Alex J. Noble

A pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry

COPII vesicles transport proteins from the endoplasmic reticulum to the Golgi apparatus. Previous COPII-cage cryo-EM structures lacked the resolution necessary to determine the residues of Sec13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12-Å structure of the human COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13–Sec31 complex to create a reliable pseudoatomic model of the COPII cage. We combined this model with hydrogen/deuterium-exchange MS analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the two-fold symmetry of the Sec31 dimeric region in Sec13–Sec31 is broken upon cage formation and that the resulting hinge is essential to form the proper edge geometry in COPII cages.

Insights into the Mechanisms of Membrane Curvature and Vesicle Scission by the Small GTPase Sar1 in the Early Secretory Pathway

The small GTPase protein Sar1 is known to be involved in both the initiation of COPII-coated vesicle formation and scission of the nascent vesicle from the endoplasmic reticulum. The molecular details for the mechanism of membrane remodeling by Sar1 remain unresolved. Here, we show that Sar1 transforms synthetic liposomes into structures of different morphologies including tubules and detached vesicles. We demonstrate that Sar1 alone is competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. Sar1 molecules align on low-curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. The three-dimensional structure of the Sar1 lattice was reconstructed by substituting spherical liposomes with galactoceramide lipid tubules of homogeneous diameter. These data suggest that Sar1 dimerization is responsible for the formation of constrictive membrane curvature. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission.

The influence of frame alignment with dose compensation on the quality of single particle reconstructions.

As direct electron detection devices in cryo-electron microscopy become ubiquitous, the field is now ripe for new developments in image analysis techniques that take advantage of their increased SNR coupled with their high-throughput frame collection abilities. In approaching atomic resolution of native-like biomolecules, the accurate extraction of structural locations and orientations of side-chains from frames depends not only on the electron dose that a sample receives but also on the ability to accurately estimate the CTF. Here we use a new 2.8Å resolution structure of a recombinant gene therapy virus, AAV-DJ with Arixtra, imaged on an FEI Titan Krios with a DE-20 direct electron detector to probe new metrics including relative side-chain density and ResLog analysis for optimizing the compensation of electron beam damage and to characterize the factors that are limiting the resolution of the reconstruction. The influence of dose compensation on the accuracy of CTF estimation and particle classifiability are also presented. We show that rigorous dose compensation allows for better particle classifiability and greater recovery of structural information from negatively charged, electron-sensitive side-chains, resulting in a more accurate macromolecular model.

Automated batch fiducial-less tilt-series alignment in Appion using Protomo.

The field of electron tomography has benefited greatly from manual and semi-automated approaches to marker-based tilt-series alignment that have allowed for the structural determination of multitudes of in situ cellular structures as well as macromolecular structures of individual protein complexes. The emergence of complementary metal-oxide semiconductor detectors capable of detecting individual electrons has enabled the collection of low dose, high contrast images, opening the door for reliable correlation-based tilt-series alignment. Here we present a set of automated, correlation-based tilt-series alignment, contrast transfer function (CTF) correction, and reconstruction workflows for use in conjunction with the Appion/Leginon package that are primarily targeted at automating structure determination with cryogenic electron microscopy.

The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide

Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ’s directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.

Routine determination of ice thickness for cryo-EM grids

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.

