Analysis of Global and Site-Specific Radiation Damage in Cryo-EM

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Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED

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A Rare Lysozyme Crystal Form Solved Using Highly Redundant Multiple Electron Diffraction Datasets from Micron-Sized Crystals

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Fabrication of carbon films with ∼500 nm holes for cryo-EM with a direct detector device

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.     

Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size

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The architecture of outer dynein arms in situ

Outer dynein arms, the force generators for axonemal motion, form arrays on microtubule doublets in situ, although they are bouquet-like complexes with separated heads of multiple heavy chains when isolated in vitro. To understand how the three heavy chains are folded in the array, we reconstructed the detailed 3D structure of outer dynein arms of Chlamydomonas flagella in situ by electron cryo-tomography and single-particle averaging. The outer dynein arm binds to the A-microtubule through three interfaces on two adjacent protofilaments, two of which probably represent the docking complex. The three AAA rings of heavy chains, seen as stacked plates, are connected in a striking manner on microtubule doublets. The tail of the alpha-heavy chain, identified by analyzing the oda11 mutant, which lacks alpha-heavy chain, extends from the AAA ring tilted toward the tip of the axoneme and towards the inside of the axoneme at 50 degrees , suggesting a three-dimensional power stroke. The neighboring outer dynein arms are connected through two filamentous structures: one at the exterior of the axoneme and the other through the alpha-tail. Although the beta-tail seems to merge with the alpha-tail at the internal side of the axoneme, the gamma-tail is likely to extend at the exterior of the axoneme and join the AAA ring. This suggests that the fold and function of gamma-heavy chain are different from those of alpha and beta-chains.     

An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.     

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.     

Protonation and inhibition of Nitrosomonas europaea cytochrome c peroxidase observed with protein film voltammetry

The impact of protonation and inhibitor binding of the diheme cytochrome c peroxidase (CCP) from Nitrosomonas europaea has been examined by the technique of catalytic protein film voltammetry (PFV). Previous efforts have shown that the low-potential heme active site (L) binds substrate and yields electrocatalysis at an pyrolytic graphite edge electrode, with properties evocative of a high-potential intermediate, with E(m)>540mV (vs. normal hydrogen electrode) [A.L. Bradley, S.E. Chobot, D.M. Arciero, A.B. Hooper, S. J. Elliott, J. Biol. Chem. 279 (2004) 13297-13300]. Here we demonstrate through similar experiments that catalytic PFV generates limiting currents which allow for electrochemically-detected enzymology of the Ne CCP: such as the demonstration that pH-dependent Michaelis-Menten constants (K(m) values) reveal a pK(a) value of 6.5 associated with the "ES" complex. Further, the direct electrocatalysis is shown in the presence of known inhibitors (cyanide and azide), indicating that inhibitor binding occurs at L, and shifts the resulting catalytic midpoint potential in a negative direction. Michaelis-Menten treatment of the limiting currents generated in the presence of variable concentrations of inhibitors showed that cyanide behaved as a competitive inhibitor with a K(i) value of 0.15muM; azide revealed a mixed-mode of inhibition. The observed data were found to support a previous model of electrocatalysis, and the role of proton transfer chemistry in the active site is discussed in terms of a structural model.     

Rapid protein identification using direct infusion nanoelectrospray ionization mass spectrometry

Current protein identification techniques are largely based on MALDI-TOF mass fingerprinting and LC-ESI MS/MS sequence tag analysis. Here we describe an improved method for rapid protein identification that uses direct infusion nanoelectrospray quadrupole time-of-flight (nanoESI QTOF) MS. Protein digests were analyzed without LC separation using nanoESI on a QSTAR XL MS/MS system in information dependent data acquisition mode. The protein identification conditions and parameters were extensively evaluated with in-solution and in-gel digested protein samples. Rapid identification of proteins was achieved and compared directly to the results obtained on the same samples using nanoflow HPLC-MS/MS on the QSTAR system. The increased throughput, reproducibility, the high data quality, and the ease of use make the direct infusion system an efficient and affordable technique for protein identification analysis.