Matthew G. Iadanza

Three-dimensional electron crystallography of protein microcrystals

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology. DOI: http://dx.doi.org/10.7554/eLife.01345.001

The structure of purified kinetochores reveals multiple microtubule-attachment sites

Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an ~126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.

Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE.

The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.

pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers

Significance Oligomers formed en route to amyloid fibrils are thought to be the perpetrators of toxicity in many amyloid disorders. How amyloid fibrils contribute to disease, however, is less clear. Here, using β2-micoglobulin (β2m) as a model system, we show that the stability of amyloid fibrils is highly pH-dependent, with mild acidification enhancing the formation of fibril-derived nonnative oligomers that disrupt membranes and alter cellular function. Enhancing fibril stability by incubation with the molecular chaperone, hsp70, or by cross-linking, protects against fibril-induced membrane disruption and cellular dysfunction. The results highlight the importance of pH in determining fibril stability and suggest that uptake of fibrils into acidic cellular compartments may contribute to amyloid disease by pH-induced molecular shedding of toxic species. Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using β2-microglobulin (β2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

Methods to account for movement and flexibility in cryo-EM data processing.

Recent advances in direct electron detectors and improved CMOS cameras have been accompanied by the development of a range of software to take advantage of the data they produce. In particular they allow for the correction of two types of motion in cryo electron microscopy samples: motion correction for movements of the sample particles in the ice, and differential masking to account for heterogeneity caused by flexibility within protein complexes. Here we provide several scripts that allow users to move between RELION and standalone motion correction and centring programs. We then compare the computational cost and improvements in data quality with each program. We also describe our masking procedures to account for conformational flexibility. For the different elements of this study we have used three samples; a high symmetry virus, flexible protein complex (∼1 MDa) and a relatively small protein complex (∼550 kDa), to benchmark four widely available motion correction packages. Using these as test cases we demonstrate how motion correction and differential masking, as well as an additional particle re-centring protocol can improve final reconstructions when used within the RELION image-processing package.

A suite of software for processing MicroED data of extremely small protein crystals

Electron diffraction of extremely small three-dimensional crystals (MicroED) allows for structure determination from crystals orders of magnitude smaller than those used for X-ray crystallography. The MicroED suite was developed to accomplish the tasks of unit-cell determination, indexing, background subtraction, intensity measurement and merging, resulting in data that can be carried forward to molecular replacement and structure determination.

Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three‐dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X‐ray crystallography. Electron crystallography employs two‐dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well‐ordered two‐dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well‐ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution. Curr. Protoc. Protein Sci. 72:17.15.1‐17.15.11.

MpUL-multi: Software for Calculation of Amyloid Fibril Mass per Unit Length from TB-TEM Images.

Structure determination for amyloid fibrils presents many challenges due to the high variability exhibited by fibrils and heterogeneous morphologies present, even in single samples. Mass per unit length (MPL) estimates can be used to differentiate amyloid fibril morphologies and provide orthogonal evidence for helical symmetry parameters determined by other methods. In addition, MPL data can provide insight on the arrangement of subunits in a fibril, especially for more complex fibrils assembled with multiple parallel copies of the asymmetric unit or multiple twisted protofilaments. By detecting only scattered electrons, which serve as a relative measure of total scattering, and therefore protein mass, dark field imaging gives an approximation of the total mass of protein present in any given length of fibril. When compared with a standard of known MPL, such as Tobacco Mosaic Virus (TMV), MPL of the fibrils in question can be determined. The program suite MpUL-multi was written for rapid semi-automated processing of TB-TEM dark field data acquired using this method. A graphical user interface allows for simple designation of fibrils and standards. A second program averages intensities from multiple TMV molecules for accurate standard determination, makes multiple measurements along a given fibril, and calculates the MPL.

A new era for understanding amyloid structures and disease

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-β structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention.The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. Recent advances in structural biology techniques have provided insight into how amyloid structure may affect the ability of fibrils to spread in a prion-like manner and into their roles in disease.