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Gold nanocrystal labels provide a sequence-to-3D structure map in SAXS reconstructions.

ABSTRACT: Small-angle x-ray scattering (SAXS) is a powerful technique to probe the structure of biological macromolecules and their complexes under virtually arbitrary solution conditions, without the need for crystallization. While it is possible to reconstruct molecular shapes from SAXS data ab initio, the resulting electron density maps have a resolution of ~1 nm and are often insufficient to reliably assign secondary structure elements or domains. We show that SAXS data of gold-labeled samples significantly enhance the information content of SAXS measurements, allowing the unambiguous assignment of macromolecular sequence motifs to specific locations within a SAXS structure. We first demonstrate our approach for site-specifically internally and end-labeled DNA and an RNA motif. In addition, we present a protocol for highly uniform and site-specific labeling of proteins with small (~1.4 nm diameter) gold particles and apply our method to the signaling protein calmodulin. In all cases, the position of the small gold probes can be reliably identified in low-resolution electron density maps. Enhancing low-resolution measurements by site-selective gold labeling provides an attractive approach to aid modeling of a large range of macromolecular systems.

SUBMITTER: Zettl T

PROVIDER: S-EPMC5969820 | biostudies-other | 2018 May

REPOSITORIES: biostudies-other

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Gold nanocrystal labels provide a sequence-to-3D structure map in SAXS reconstructions.

Zettl Thomas T Mathew Rebecca S RS Shi Xuesong X Doniach Sebastian S Herschlag Daniel D Harbury Pehr A B PAB Lipfert Jan J

Science advances 20180525 5

Small-angle x-ray scattering (SAXS) is a powerful technique to probe the structure of biological macromolecules and their complexes under virtually arbitrary solution conditions, without the need for crystallization. While it is possible to reconstruct molecular shapes from SAXS data ab initio, the resulting electron density maps have a resolution of ~1 nm and are often insufficient to reliably assign secondary structure elements or domains. We show that SAXS data of gold-labeled samples signifi ...[more]

PMID: 29806025

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Project description:BACKGROUND:Three dimensional (3D) genome spatial organization is critical for numerous cellular functions, including transcription, while certain conformation-driven structural alterations are frequently oncogenic. Genome conformation had been difficult to elucidate but the advent chromatin conformation capture assays, notably Hi-C, has transformed understanding of chromatin architecture and yielded numerous biological insights. Although most of these findings have flowed from analysis of proximity data produced by these assays, added value in generating 3D reconstructions has been demonstrated, deriving, in part, from superposing genomic features on the reconstruction. However, advantages of 3D structure-based analyses are clearly conditional on the accuracy of the attendant reconstructions, which is difficult to assess. Proponents of competing reconstruction algorithms have evaluated their accuracy by recourse to simulation of toy structures and/or limited fluorescence in situ hybridization (FISH) imaging that features a handful of low resolution probes. Accordingly, new methods of reconstruction accuracy assessment are needed. RESULTS:Here we utilize two recently devised assays to develop methodology for assessing 3D reconstruction accuracy. Multiplex FISH increases the number of probes by an order of magnitude and hence the number of inter-probe distances by two orders, providing sufficient information for structure-level evaluation via mean-squared deviations (MSD). Crucially, underscoring multiplex FISH applications are large numbers of coordinate-system aligned replicates that provide the basis for a referent distribution for MSD statistics. Using this system we show that reconstructions based on Hi-C data for IMR90 cells are accurate for some chromosomes but not others. The second new assay, genome architecture mapping, utilizes large numbers of thin cryosections to obtain a measure of proximity. We exploit the planarity of the cryosections - not used in inferring proximity - to obtain measures of reconstruction accuracy, with referents provided via resampling. Application to mouse embryonic stem cells shows reconstruction accuracies that vary by chromosome. CONCLUSIONS:We have developed methods for assessing the accuracy of 3D genome reconstructions that exploit features of recently advanced multiplex FISH and genome architecture mapping assays. These approaches can help overcome the absence of gold standards for making such assessments which are important in view of the considerable uncertainties surrounding 3D genome reconstruction.

Project description:The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing.In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy.We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues, including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7-4.8 Å C(?)-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org). This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of protein structures, new strategies in protein and drug design, and the identification of functional genetic variants in normal and disease genomes.

Dataset Information

Gold nanocrystal labels provide a sequence-to-3D structure map in SAXS reconstructions.

Publications

Gold nanocrystal labels provide a sequence-to-3D structure map in SAXS reconstructions.

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