Abstract P-6: Automated Pipeline for Parametrization of the Coarse-Grained Models for Biomolecular Simulations

Background: Antibiotic-resistant strains of Staphylococcus aureus cause human infections that are difficult to treat and can lead to death. Bacteriophage (phage) phi812K1/420 from the family Myoviridae infects 95% of clinical isolates of S. aureus and therefore is a promising candidate for a phage therapy agent. As the native phage particle approaches its host cell, phage receptor-binding proteins make a contact with the host cell wall. This interaction triggers a cascade of structural changes in the baseplate resulting in phage tail contraction and genome ejection. Mechanistic description of the baseplate re-organization, however, remains unknown. Methods: Using cryo-electron microscopy (cryo-EM), we studied the baseplate of the phage phi812K1/420. Also, selected proteins involved in the host cell wall binding and penetration were produced in recombinant form and their structures were solved using X-ray crystallography and cryo-EM single-particle reconstruction. Results: We reconstructed the phage baseplate in native and contracted states. The reconstruction of the native baseplate reaches a resolution of 4 Å, which enables us to discern individual protein structures. Solved protein structures will be fitted into the reconstruction of the contracted baseplate. Conclusion: Our results provide the first structural characterization of contractile phage infecting a Gram-positive bacterium. Comparison of the two distinct baseplate states will allow us to describe the molecular mechanism of the initial stage of phage infection in detail.

Abstract OR-9: Cryo-EM Structure of the Reconstituted Human γ-Tubulin Ring Complex

Background: Microtubules (MTs) are essential cytoskeletal polymers that provide structural support for the cell and play important roles in cell division, motility, and intracellular transport. The γ-tubulin ring complex (γTuRC) is the major MT nucleator in animal cells. The molecular mechanism by which the γTuRC promotes MT nucleation remains poorly understood although a template-based mechanism, remains the most widely accepted (Moritz et al., 2000, Kollman et al., 2010). According to this model γTuRC, a 2 MDa multi-subunit protein complex, forms a lock washer-like structure, in which γ-tubulin molecules are arranged in a ring-shaped structure that serves as a template for the assembly of αβ-tubulin heterodimers. Methods: We have set up an in vitro system to purify the human γTuRC using infected insect cells with recombinant baculoviruses. This complex sample was subjected to cryo-EM analysis and single-particle reconstruction. Results: We have demonstrated that RUVBL1-RUVBL2 AAA-ATPase complex (RUVBL) controls the assembly and composition of γTuRC in human cells both in vivo and in vitro. Likewise, RUVBL assembles γTuRC from a minimal set of core subunits in a heterologous co-expression system. Purified, reconstituted γTuRC has nucleation activity and resembles native γTuRC (Consolati et al., 2020, Liu et al., 2020, Wieczorek et al., 2020), as revealed by its cryo-EM structure at ~4.0 Å resolution. Conclusion: We have been able to identify novel mechanistic and structural features that determine the intricate, higher-order γTuRC architecture (Zimmermann, Serna et al., 2020).

Abstract OR-6: Baseplate Structure of Bacteriophage Phi812 and Mechanism of Cell Wall Binding and Penetration

Background: Antibiotic-resistant strains of Staphylococcus aureus cause human infections that are difficult to treat and can lead to death. Bacteriophage (phage) phi812K1/420 from the family Myoviridae infects 95% of clinical isolates of S. aureus and therefore is a promising candidate for a phage therapy agent. As the native phage particle approaches its host cell, phage receptor-binding proteins make a contact with the host cell wall. This interaction triggers a cascade of structural changes in the baseplate resulting in phage tail contraction and genome ejection. Mechanistic description of the baseplate re-organization, however, remains unknown. Methods: Using cryo-electron microscopy (cryo-EM), we studied the baseplate of the phage phi812K1/420. Also, selected proteins involved in the host cell wall binding and penetration were produced in recombinant form and their structures were solved using X-ray crystallography and cryo-EM single-particle reconstruction. Results: We reconstructed the phage baseplate in native and contracted states. The reconstruction of the native baseplate reaches a resolution of 4 Å, which enables us to discern individual protein structures. Solved protein structures will be fitted into the reconstruction of the contracted baseplate. Conclusion: Our results provide the first structural characterization of contractile phage infecting a Gram-positive bacterium. Comparison of the two distinct baseplate states will allow us to describe the molecular mechanism of the initial stage of phage infection in detail.

