Sequence assignment validation in cryo-EM models with checkMySequence

The availability of new AI-based protein structure prediction tools radically changed the way cryo-EM maps are interpreted, but it has not eliminated the challenges of map interpretation faced by a microscopist. Models will continue to be locally rebuilt and refined using interactive tools. This inevitably results in occasional errors, among which register-shifts remain one of the most difficult to identify and correct. Here we introduce checkMySequence; a fast, fully automated and parameter-free method for detecting register-shifts in protein models built into cryo-EM maps. We show that the method can assist model building in cases where poorer map resolution hinders visual interpretation. We also show that checkMySequence could have helped avoid a widely discussed sequence register error in a model of SARS-CoV-2 RNA-dependent RNA polymerase that was originally detected thanks to a visual residue-by-residue inspection by members of the structural biology community.

Sequence assignment for low-resolution modelling of protein crystal structures

The performance of automated model building in crystal structure determination usually decreases with the resolution of the experimental data, and may result in fragmented models and incorrect side-chain assignment. Presented here are new methods for machine-learning-based docking of main-chain fragments to the sequence and for their sequence-independent connection using a dedicated library of protein fragments. The combined use of these new methods noticeably increases sequence coverage and reduces fragmentation of the protein models automatically built with ARP/wARP.

Automated sequence-level analysis of kinetics and thermodynamics for domain-level DNA strand-displacement systems

As an engineering material, DNA is well suited for the construction of biochemical circuits and systems, because it is simple enough that its interactions can be rationally designed using Watson–Crick base pairing rules, yet the design space is remarkably rich. When designing DNA systems, this simplicity permits using functional sections of each strand, called domains, without considering particular nucleotide sequences. However, the actual sequences used may have interactions not predicted at the domain-level abstraction, and new rigorous analysis techniques are needed to determine the extent to which the chosen sequences conform to the system’s domain-level description. We have developed a computational method for verifying sequence-level systems by identifying discrepancies between the domain-level and sequence-level behaviour. This method takes a DNA system, as specified using the domain-level tool Peppercorn, and analyses data from the stochastic sequence-level simulator Multistrand and sequence-level thermodynamic analysis tool NUPACK to estimate important aspects of the system, such as reaction rate constants and secondary structure formation. These techniques, implemented as the Python package KinDA, will allow researchers to predict the kinetic and thermodynamic behaviour of domain-level systems after sequence assignment, as well as to detect violations of the intended behaviour.

Using selenomethionyl derivatives to assign sequence in low-resolution structures of the AP2 clathrin adaptor

Selenomethionine incorporation is a powerful technique for assigning sequence to regions of electron density at low resolution. Genetic introduction of methionine point mutations and the subsequent preparation and crystallization of selenomethionyl derivatives permits unambiguous sequence assignment by enabling the placement of the anomalous scatterers (Se atoms) thus introduced. Here, the use of this approach in the assignment of sequence in a part of the AP2 clathrin adaptor complex that is responsible for clathrin binding is described. AP2 plays a pivotal role in clathrin-mediated endocytosis, a tightly regulated process in which cell-surface transmembrane proteins are internalized from the plasma membrane by incorporation into lipid-enclosed transport vesicles. AP2 binds cargo destined for internalization and recruits clathrin, a large trimeric protein that helps to deform the membrane to produce the transport vesicle. By selenomethionine labelling of point mutants, it was shown that the clathrin-binding site is buried within a deep cleft of the AP2 complex. A membrane-stimulated conformational change in AP2 releases the clathrin-binding site from autoinhibition, thereby linking clathrin recruitment to membrane localization.

Model morphing and sequence assignment after molecular replacement

A procedure termed `morphing' for improving a model after it has been placed in the crystallographic cell by molecular replacement has recently been developed. Morphing consists of applying a smooth deformation to a model to make it match an electron-density map more closely. Morphing does not change the identities of the residues in the chain, only their coordinates. Consequently, if the true structure differs from the working model by containing different residues, these differences cannot be corrected by morphing. Here, a procedure that helps to address this limitation is described. The goal of the procedure is to obtain a relatively complete model that has accurate main-chain atomic positions and residues that are correctly assigned to the sequence. Residues in a morphed model that do not match the electron-density map are removed. Each segment of the resulting trimmed morphed model is then assigned to the sequence of the molecule using information about the connectivity of the chains from the working model and from connections that can be identified from the electron-density map. The procedure was tested by application to a recently determined structure at a resolution of 3.2 Å and was found to increase the number of correctly identified residues in this structure from the 88 obtained usingphenix.resolvesequence assignment alone (Terwilliger, 2003) to 247 of a possible 359. Additionally, the procedure was tested by application to a series of templates with sequence identities to a target structure ranging between 7 and 36%. The mean fraction of correctly identified residues in these cases was increased from 33% usingphenix.resolvesequence assignment to 47% using the current procedure. The procedure is simple to apply and is available in thePhenixsoftware package.