A Rapid Cloning Method Employing Orthogonal End Protection

We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)n-VWFA2-(FNIII)n constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors

Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1

Cryo-EM structure of gas vesicles - supplementary data

This deposition contains supplementary data from our preprint: Huber et. al, Cryo-EM structure of gas vesicles for buoyancy-controlled motility, BioRxiv (2022) (Update 26.04.2023), Now in: Huber et. al, Cryo-EM structure of gas vesicles for buoyancy-controlled motility, Cell (2023) 2Dclasses_AnaMega_SeamsEdgesWallTips.zip This folder contains 2D class averages of different features of A.flos-aquae and B.megaterium gas vesicles (GVs) such as the seams between both GV halves, GV wall edges, collapsed walls and tips. We use this data to inform our pseudo-atomic model of an entire gas vesicle, and derive a model for biogenesis and growth. Some 2D classes provide high-resolution views on the GV wall in projection with a resolution that show the α-helical pitch and large side-chain densities. We use those views to confirm the evolutionarily conserved fold of the gas vesicle wall protein. The box size and pixel size of the 2D classes is contained in the file names. A selected and sharpened 2D class for display in the manuscript is included in .png format. AlphaFold2_Models_5merRib_DifferentOrganisms.zip This folder contains five AlphaFold2 predictions of a single rib of the gas vesicle wall for organisms that are evolutionarily only little related (GvpA1 and GvpA2 from B.megaterium, GvpA from A.flos-aquae and GvpA1 and GvpA2 from H.salinarum). AF2 predicts very similar structure for these organisms, further supporting the high conservation of the gas vesicle wall. AnaGvpA_GvpC_ComputationalDocking_HADDOCK.zip In the manuscript, we propose a binding mode of the secondary protein GvpC to A.flos-aquae gas vesicles using computational docking with HADDOCK. We present two possible solutions with opposite orientation. The cif-files of the docking solution is contained in this folder. GasPoreAnalysis.zip We analysed gas pores between α-helix 1 of adjacent GvpA monomers using MOLE2.5. The three resulting tunnels are in this folder. The respective pdb files contain the constriction in Ångstrom in each line. A Chimera 1.13.1 session is included for visualisation

Thresholding of cryo-EM density maps by false discovery rate control

Cryo-EM now commonly generates close-to-atomic resolution as well as intermediate resolution maps from macromolecules observed in isolation and in situ. Interpreting these maps remains a challenging task owing to poor signal in the highest resolution shells and the necessity to select a threshold for density analysis. In order to facilitate this process, a statistical framework for the generation of confidence maps by multiple hypothesis testing and false discovery rate (FDR) control has been developed. In this way, three-dimensional confidence maps contain signal separated from background noise in the form of local detection rates of EM density values. It is demonstrated that confidence maps and FDR-based thresholding can be used for the interpretation of near-atomic resolution single-particle structures as well as lower resolution maps determined by subtomogram averaging. Confidence maps represent a conservative way of interpreting molecular structures owing to minimized noise. At the same time they provide a detection error with respect to background noise, which is associated with the density and is particularly beneficial for the interpretation of weaker cryo-EM densities in cases of conformational flexibility and lower occupancy of bound molecules and ions in the structure

Apparent melting temperature of concatameric proteins.

<p>Apparent melting temperature of concatameric proteins.</p

Expression and characterization of ¹³FNIII and VWFA2 tandem repeat proteins.

<p>Superposed elution profiles from size exclusion chromatography of (¹³FNIII)_2–8 proteins (A) and (VWFA2)_6–10 (B). (C,D) Coomassie stained SDS-PAGE of the purified proteins. (E,F) Unfolding curves from Thermofluor analysis suggest that the concatameric constructs are properly folded. Note the consistent shift of the (VWFA2)_n unfolding curves in the presence and absence of Ca²⁺.</p

Assembly of chimeric constructs and one-pot concatamer formation.

<p>(A) Assembly of (FNIII)₂-VWFA2-(FNIII)₂ sandwich constructs from a modular assembly vector (top). PCR amplification with specific primers (indicated above the lanes; <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037617#pone.0037617.s002" target="_blank">Table S1</a></b>) show that the A2 synthon is sandwiched between two ¹³FNIII repeats. (B) One-pot concatamer formation with orthogonal chain stoppers. Fully deprotected synthons are mixed in different molar ratios with orthogonal chain stoppers (equivalent synthons with protecting groups on one end). Increasing the concentration of chain stoppers shifts the size distribution towards shorter concatamers. Molar ratios of unprotected synthons:chain stoppers are indicated at the top of the lanes. Bands marked with asterisks presumably correspond to circularized dimers.</p

Efficient synthon assembly with split-and-pool reactions.

<p>(A) Equimolar amounts of BsaI or BsmBI deprotected ¹³FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of (¹³FNIII)₁ to (¹³FNIII)₈ repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the (¹³FNIII)₈ construct.</p

Vectors for synthon recombination and transfer to expression plasmids.

<p>(A) Deprotected synthons can be ligated into a BsaI-digested shuttle vector (pShuttle) that contains 5′-BamHI and 3′-NotI restriction sites compatible with our in-house collection of expression vectors. (B) For applications that require modular recombination or insertion of individual elements, synthons can be ligated into the modular assembly vectors pDA-N or pDA-C. pDA-N vectors that carry synthons encoding amino-terminal elements of protein constructs (<b>1</b>) can be combined with synthons encoding carboxyl-terminal elements from pDA-C vectors (<b>2</b>) to yield a composite product (<b>3a</b>), optionally leaving an additional entry point via BsaI restriction sites to insert additional synthons (<b>3b</b>). Final assemblies (<b>3a</b> and <b>4</b>) contain 5′-BamHI and 3′-NotI restriction sites for transfer into expression vectors.</p