Steven J. Ludtke

Outcome of the first electron microscopy validation task force meeting

This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.

Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids

Synaptotagmin acts as a Ca2+ sensor in neurotransmitter release through its two C2 domains. Ca2+-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca2+ binding to the C2B domain is more crucial for release than Ca2+ binding to the C2A domain. Here we show that Ca2+ induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C2B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C2B domain.

Mechanism of Folding Chamber Closure in a Group II Chaperonin

Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, ∼1 megadalton, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis. The structural rearrangements and molecular events leading to lid closure are still unknown. Here we report four single particle cryo-electron microscopy (cryo-EM) structures of Mm-cpn, an archaeal group II chaperonin, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 Å resolution of the closed conformation allowed building of the first ever atomic model directly from the single particle cryo-EM density map, in which we were able to visualize the nucleotide and more than 70% of the side chains. The model of the open conformation was obtained by using the deformable elastic network modelling with the 8 Å resolution open-state cryo-EM density restraints. Together, the open and closed structures show how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analyses show that there is an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signalling, driving the conformational cycle of group II chaperonins. Beyond this, we anticipate that our methodology of combining single particle cryo-EM and computational modelling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.

Superparamagnetic gadonanotubes are high-performance MRI contrast agents

We report the nanoscale loading and confinement of aquated Gd3+n-ion clusters within ultra-short single-walled carbon nanotubes (US-tubes); these Gd3+n@US-tube species are linear superparamagnetic molecular magnets with Magnetic Resonance Imaging (MRI) efficacies 40 to 90 times larger than any Gd3+-based contrast agent (CA) in current clinical use.

4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ∼5–10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC’s unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-Å resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-Å resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Cα backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed ∼95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC’s cellular substrate specificity.

Interprotofilament interactions between Alzheimer's Aβ1–42 peptides in amyloid fibrils revealed by cryoEM

Alzheimers disease is a neurodegenerative disorder characterized by the accumulation of amyloid plaques in the brain. This amyloid primarily contains amyloid-β (Aβ), a 39- to 43-aa peptide derived from the proteolytic cleavage of the endogenous amyloid precursor protein. The 42-residue-length Aβ peptide (Aβ1–42), the most abundant Aβ peptide found in plaques, has a much greater propensity to self-aggregate into fibrils than the other peptides and is believed to be more pathogenic. Synthetic human Aβ1–42 peptides self-aggregate into stable but poorly-ordered helical filaments. We determined their structure to ≈10-Å resolution by using cryoEM and the iterative real-space reconstruction method. This structure reveals 2 protofilaments winding around a hollow core. Previous hairpin-like NMR models for Aβ17–42 fit well in the cryoEM density map and reveal that the juxtaposed protofilaments are joined via the N terminus of the peptide from 1 protofilament connecting to the loop region of the peptide in the opposite protofilament. This model of mature Aβ1–42 fibrils is markedly different from previous cryoEM models of Aβ1–40 fibrils. In our model, the C terminus of Aβ forms the inside wall of the hollow core, which is supported by partial proteolysis analysis.

Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel

The skeletal muscle Ca2+ release channel (RyR1), a homotetramer, regulates the release of Ca2+ from the sarcoplasmic reticulum to initiate muscle contraction. In this work, we have delineated the RyR1 monomer boundaries in a subnanometer-resolution electron cryomicroscopy (cryo-EM) density map. In the cytoplasmic region of each RyR1 monomer, 36 α-helices and 7 β-sheets can be resolved. A β-sheet was also identified close to the membrane-spanning region that resembles the cytoplasmic pore structures of inward rectifier K+ channels. Three structural folds, generated for amino acids 12–565 using comparative modeling and cryo-EM density fitting, localize close to regions implicated in communication with the voltage sensor in the transverse tubules. Eleven of the 15 disease-related residues for these domains are mapped to the surface of these models. Four disease-related residues are found in a basin at the interfaces of these regions, creating a pocket in which the immunophilin FKBP12 can fit. Taken together, these results provide a structural context for both channel gating and the consequences of certain malignant hyperthermia and central core disease-associated mutations in RyR1.

Structure of the SecY channel during initiation of protein translocation

Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during their synthesis. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from Methanocaldococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome–channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the channel directly.

Gating machinery of InsP3R channels revealed by electron cryomicroscopy

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca2+ signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure–function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ∼85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca2+-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals.

An atomic model of brome mosaic virus using direct electron detection and real-space optimization

Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.