Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy.

Cryo-EM samples prepared using traditional methods often suffer from too few particles, poor particle distribution, strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.

A simple and robust procedure for preparing graphene-oxide cryo-EM grids.

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited their widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids can be used for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and provide partial protection from the air-water interface. Additionally, the signal of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.

Assemblathon 1: a competitive assessment of de novo short read assembly methods.

Low-cost short read sequencing technology has revolutionized genomics, though it is only just becoming practical for the high-quality de novo assembly of a novel large genome. We describe the Assemblathon 1 competition, which aimed to comprehensively assess the state of the art in de novo assembly methods when applied to current sequencing technologies. In a collaborative effort, teams were asked to assemble a simulated Illumina HiSeq data set of an unknown, simulated diploid genome. A total of 41 assemblies from 17 different groups were received. Novel haplotype aware assessments of coverage, contiguity, structure, base calling, and copy number were made. We establish that within this benchmark: (1) It is possible to assemble the genome to a high level of coverage and accuracy, and that (2) large differences exist between the assemblies, suggesting room for further improvements in current methods. The simulated benchmark, including the correct answer, the assemblies, and the code that was used to evaluate the assemblies is now public and freely available from http://www.assemblathon.org/.

The 3A protein from multiple picornaviruses utilizes the golgi adaptor protein ACBD3 to recruit PI4KIIIß.

The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIß) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIß and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIß by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIIIß. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIIIß in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIIIß could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIIIß. The dependence of Aichi virus replication on the activity of PI4KIIIß was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus. Point mutations in 3A that eliminate PI4KIIIß association sensitized Aichi virus to PIK93, suggesting that disruption of the 3A/ACBD3/PI4KIIIß complex may represent a novel target for therapeutic intervention that would be complementary to the inhibition of the kinase activity itself.

Competing protein-protein interactions regulate binding of Hsp27 to its client protein tau.

Small heat shock proteins (sHSPs) are a class of oligomeric molecular chaperones that limit protein aggregation. However, it is often not clear where sHSPs bind on their client proteins or how these protein-protein interactions (PPIs) are regulated. Here, we map the PPIs between human Hsp27 and the microtubule-associated protein tau (MAPT/tau). We find that Hsp27 selectively recognizes two aggregation-prone regions of tau, using the conserved ß4-ß8 cleft of its alpha-crystallin domain. The ß4-ß8 region is also the site of Hsp27-Hsp27 interactions, suggesting that competitive PPIs may be an important regulatory paradigm. Indeed, we find that each of the individual PPIs are relatively weak and that competition for shared sites seems to control both client binding and Hsp27 oligomerization. These findings highlight the importance of multiple, competitive PPIs in the function of Hsp27 and suggest that the ß4-ß8 groove acts as a tunable sensor for clients.

Symmetry broken and rebroken during the ATP hydrolysis cycle of the mitochondrial Hsp90 TRAP1.

Hsp90 is a homodimeric ATP-dependent molecular chaperone that remodels its substrate 'client' proteins, facilitating their folding and activating them for biological function. Despite decades of research, the mechanism connecting ATP hydrolysis and chaperone function remains elusive. Particularly puzzling has been the apparent lack of cooperativity in hydrolysis of the ATP in each protomer. A crystal structure of the mitochondrial Hsp90, TRAP1, revealed that the catalytically active state is closed in a highly strained asymmetric conformation. This asymmetry, unobserved in other Hsp90 homologs, is due to buckling of one of the protomers and is most pronounced at the broadly conserved client-binding region. Here, we show that rather than being cooperative or independent, ATP hydrolysis on the two protomers is sequential and deterministic. Moreover, dimer asymmetry sets up differential hydrolysis rates for each protomer, such that the buckled conformation favors ATP hydrolysis. Remarkably, after the first hydrolysis, the dimer undergoes a flip in the asymmetry while remaining in a closed state for the second hydrolysis. From these results, we propose a model where direct coupling of ATP hydrolysis and conformational flipping rearranges client-binding sites, providing a paradigm of how energy from ATP hydrolysis can be used for client remodeling.

Genome-wide regulatory dynamics of translation in the Plasmodium falciparum asexual blood stages.

The characterization of the transcriptome and proteome of Plasmodium falciparum has been a tremendous resource for the understanding of the molecular physiology of this parasite. However, the translational dynamics that link steady-state mRNA with protein levels are not well understood. In this study, we bridge this disconnect by measuring genome-wide translation using ribosome profiling, through five stages of the P. falciparum blood phase developmental cycle. Our findings show that transcription and translation are tightly coupled, with overt translational control occurring for less than 10% of the transcriptome. Translationally regulated genes are predominantly associated with merozoite egress functions. We systematically define mRNA 5' leader sequences, and 3' UTRs, as well as antisense transcripts, along with ribosome occupancy for each, and establish that accumulation of ribosomes on 5' leaders is a common transcript feature. This work represents the highest resolution and broadest portrait of gene expression and translation to date for this medically important parasite.

ACBD3 interaction with TBC1 domain 22 protein is differentially affected by enteroviral and kobuviral 3A protein binding.

UNLABELLED: Despite wide sequence divergence, multiple picornaviruses use the Golgi adaptor acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GCP60) to recruit phosphatidylinositol 4-kinase class III beta (PI4KIIIß/PI4KB), a factor required for viral replication. The molecular basis of this convergent interaction and the cellular function of ACBD3 are not fully understood. Using affinity purification-mass spectrometry, we identified the putative Rab33 GTPase-activating proteins TBC1D22A and TBC1D22B as ACBD3-interacting factors. Fine-scale mapping of binding determinants within ACBD3 revealed that the interaction domains for TBC1D22A/B and PI4KB are identical. Affinity purification confirmed that PI4KB and TBC1D22A/B interactions with ACBD3 are mutually exclusive, suggesting a possible regulatory mechanism for recruitment of PI4KB. The C-terminal Golgi dynamics (GOLD) domain of ACBD3 has been previously shown to bind the 3A replication protein from Aichi virus. We find that the 3A proteins from several additional picornaviruses, including hepatitis A virus, human parechovirus 1, and human klassevirus, demonstrate an interaction with ACBD3 by mammalian two-hybrid assay; however, we also find that the enterovirus and kobuvirus 3A interactions with ACBD3 are functionally distinct with respect to TBC1D22A/B and PI4KB recruitment. These data reinforce the notion that ACBD3 organizes numerous cellular functionalities and that RNA virus replication proteins likely modulate these interactions by more than one mechanism. IMPORTANCE: Multiple viruses use the same Golgi protein (ACBD3) to recruit the lipid kinase phosphatidylinositol 4-kinase class III beta (PI4KB) in order to replicate. We identify a new binding partner of ACBD3 in the evolutionarily conserved Rab GTPase-activating proteins (RabGAPs) TBC1D22A and -B. Interestingly, TBC1D22A directly competes with PI4KB for binding to the same location of ACBD3 by utilizing a similar binding domain. Different viruses are able to influence this interaction through distinct mechanisms to promote the association of PI4KB with ACBD3. This work informs our knowledge of both the physical interactions of the proteins that help maintain metazoan Golgi structure and how viruses subvert these evolutionarily conserved interactions for their own purposes.