NetNotes


IF this complex is located inside the cytoplasm of any given cell, the optimal pH would be close to pH 7.0 (with quite some variations, de- pending on the physiology of the cell!). Tis means that the conc of H+ ions ideally might be 10 times higher than in the buffer you are using. Second: NaCl as the main salt used in your buffer (150 mM) is close to what is optimal for extracellular environments but NOT to intra- cellular conditions. I doubt that this is ideal for your complex under investigation. Inside cells, you have a balance of K+ and Na+ ions, with 3 to 10(0)×higher conc of K+ (!!). In addition, there is some Cl- found inside cells, but the dominant counterions are phosphate groups from nucleic acids and metabolites, and carboxylates (from metabolites). Can Cl- be used? Tey may, but if you fail, you have to think about this. Tus, I would use HEPES at pH 7; use the optimal temp for your complex before starting the freezing process (which organism at which “optimal” temp? this influences the real pH); and a balanced amount of lots of K+, little Na+, and counterions (you may start with Cl- salts, simply for ease of use; but later, you may think about phosphates and carbonates). Adding EDTA and DTT is common, but may not be physiological? However, it might help. EDTA substitutes for the fact that inside a cell there are many metabolites with similar “complexing” functions; DTT substitutes for the fact that inside a cell there are usu- ally quite complex systems for keeping the Redox state in balance (i.e., reduced). Tis again depends on your type of cell. Reinhard Rachel reinhard.rachel@biologie.uni-regensburg.de


I would try Quantifoil carbon grids R2/4+ or R3/3+2 nm addi-


tional carbon film. Always check NS first and then go into cryo. Use 100% humidity and 4°C. Blot for only 2 or 4 seconds. If you succeed, then you can increase the sample concentration by a factor of 10 and freeze 1 or 2 sec R1.2/1.3 without carbon. Good luck. I know every grid looks different. Jorg Buerger buerger@molgen.mpg.de


Are you sure the particles are intact aſter freezing? If the protein


complex dissociates on the air-water interface, it might look like poor particle spread. Also be aware that textbook-like particle spread is de- sirable, but by no means a necessary requirement to obtain high-reso- lution reconstructions. If you can clearly see the particles, try collecting a small dataset on a better microscope. You might be surprised. Other ideas to improve your sample: Crosslinking (this might not


be ideal for nucleic acid—protein complexes because the crosslinker can modify lysine residues in the DNA binding interface, so check the crosslinked sample with an EMSA. Crosslinking has the advantage of stabilizing the complex through intermolecular crosslinks and pas- sivating the surface through mono-crosslinks) and detergents (when detergents are used increase the protein concentration, aim for around 2 mg/mL at least). Matthias Vorlaender matthias.vorlaender@embl.de


Orientation Issue with Membrane Protein Structure 3DEM Listserver I am trying to solve the structure of a tetrameric membrane protein


complex with the protein embedded in detergent micelle (0.05% GDN) and a soluble accessory protein attached to it. Following 2D classification, while the side views and oblique views are easily visible, the top and bottom views are few (∼1–2%) and further diminished in subsequent rounds of 2D classification. While we have tried different grid types to overcome the orientation problem at the sample level, I was wondering if there are certain tweaks we can make to the analysis parameters (particle picking, box size, mask, contrast transfer function, etc.) to enhance the signal of a protein embedded within a micelle in the current data set. We are using Relion 3.1 for SPA. Any suggestions to salvage this set of data will be very helpful. Saumya Bajaj saumya.bajaj@ntu.edu.sg


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Assuming this is a symmetric membrane protein with at least


3-fold rotational symmetry, you don’t need the top and bottom views to fully sample Fourier space and arrive at a high-quality reconstruc- tion. Side-views of a rotationally symmetric particle are sufficient, and the reconstruction will be complete. Dmitry Lyumkis dlyumkis@ salk.edu


Why do you say that the symmetry has to be at least C3? Philip


Koeck koeck@kth.se I am also confused by this. Shouldn’t a tomographic series (180


degrees worth of side views) do it for a C1 particle? Basil Greber basil- greber@gmx.net


If it is a two-fold rotationally symmetric object, adhered to the


air-water interface along a single side view, then a projection along, e.g., phi=0°/theta=90° samples the same Fourier plane as the sec- ond projection along phi=180°/theta=90°. Te reconstruction will behave in a manner that is identical to one that is composed of exclu- sively top views of a rotationally symmetric object (or, more simply, an asymmetric particle with one preferential orientation, normal- izing for the number of asymmetric units). Basically, you will end up hyper-sampling around one plane in both cases, except that the planes will lie along distinct axes of the transform. Both are bad cases and, in the absence of other views, will lead to a bad reconstruction. To be clear: this is assuming that there is only one preferential orien- tation along the side view of a two-fold rotationally symmetric par- ticle, and not more. If you have 3-fold rotational symmetry, and the sample is adhered to its side view, projections separated by phi=120° spacing will lead to sampling of Fourier planes separated by phi=60° (due to symmetry). Effectively, you add two additional planes. As- suming you have enough particles, and there is a bit of spread in the phi angle, in most practical cases, the reconstruction of a 3-fold rota- tionally symmetric object will be complete (or nearly so). Te higher the rotational symmetry, obviously the better. To answer the origi- nal question, adding top views for your membrane protein will be negligible in the reconstruction. Looks like you should already get a very nice map from the current data. Give it a try. Dmitry Lyumkis dlyumkis@salk.edu


You are completely right. 180° of side views about a single axis


samples 100% of 3D Fourier space with a C1 particle. Ed Morris ed.morris@icr.ac.uk


Tis is done all the time in helical reconstruction, to resolutions


better than 3.0 angstroms. Having all projections of side views is a sin- gle-axis tilt series, which yields all information needed in the absence of any symmetry. Edward Egelman egelman@virginia.edu


To be honest, I’m a little surprised by this discussion, as it is


part of most basic Cryo-EM intro courses. While Ed is correct for helices, of course, and while having all possible side views does, indeed, yield a complete data set, there is a problem in the case of single particle analysis. In helical reconstruction, once the symme- try is known, there is a relationship between linear position along the length of the helix and orientation about the helical axis. In the case of single particle analysis, with only pure “side views” (perpen- dicular to some axis), there is no information available to accurately determine the angle about the symmetry axis, since the only com- mon line the projections share lies on the helical axis. While there are approaches to come up with reasonable results (for example the “sidewinder” program developed by Penczek) to achieve good


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