The synthesis of Au(102)(p-MBA)(44) nanoparticles on a preparative scale in high yield is described. Various analytical methods are shown to give results consistent with the composition and known structure of the particles, showing the preparation is essentially homogeneous, and attesting to the validity of the methods as well. Derivatization of the particles with proteins and DNA is demonstrated, and conditions are described for imaging individual particles by cryo-EM at low electron dose, close to focus, conditions optimal for recording high-resolution details.

The TRiC/CCTchaperonin is a 1-MDa hetero-oligomer of 16 subunits that assists the folding of proteins in eukaryotes. Low-resolution structural studies confirmed the TRiC particle to be composed of two stacked octameric rings enclosing a folding cavity. The exact arrangement of the different proteins in the rings underlies the functionality of TRiC and is likely to be conserved across all eukaryotes. Yet despite its importance it has not been determined conclusively, mainly because the different subunits appear nearly identical under low resolution. This work successfully addresses the arrangement problem by the emerging technique of cross-linking, mass spectrometry, and modeling. We cross-linked TRiC under native conditions with a cross-linker that is primarily reactive toward exposed lysine side chains that are spatially close in the context of the particle. Following digestion and mass spectrometry we were able to identify over 60 lysine pairs that underwent crosslinking, thus providing distance restraints between specific residues in the complex. Independently of the cross-link set, we constructed 40,320 (¼8 factorial) computational models of the TRiC particle, which exhaustively enumerate all the possible arrangements of the different subunits. When we assessed the compatibility of each model with the cross-link set, we discovered that one specific model is significantly more compatible than any other model. Furthermore, bootstrapping analysis confirmed that this model is 10 times more likely to result from this cross-link set than the next best-fitting model. Our subunit arrangement is very different than any of the previously reported models and changes the context of existing and future findings on TRiC.group II chaperonins | protein folding | violation distances I n eukaryotes and archaea the folding of nascent and mis-folded polypeptide chains is assisted by a group II chaperonin system known as the thermosome in archea and TRiC (or CCT) in eukaryotes (1). These large protein complexes consist of two stacked octameric rings (2) The open state of the complex binds the polypeptide substrate (4, 5), whereupon closing the substrate is sequestered into a large interior cavity where folding can occur. TRiC has been implicated in the folding pathways of many cytosolic proteins (6), most notably actin and tubulin (7,8).Many archaeal species have just one thermosome gene and a simple homo-oligomeric architecture (9). Other archaeal species have at most three different types of subunits, and there is evidence that the multiple genes are redundant (10). In stark contrast, the eukaryotic TRiC consists of eight different subunit types (CCT1 to CCT8), all of which are essential. The subunit specialization occurred very early in eukaryote evolution (11) and is conserved to such an extent that the sequence identity between mammalian and yeast subunits of the same type is nearly 60%, whereas the sequence identity between the different subunit types in the same organism is only 30%. The diversity of eight distinct subun...

The neocortex has a high capacity for plasticity. To understand the full scope of this capacity, it is essential to know how neurons choose particular partners to form synaptic connections. By using multineuron whole-cell recordings and confocal microscopy we found that axons of layer V neocortical pyramidal neurons do not preferentially project toward the dendrites of particular neighboring pyramidal neurons; instead, axons promiscuously touch all neighboring dendrites without any bias. Functional synaptic coupling of a small fraction of these neurons is, however, correlated with the existence of synaptic boutons at existing touch sites. These data provide the first direct experimental evidence for a tabula rasa-like structural matrix between neocortical pyramidal neurons and suggests that pre-and postsynaptic interactions shape the conversion between touches and synapses to form specific functional microcircuits. These data also indicate that the local neocortical microcircuit has the potential to be differently rewired without the need for remodeling axonal or dendritic arbors.connectivity ͉ neocortex ͉ spines A hallmark of the neocortex is its sensitivity to environmental conditions that can drive profound structural and functional changes (1-4). At the microcircuit level, these changes include the creation, elimination, and modification of synaptic connections between neurons (5-10). To understand the mechanisms and full potential of cortical plasticity, it is essential to answer a fundamental question: How does a neuron choose to connect to only a small fraction of its neighboring neurons in a cortical volume packed with intertwined dendrites? This question has been debated extensively for more than a decade (11)(12)(13)(14)(15). At the heart of the debate is the question of whether recurrent synapses between pyramidal neurons, which are the vast majority of neocortical synapses (14, 16), are placed randomly to form an arbitrary functional microcircuit or deterministically to form a highly specific functional microcircuit.Theoretical work suggests that it is essentially impossible for an axon of a pyramidal neuron to avoid touching any other pyramidal neuron within the local microcircuit (up to Ϸ150 m from the soma) (see Fig. 1C) (14,17,18). However, experimental studies with paired recordings clearly indicate that pyramidal neurons are synaptically connected to only a small fraction of their neighboring neurons (19)(20)(21)(22)(23)(24). Even more puzzling is that when a functional connection is established, multiple synaptic contacts are observed (22,23,25,26). Furthermore, there are only enough synaptic boutons on a local pyramidal axon (Ϸ1,000-1,500) to form multiple synaptic contacts with a small fraction of the 1,000-2,000 neighboring pyramidal neurons (14,27). A form of target neuron selection must therefore take place at some level. To resolve this question, we undertook a combined multineuron recording and confocal study to compare structural and functional properties of connections between neocort...

Summary The protein density and arrangement of subunits of a complete, 31-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by cryo-electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs), and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.