Computational protocol: Polymer Nanodiscs: Discoidal Amphiphilic Block Copolymer Membranes as a New Platform for Membrane Proteins

Similar protocols

Protocol publication

[…] The morphology of polymersomes was characterized on a Hitachi H-8100 electron microscope equipped with an AMT digital side mount camera and operated at an accelerating voltage of 75 kV. The polymersomes were stained with 1% uranyl acetate on the TEM grid immediately before taking measurements. To observe PNDs serial dilutions of the sample were stained with uranyl formate as described, using sample buffer (200 mM NaCl, 20 mM Tris/HCl pH 7.5) instead of water for the washes. Specimens were then imaged in a Tecnai 12 electron microscope (FEI Company, Hillsboro, OR) equipped with a Lab6 electron source operated at 120 kV. Micrographs were automatically collected under low-dose conditions using EPU (FEI Company, Hillsboro, OR) at a nominal magnification of 67,000 X. Under-focused images (1 to 3 μm) were recorded on a US4000 CCD camera (Gatan, Pleasanton, CA) with a pixel size at the specimen level of 1.77 Å. The contrast transfer function (CTF) of the images was determined using ctffind. Images were selected based on the following criteria: visual assessment of particle dispersion, quality of stain and background, low astigmatism, and amplitude of signal and correlation with the expected CTF in the frequency range of 50 to 10 Å. All further processing was performed within the framework of EMAN 2.12.66 Particles were extracted with a box size of 150 pixels, CTF corrected and pooled in a set. Reference free classification of down-sampled and low-pass filtered (16 Å) images was used to eliminate “bad” particles and false positives, resulting in a “cleaned” dataset. This set was subject to a second round of classification. […]

Pipeline specifications

Software tools CTFFIND, EMAN
Application cryo-EM
Chemicals Adenosine Triphosphate