From cryo-em images of protein complexes to high-resolution 3D structures

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1 From cryo-em images of protein complexes to high-resolution 3D structures Rouslan Efremov /01/23 Gent

2 What you should do in order for us to make more rapid progress is to make the electron microscope 100 times better Richard Feynman Plenty of Room at the Bottom, 1959

4 Outline Basic concepts of image processing in cryo-em A real-life example BECM: Cryo-EM facility

5 Outline Basic concepts of image processing in cryo-em A real-life example BECM: Cryo-EM facility

6 How to go from 2D images to 3D density map?

7 Central section theorem Fourier transformation of a projection of a 3D object = central section of 3D Fourier transformation of an object perpendicular direction of projection

8 Central section theorem: application in practice

9 Central section theorem: take home message Single particle Tomography complete 180 degrees rotation around at leas one axis is needed relative orientations of the projections have to be known

Highest CC Prn q 2n,f 2n Alignment and CC of

10 In single particle analysis particle orientations are refined iteratively by method of projection matching Stack 3D reconstruction New 3D reconstruction Pr1 q 1,f 1 Pr2 q 2,f 2 Highest CC Prn q 2n,f 2n Alignment and CC of each image to each projection Orientation assignment Y, q 2, y 2

11 Where do I get initial model from? From common line theorem

12 2D alignment and classification

13 How do we compare images? distance = (I1 ij I2 ij ) 2 ij 1 2 CC = ij I1 ij * I2 ij Search for Dx, Dy and Da that maximize cross correlation between 2 images

14 Why is it difficult? original image low dose image low dose defocused image Poisson noise CTF modulaiton

15 Fourier spectrum of EM image is modulated by contrast transfer function Point spread function contrast-transfer function

16 Dose fragmentation and correction of Beaminduced movement

17 Where do we start? To align particles one needs a reference image stack Average all particles in initial orientation Average Align particles to average and average all of them in the new orientation Stack with new orientation parameters: Dx, Dy, a Repeat until convergence

initial N/n particles and averaging them (N-total number of

18 Classification: separation of the stack into classes of particles Seed images are generated Can be generated by selecting randomly initial N/n particles and averaging them (N-total number of particles, n number of classes) Reference 1 Reference Reference n

19 Classification: separation of the stack into classes of particles Particles are compared without alignment to each seed class average and assigned to a class with highest CC CC1 CC Reference 1 Reference Reference n The procedure is repeated until it converges

2,f 2 Highest CC Prn q 2n,f 2n Alignment and CC of

21 Classification can also be done in 3D 3D reconst ruction New 3D reconstruction Stack Pr1 q 1,f 1 Pr2 q 2,f 2 Highest CC Prn q 2n,f 2n Alignment and CC of each image to each projection Orientation assignment Y, q 2, y 2

22 Multiple 3D reconstrucitons visualize dynamics of protein complexes retro translocation by ribosome Fischer et.al., Nature 2010

23 Outline Basic concepts of image processing in cryo-em A real-life example BECM: Cryo-EM facility

24 Software for image processing RELION SPHIRE

25 Individual particles are extracted form EM images

26 and assembled in a single file called stack

27 Particles are aligned and classified

28 Initial model

29 3D reconstruction is obtained by iterative refinement of particle s orientation

30 3D map reveals secondary structure elements Efremov et.al., Nature, 2015

31 Complete 3D map was modelled Efremov et.al., Nature, 2015

32 Multiple conformations of RyR are observed with and without calcium

33 Outline Basic concepts of image processing in cryo-em A real-life example BECM: Cryo-EM

34 Three components are required to do a state-of-the-art cryo-em 1. Electron microscope operating at accelerating voltage of 300 kv with Field Emission Gun (FEG) and cryo-stage 2. Direct Electron Detector (DED) 3. Automation

35 JEOL CryoARM300 Cold Field Emission Gun, 300 kv Four-lenses condenser Phase plate Cryogenic stage Sample autoloader for 12 grids Electron energy filter direct electron detector (Gatan)

36 2 x JEOL JEM-1400: 120 kv

37 CryoBECM: Building

38 Key points Based in Brussels at VUB Open to all Flemish/Belgian/European research groups and industry Core expertize: single particle cryo-em and later cryotomography Dedicated microscopists and manager will provide full support with data collection, sample preparation and training

September 2018 Active development of cryo-tomography expertize 2020 2017

39 BECM: VIB-VUB Biological Electron Cryogenic Microscopy Center Timeline Tender & ordering 2016 Commissioning of Flanders cryo-em Center 19 September 2018 Active development of cryo-tomography expertize Hire microscopist Microscope installed 2019 Access for external academic and industrial users

40 Hemoglobin 64 kda, 3.2 Å?

Transmission Electron Microscopy 3D Construction by IPET Method TCBG GPU Workshop

Transmission Electron Microscopy 3D Construction by IPET Method TCBG GPU Workshop Xing Zhang 8/4/2013 Molecular Foundry Lawrence Berkeley National Laboratory Department of Applied Physics Xi an Jiaotong