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment revealed by Cryo-EM and hydrogen/deuterium exchange mass spectrometry

Encapsulins (Enc) are protein nanocompartments which house various cargo enzymes, including a family of decameric ferritin-like proteins. Previously, we elucidated the structure and activity of these ferritin-like proteins (EncFtn) and demonstrated that they must be encapsulated in a nanocompartment for iron storage. Here, we study a recombinant Haliangium ochraceum Enc:EncFtn complex using electron cryo-microscopy (Cryo-EM) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to gain insight into the structural relationship between Enc and EncFtn. An asymmetric single particle reconstruction reveals four EncFtn decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the EncFtn cargo and the icosahedral encapsulin shell. The EncFtn decamers are offset from the interior face of the encapsulin shell and are resolved at a much lower overall resolution in the final reconstruction. This flexibility, and the fixed number of EncFtn decamers sequestered within, implies that the loading of the encapsulin nanocompartment is limited by the steric effect of both engaged and free encapsulin localization sequences. Using a combination of focused refinements and HDX-MS, we observed dynamic behavior of the major five-fold pore, and show the pore opening via the movement of the encapsulin A-domain. These data can accelerate efforts to engineer the sequestration of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation

Structure-determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure, but is constrained by the need for large, well ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular-weight threshold required for single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure-determination methods. Here, a data-processing scheme is presented that combines routines from X-ray crystallography and new algorithms that have been developed to solve structures from tomograms of nanocrystals. This pipeline handles image-processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. The tolerance of this workflow to the effects of radiation damage is also assessed. The simulations indicate a trade-off between a wider tilt range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors, but not merging errors, can be overcome with additional data sets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

Sparseness and Smoothness Regularized Imaging for improving the resolution of Cryo-EM single-particle reconstruction

In this paper, we present a refinement method for cryo-electron microscopy (cryo-EM) single-particle reconstruction, termed as OPUS-SSRI (Sparseness and Smoothness Regularized Imaging). In OPUS-SSRI, spatially varying sparseness and smoothness priors are incorporated to improve the regularity of electron density map, and a type of real space penalty function is designed. Moreover, we define the back-projection step as a local kernel regression and propose a first-order method to solve the resulting optimization problem. On the seven cryo-EM datasets that we tested, the average improvement in resolution by OPUS-SSRI over that from RELION 3.0, the commonly used image-processing software for single-particle cryo-EM, was 0.64 Å, with the largest improvement being 1.25 Å. We expect OPUS-SSRI to be an invaluable tool to the broad field of cryo-EM single-particle analysis. The implementation of OPUS-SSRI can be found at https://github.com/alncat/cryoem.

Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for carbohydrate binding

VAR2CSA is the placental-malaria–specific member of the antigenically variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. It is expressed on the surface of Plasmodium falciparum-infected host red blood cells and binds to specific chondroitin-4-sulfate chains of the placental proteoglycan receptor. The functional ∼310 kDa ectodomain of VAR2CSA is a multidomain protein that requires a minimum 12-mer chondroitin-4-sulfate molecule for specific, high affinity receptor binding. However, it is not known how the individual domains are organized and interact to create the receptor-binding surface, limiting efforts to exploit its potential as an effective vaccine or drug target. Using small angle X-ray scattering and single particle reconstruction from negative-stained electron micrographs of the ectodomain and multidomain constructs, we have determined the structural architecture of VAR2CSA. The relative locations of the domains creates two distinct pores that can each accommodate the 12-mer of chondroitin-4-sulfate, suggesting a model for receptor binding. This model has important implications for understanding cytoadherence of infected red blood cells and potentially provides a starting point for developing novel strategies to prevent and/or treat placental malaria.