Biology subjects

Lander, G. C.

Publications and source records attributed to Lander, G. C..

45 records · Page 3Linked to original sources

bioRxiv2018

High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our [~]2.9 [A] structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our [~]2.8 [A] and [~]3.2 [A] resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

biophysics↗

bioRxiv2018

Structure reveals mechanism of CRISPR RNA-guided nuclease recruitment and anti-CRISPR viral mimicry

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here we present the cryo-EM structure of the type I-F CRISPR RNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a I180-degree rotation of the C-terminal helical bundle on the \"large\" Cas8f subunit. We show that the dsDNA-induced conformational change in Cas8f exposes a Cas2/3 \"nuclease recruitment helix\" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f \"nuclease recruitment helix\", implying that cas genes may sometimes serve as genetic fodder for the evolution of anti-CRISPRs.

molecular biology↗

bioRxiv2018

Conformational ensemble of the human TRPV3 ion channel

Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated simulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show -to-{pi}-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker {pi}-helix during ligand gating.

biochemistry↗

bioRxiv2018

Structures of the substrate-engaged 26S proteasome reveal the mechanisms for ATP hydrolysis-driven translocation

The 26S proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core, while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-EM structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination, and how ATP-binding, hydrolysis, and phosphate-release events are coordinated within the AAA+ motor to induce conformational changes and propel the substrate through the central pore.

molecular biology↗

bioRxiv2017

Exploring Applications of Crowdsourcing to Cryo-EM

Extraction of particles from cryo-electron microscopy (cryo-EM) micrographs is a crucial step in processing single-particle datasets. Although algorithms have been developed for automatic particle picking, these algorithms generally rely on two-dimensional templates for particle identification, which may exhibit biases that can propagate artifacts through the reconstruction pipeline. Manual picking is viewed as a gold-standard solution for particle selection, but it is too time-consuming to perform on data sets of thousands of images. In recent years, crowdsourcing has proven effective at leveraging the open web to manually curate datasets. In particular, citizen science projects such as Galaxy Zoo have shown the power of appealing to users' scientific interests to process enormous amounts of data. To this end, we explored the possible applications of crowdsourcing in cryo-EM particle picking, presenting a variety of novel experiments including the production of a fully annotated particle set from untrained citizen scientists. We show the possibilities and limitations of crowdsourcing particle selection tasks, and explore further options for crowdsourcing cryo-EM data processing.

bioinformatics↗

bioRxiv2017

Atomic structure of the mitochondrial inner membrane AAA+ protease YME1 reveals the mechanism of substrate processing

We present the first atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease, YME1. Our ~3.4 [A] cryo-EM structure reveals how the ATPases form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Importantly, the structure reveals how three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring ATP hydrolysis cycle that results in step-wise substrate translocation. Furthermore, we identify a hinge-like linker that accommodates the large-scale nucleotide-driven motions of the ATPase spiral independently of the contiguous planar proteolytic base. These results define the first molecular mechanism for a mitochondrial inner membrane AAA+ protease and reveal a translocation mechanism likely conserved for other AAA+ ATPases.

molecular biology↗

bioRxiv2017

Structural analyses reveal that the ATG2A-WIPI4 complex functions as a membrane tether for autophagosome biogenesis

Autophagy is an enigmatic cellular process in which double-membrane compartments, called autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the phosphatidylinositol-3-phosphate (PI3P)-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

biochemistry↗

bioRxiv2017

Achieving Better Than 3 A Resolution By Single Particle Cryo-EM At 200 keV

Technical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 [A] resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 [A] resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.

biophysics↗

bioRxiv2017

A multi-model approach to assessing local and global cryo-EM map quality

There does not currently exist a standardized indicator of how well a cryo-EM-derived model represents the density from which it was generated. We present a straightforward methodology that utilizes freely available tools to generate a suite of independent models and to evaluate their convergence in an EM density. These analyses provide both a quantitative and qualitative assessment of the precision of the models and their representation of the density, respectively, while concurrently providing a platform for assessing both global and local EM map quality. We further use standardized datasets to provide an expected model-model agreement criterion for EM maps reported to be at 5 [A] resolution or better. Associating multiple atomic models with a deposited EM map provides a rapid and accessible reporter of convergence, a strong indicator of highly resolved molecular detail, and is an important step toward an FSC-independent assessment of map and model quality.

biophysics↗