Imaging thin organic specimens with Transmission Electron Microscopy (TEM) presents a significant challenge due to their inherently weak contrast. An additional electron optical element, placed in a focal plane of the objective lens, such as a phase plate (PP) can improve the contrast by inducing a relative phase shift between scattered and unscattered electrons. However, any additional optical element can also lead to additional noise due to random and beam-induced variations. To address this problem we have, as a first example, simulated the dynamic image formation of a weak phase object in a TEM equipped with a proposed PP consisting of two electron beams orthogonal to the optical axis of the TEM. The random and beam-induced variation of the PP is simulated with particle dynamics including all pairwise interactions among the electrons of the PP and the TEM electron. The resulting three-dimensional PP potential, which now includes these variations, is then used in a variant of the multislice algorithm to compute the exit wave's interaction with the PP. The quality of the simulation was validated against previous theoretical calculations and the simulated images were quantitatively compared to the projected potential of the specimen using Fourier ring correlation. These simulations indicate that a TEM equipped with this type of PP could produce images with consistent contrast in a resolution band up to about 4 Å. This range could be extended to higher resolutions by a modified CTF-correction including the effect of the PP. The underlying idea of dynamic simulations taking the variation of optical elements and maybe even the specimen into account could be generalized to many other imaging situations.

Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.

I have investigated two different forward models for image formation in transmission electron microscopy of thick specimens, the 3DCtf model, which introduces a defocus gradient in the linear approximation, and the multislice model. An important result is that the 3DCtf model does not seem to be compatible with the multislice image formation model. A second very useful finding is that the exit wave in the multislice model has an imaginary part, which, in first-order approximation, is a pure projection of the specimen and is not affected by the defocus gradient. The defocus gradient only comes into play in real valued and higher-order imaginary terms. If the multislice model is closer to reality than the 3DCtf-model, then the best way to retrieve the specimen projection for thicker specimens should be a procedure for retrieving the exit wave's imaginary term, for example using images recorded at different defocus values.

BRICHOS domain oligomerization exposes three short hydrophobic motifs that are necessary for efficient chaperone activity against amorphous protein aggregation. ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of ``life on the edge of solubility.'' Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT*). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT* domain. We conclude that interactions with NT* help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT*. In summary, we demonstrate that inducing co-translational folding via a molecular ``spindle and thread'' mechanism unblocks protein translation in vitro.

Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a pKa value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations. 

Amyloid-β peptide (Aβ) aggregation is one of the hallmarks of Alzheimer's disease (AD). Mutations in Aβ are associated with early onset familial AD, and the Arctic mutant E22G (Aβarc) is an extremely aggregation-prone variant. Here, we show that BRICHOS, a natural anti-amyloid chaperone domain, from Bri2 efficiently inhibits aggregation of Aβarcby mainly interfering with secondary nucleation. This is qualitatively different from the microscopic inhibition mechanism for the wild-type Aβ, against which Bri2 BRICHOS has a major effect on both secondary nucleation and fibril end elongation. The monomeric Aβ42arcpeptide aggregates into amyloid fibrils significantly faster than wild-type Aβ (Aβ42wt), as monitored by thioflavin T (ThT) binding, but the final ThT intensity was strikingly lower for Aβ42arccompared to Aβ42wtfibrils. The Aβ42arcpeptide formed large aggregates, single-filament fibrils, and multiple-filament fibrils without obvious twists, while Aβ42wtfibrils displayed a polymorphic pattern with typical twisted fibril architecture. Recombinant human Bri2 BRICHOS binds to the Aβ42arcfibril surface and interferes with the macroscopic fibril arrangement by promoting single-filament fibril formation. This study provides mechanistic insights on how BRICHOS efficiently affects the aggressive Aβ42arcaggregation, resulting in both delayed fibril formation kinetics and altered fibril structure. 

I suggest an electrostatic phase plate designed to broaden the contrast transfer function of a transmission electron microscope operated close to Scherzer defocus primarily in the low resolution direction. At higher defocus the low frequency behavior is equal to that close to Scherzer defocus, but CTF-correction becomes necessary to extend image interpretation to higher resolution. One simple realization of the phase plate consists of two ring shaped electrodes symmetrically surrounding the central beam. Since no physical components come into contact with the central beam and charge on the electrodes is controlled by an external voltage supply, problems with uncontrolled charging are expected to be reduced.