478. An Essential and Ubiquitous Protein Receptor for AAV; Glycans as Attachment Receptors

Motivated by unsuccessful attempts to observe physical binding between AAV-2 and heterologously expressed domains of previously reported co-receptors, we set out to identify novel protein receptor(s) for AAV2 through an unbiased genome-wide knockout screen in human cells. Using an mCherry AAV vector, resistant cells were iteratively selected by FACS for gene trap screening in a library of mutagenized haploid cells. Upon deep sequencing, refractory cells had significantly high frequencies of mutation in genes encoding glycan synthesis and retrograde transport, but most significantly in a hitherto poorly characterized transmembrane protein, now termed AAVR. Genetic confirmation of AAVRs role in the entry of multiple AAV serotypes has come through CRISPR-Cas9 knockouts in multiple cell lines then restoration of susceptibility through complementation; infection of poorly permissive cells following AAVR transduction; and creation of a mouse knockout with greatly diminished susceptibility.Various AAVR ectodomain constructs have been heterologously expressed and purified as fusion proteins, and these have been shown to inhibit in vitro viral transduction at concentrations consistent with effective nM binding constants (between AAV & AAVR) measured by surface plasmon resonance (SPR). Pre-incubation with antibodies to AAVR also inhibits infection or transduction. AAVR is transiently expressed on the plasma membrane. Expression of chimeric constructs suggests that AAV takes advantage of its trafficking to the perinuclear trans Golgi network as the dominant, but non-exclusive, entry pathway. Identification of AAVR and its apparently ubiquitous use has interesting implications for AAVs cell specificity. Progress towards structure of complexes will be reported.AAVR exhibits the classic characteristics of a viral receptor, casting the roles ascribed to glycan “primary” receptors in new light. Electron microscopy has been used to visualize AAV-DJ in complex with various heparin analogs at increasingly high resolution. A structure at 2.8 A resolution, as a pentasaccharide complex, shows some disorder in the glycan, but the side chains of viral amino acids are clearly resolved and in different conformations from those seen in a sucrose octasulfate complex. With little change to the backbone, the binding site accommodates diverse glycan sequences through adjustments to side chains, consistent with SPR binding assays of AAV-2 to a library of heparanoids. This, together with comparisons of heparan and AAVR cell knock-outs, indicates a more accessory role for glycans than is implied by the term “primary”. As for several other viruses, in AAV-2 at least, the glycan is an attachment receptor that likely elevates the AAV concentration proximal to the membrane, improving the efficiency with which the virus then binds to AAVR.

COPI gets a fancy new coat

An interconnected scaffolding of proteins bends the membrane to form vesicles [Also see Report by Dodonova et al.] A defining characteristic of eukaryotic cells is their numerous membrane-bound compartments, including the endoplasmic reticulum (ER), Golgi apparatus, and other organelles. Proteins and lipids are transported between these compartments and the cell surface by vesicles about 100 nm in diameter. Three canonical protein coat complexes facilitate the formation of these small vesicular carriers: the clathrin coat, coat protein 1 (COPI), and coat protein 2 (COPII) complexes. The COPII coat mediates vesicle formation at the ER for transport to the Golgi; the COPI coat forms vesicles at the cis-Golgi for transport to the ER; and clathrin has a dual role, forming vesicles at the trans-Golgi for transport to the plasma membrane and forming vesicles at the plasma membrane for endocytosis. Each of these coat complexes is composed of numerous proteins that bind to membrane, recognize cargo proteins, bend the parent membranes into a bud, and pinch the buds off into vesicles (1). On page 195 of this issue, Dodonova et al. (2) have created an atomic model of the COPI coat by using cryogenic electron tomography (cryo-ET) combined with labeling and cross-linking mass spectrometry. Their model gives new insights into the mechanisms by which the COPI proteins assemble into highly interconnected scaffoldings and work together to bend membrane.

Sar1 Forms Ordered Arrays That Can Facilitate Vesicle Scission in the COPII Pathway

Secretion is a fundamental process in eukaryotes. It’s disruption can lead to one of several diseases including chylomicron retention disease 1 , cranio-lenticulo-sutural dysplasia 2 , or congenital dyserythropoietic anemia 3 . Transport of secreted cargo such as membrane proteins or soluble secreted cargos is initiated at the endoplasmic reticulum (ER). There proteins are co-translationally translocated into the lumen of the ER. These cargo proteins are then concentrated at specific sites on the ER called exit sites, and are collected into a membrane-bound vesicle that is transported to the Golgi apparatus and on to the cell surface. The coat protein complex II (COPII) proteins Sar1, Sec23/24, and Sec13/31 facilitate the formation of transport vesicles at the ER exit sites 4 . Vesicle formation is completed when the vesicle detaches from the parent membrane in a process called scission. It has been shown that Sar1 is required for scission though the mechanism by which the bud neck is collapsed and fused remains unclear.

ResLog Plots: A New Metric for the Quality of Cryo-EM Reconstructions

1 Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, FL 32306. 2 Institute of Molecular Biophysics, 91 Chieftain Way, Florida State University, Tallahassee, FL 32306. 3. Department of Physics, 77 Chieftan Way, Florida State University, Tallahassee, FL 32306-4350 4. Department of Biochemistry & Molecular Biology, School of Medicine, Oregon Health & Science University, Portland, OR 97239-3098