Homework Q&E 12

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Feb 20, 2024

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BCH 370 - Physical Methods of Biochemistry (Fall 2023) 1 Homework Q&E 12 Related to Text 9: Single-particle cryo-EM – How did it get there and where will it go. ► Read the parts of the paper that are highlighted in yellow, including the figures that are referenced in the highlighted text. You do NOT need to read the entire text. Key concepts from this paper and Homework related to Physical Methods of Biochemistry are listed below, together with links to videos or reading materials to help you understand each topic. There are additional links and resources on the Text 9 file. X-ray crystallography & Cryo-EM in perspective: https://pubs.acs.org/doi/10.1021/acs.biochem.7b01031 Cryo-EM: For those interested in going deep into this method, this lecture series is highly recommended: https://www.thermofisher.com/us/en/home/electron-microscopy/life-sciences/learning- center/cryo-em-university/getting-started-cryo-em.html Shorter videos, also included in the comments on Text 9: - 2D projections from negative stain images: https://vimeo.com/jiwasa/download/303387067/982bd370cd - The cryo-EM sample grid: https://vimeo.com/jiwasa/download/369583017/953ecaeb65 - Cryo-EM grid preparation: https://vimeo.com/jiwasa/download/369595743/fdd5cf79a6 - What is negative stain EM: https://vimeo.com/jiwasa/download/303930599/821b343918 - 3D reconstruction from averaged 2D projections: https://vimeo.com/543474043 - Direct electron-detection cameras vs CCD cameras: https://fr.coursera.org/lecture/cryo- em/detectors-Gy1g3 - Beam-induced motion: https://vimeo.com/543472124

BCH 370 - Physical Methods of Biochemistry (Fall 2023) 2 The text mentions a series of challenges that had to be overcome to obtain near atomic-resolution structures of proteins without crystals using single-particle cryo-electron microscopy. Answer each of the questions below in one brief sentence: One of the mentioned challenges was the need to keep samples in a vacuum. ► 1.1. Why is the vacuum needed? ( 0.1 points ) The vacuum is needed due to the strong scattering. ► 1.2. Why is a vacuum deleterious for biological macromolecules? ( 0.1 points ) A vacuum is deleterious for biological macromolecules because vacuum-caused dehydration would destroy the sample’s integrity. ► 1.3. What was the solution that solved this challenge? ( 0.2 points ) Taylor and Glaeser recorded better than 3-Å resolution electron diffraction patterns from frozen hydrated catalase crystals, demonstrating that the structural integrity of biological macromolecules in a high vacuum can be maintained through frozen hydration. ► 1.4. Another challenge mentioned in the text is that cryo-EM images are 2D projections of the imaged object. How can a 3D structure be determined from 2D projections? ( 0.1 points ) The use of the direct detection camera, improved motion correction, and the ability to record images at a high electron dose make the reconstruction of 3D density maps at atomic resolution possible for many proteins. ► 1.5. What is the relation between contrast and defocus? ( 0.1 points ) Defocus is how much off the focus an image is recorded and the contrast is limited in images that are recorded very close to focus. ► 1.6. List the 5 factors mentioned in the text that determine the resolution of a cryo-EM structure. ( 0.5 points ) The resolution of a single-particle cryo-EM structure depends on many factors, including the resolution and contrast of individual particle images, accuracy of aligning these images with each other, obtaining a sufficient number of images from all necessary views of the macromolecule within a reasonable time frame, the conformational and compositional homogeneity of these particles, and access to powerful enough computers with which to process images efficiently. ► 1.7. Why can the position of an electron be detected more precisely with a direct electron- detection camera than with a CCD camera? ( 0.1 points ) Direct electron-detection cameras detect charges generated directly from electrons striking the camera sensor, thus localizing the electron with much greater precision and resulting in substantially higher DQE than that of scintillator-based cameras.

BCH 370 - Physical Methods of Biochemistry (Fall 2023) 3 ► 1.8. What is one additional advantage of direct electron-detection cameras over CCD cameras that is associated with their image acquisition rate , and why is that advantageous for determining structures of proteins? ( 0.2 points ) Images recorded with direct-electron-detection cameras retain signal both at high frequency, for high-resolution structure determination, and at low frequency, for contrast required for image alignment. ► 2. Below are four figures showing the maps for a protein determined using cryo-EM at resolutions of 2 Å, 3 Å, 4 Å, and 5 Å. Write down the corresponding resolution for each of the maps. ( 0.4 points ) 3. This is related to the PDB’s June 2023 molecule of the month the human olfactory receptor that gives its stinky smell to aged cheese and its pungent smell to vinegar: https://pdb101.rcsb.org/motm/motm-by-date ► 3.1. Access the entry on the PDB for the olfactory receptor (PDB ID 8F76), and indicate the total number of unique protein chains in the asymmetric unit, and the name of each unique chain. ( 0.3 points ) ► 3.2. Download the PDB and emd map, open both on the same Chimera window. Color the background white, and each of the chains in a different color. Show side-chains for each of the chains. Display the map on ‘surface’ style with 0.5 transparency and a step of 1, and select the ‘threshold’ level such that only the map is displayed without much of the noise. Save an image (PNG, 300 dpi) of the complex from an angle where you can see all the chains, and paste the file or upload with your homework. ( 0.9 points )

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