination requires much higher (better than 1.5 Å) resolution, which so far has not been attained by cryo-EM. report methods to account for radiation damage and local changes in defocus and image drift, enabling visualization of atomic resolution features in a cryo-EM density map of inhibitor-bound β-galactosidase, and measuring of local flexibility of the bound inhibitor using constrained molecular dynamics simulations. The long-range goal of the Protein Structure Initiative (PSI) is to make the three-dimensional atomic-level structures of most proteins easily obtainablefrom knowledge of their corresponding DNA sequences. Deposition of atomic or near-atomic-resolution structures in the Protein Data Bank according to different experimental methods. It can determine structural information for complexes and crystallization-resistant samples, as well as vital cellular context. This helps to generate the topological map and study mode of action . 39 Recent advances in single particle cryo-EM1,2 have enabled near-atomic resolution structure 40 determination of biomedically important protein complexes3-5, bringing the technique to the 41 attention of the general biological research community and pharmaceutical companies. This high quality data obtained on the SuperNova allows the determination of novel structures using the Sulphur-SAD method and, with suitable crystals, makes atomic-resolution protein structures possible in the home laboratory . This paper presents my recollections on the development of protein structure determination by NMR in solution from 1968 to 1992. The long-range goal of the Protein Structure Initiative (PSI) is to make the three-dimensional atomic-level structures of most proteins easily obtainablefrom knowledge of their corresponding DNA sequences. Two-dimensional crystals and helices Until very recently, electron crystallography has been the only EM technique that had reached sufficient resolution to produce atomic models of proteins (soluble or membrane). Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimized for . to produce atomic models of proteins (soluble or mem- brane). Cryo-electron microscopy provides near-atomic resolution 3D protein structure. Our protocol consists of two steps: 1) determination of protein topology with an improved version of EM-Fold ( Lindert et al., 2009 ) and 2) refinement to atomic detail accuracy . High-Resolution Protein Structure Determination by Serial This copy is for your personal, non-commercial use only. Fan et al. determination at atomic resolution, X-ray crystallography and NMR spectroscopy. Initiated in 1999 b y NIH Phase I included 9 large centers for high throughput structure determination Phase I ran from ~2000 - 2005 Resolution is determined from these farthest spots based on their angle θ from the X-ray beam, using the Bragg equation solved for d : d = λ / (2 sin θ) with resolution of around 2.3Å or better). Figure 1. The eukaryotic organisms, . structure determination of proteins by X -ray crystallography and NMR methods and to apply these strategies to scan the entire genome of an organism at a rapid pace. Determination of Resolution The portion of the macromolecule that is best ordered in the crystal is responsible for diffraction spots that are farthest from the axis of the X-ray beam. 1 presents an outline of the method (4, 5) that covers the preparation of the protein for the NMR experiments, the @article{osti_1425979, title = {Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED}, author = {Sawaya, Michael R. and Rodriguez, Jose and Cascio, Duilio and Collazo, Michael J. and Shi, Dan and Reyes, Francis E. and Hattne, Johan and Gonen, Tamir and Eisenberg, David S.}, abstractNote = {Electrons, because of their strong interaction with matter, produce . Structure Article EM-Fold: De Novo Atomic-Detail Protein Structure Determination from Medium-Resolution Density Maps Steffen Lindert,1 Nathan Alexander,1 Nils Wo¨tzel,1 Mert Karakasx,1 Phoebe L. 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