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Tunable Substrates


Figure 3 : Structural biology interpretation of the isolated BRCA1 assemblies. (a) The 3D density map calculated from cryo-EM images of BRCA1 assemblies shown in different orientations. The structure contains the RNAP II core (yellow; pdbcode, 4A93) [ 12 ] and the BRCA1 C-terminal domain (gray; pdbcode, 1JNX) [ 13 ] adjacent to the RNAP II stalk. Scale bar is 10 nm. (b) Cross sections (1–6) are shown in the region of the density map that contains the BRCT domain. (c) A close-up view of the BRCA1 C-terminal domain (gray) in the left panel shows the peptide-binding pocket (gray rectangle). The homology model of the mutated BRCA1 5382insC protein (red) reveals the binding pocket signifi cantly disrupted in the mutated domain. A red star indicated the location of the mutated amino acid adjacent to the RNAP II core (black arrow) [ 8 ].


Harvesting BRCA1 from breast cancer cells . Major limitations in our understanding of the BRCA1 tumor suppressor are due to insuffi cient structural studies on human samples. We recently used SiN microchips to harvest native BRCA1 proteins from patient-derived breast cancer cells ( Figure 2 ) [ 8 , 9 ]. Specifi cally, cancer cells (HCC70 line) were cultured and approximately 2,000,000 cells were collected for biochemical and structural analysis of the internal protein assemblies. T e nuclear material was separated from other cellular components, and the nuclear fraction was further enriched for the RNA Polymerase II (RNAP II) core complex ( Figure 2a ). T e RNAP II complex is responsible for transcribing DNA to mRNA messages in all human cells, and it is known to interact with BRCA1 in the cell’s nucleus [ 10 ]. T e RNAP II-enriched fractions were applied to microchips decorated with antibodies against BRCA1 ( Figure 2b ). T is step selects for active RNAP II assemblies associated with BRCA1. T e microchip samples were then plunge-frozen for cryo-EM imaging ( Figure 2c ).


Analysis of BRCA1 complexes tethered to SiN microchips .


Specimens were examined using a FEI Spirit BioTwin TEM (FEI Company) equipped with an LaB 6 emitter operating at 120 kV under low-dose conditions (~5 electrons / Å 2 ). We collected EM images and selected >20,000 individual particles from the images using the program PARTICLE ( http://www. image-analysis.net/EM/ ). T e selected particles were exported into the RELION soſt ware package [ 11 ] that was used to compute and refi ne 3D reconstructions of the frozen-hydrated assemblies.


2017 July • www.microscopy-today.com


Based on computed compar- isons between the experimental particle images and that of the RNAP II initial model (pdb code, 4A93 [ 12 ]), multiple structures were output by RELION independent of the starting parameters. As each reconstruction was highly similar in its 3D features, we calculated a composite structure by combining all of the particles ( Figure 3a ). T e resolution of the fi nal structure was ~2.2 nm using the 0.5-Fourier shell correlation (FSC) criteria [ 11 ]. In comparing the composite structure to the RNAP II model, we confi rmed two extra densities in the EM map that were not present in the RNAP II structure lacking BRCA1 ( Figure 3 , yellow [ 12 ]). In conjunction with BRCA1 antibody-labeling results, each of the additional densities in the 3D map were attributed to either the BRCA1 N-terminal (RING) or C-terminal (BRCT) domains. T e unoccupied density proximal to the C-terminus of RNAP II was


assigned to the BRCT domain ( Figure 3b , gray; pdb code, 1JNX) [ 13 ]. Representative cross-sections (1–6) through the BRCT region in the EM map show that the atomic model fi ts well within the density. As one traverses through the cross sections, the atomic model and EM density disappear at the same rate. T is eff ect indicates that the atomic model fully occupies its assigned location and that there is no unassigned density leſt over, an indicator of a suitable model fi t in the density map.


Improving our knowledge of how the BRCT domain interacts with other proteins is important, considering many cancer-related mutations in BRCA1 reside in this region of the protein. T erefore, we calculated a homology model for the notable BRCA1 frame-shiſt mutation, BRCA1 5382insC . Comparing the structure of the wild type BRCT ( Figure 3c , leſt panel) with a homology-based model of the mutated BRCA1 5382insC ( Figure 3c , right panel), we found S1755 was mutated to L1755 ( Figure 3 , red star) due to a frame-shiſt in the DNA sequence [ 8 ]. Likewise, we found no secondary structure beyond G1763, and the BRCA1 binding pocket was signifi cantly disrupted in the mutated model compared to the wild type binding site ( Figure 3c ). T ese modeling results suggested that the BRCA1 5382insC mutation alters interactions with proteins that bind in this region, including RNAP II [ 9 , 10 ]. With this new information in hand we expect the continued investigation of these new structures, along with complementary biochemical analysis, to shed light on the molecular underpinnings of BRCA1 disease mechanisms in the growing fi eld of structural oncology.


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