Measurement of Dynamic trna Movement and Visualizing Ribosome Biogenesis Applications of Time resolved Electron Cryo microscopy

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1 2011 TIGP CBMB Seminar Measurement of Dynamic trna Movement and Visualizing Ribosome Biogenesis Applications of Time resolved Electron Cryo microscopy Ribosome dynamics and trna movement by time resolved electron cryomicroscopy, 2010 Nature, 466: , 2010 (Holger Stark s group, Max Plank Institute, Germany) "Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit " Science, 330:673 7 (James Williamson s group, Scripps Institute, USA) Speaker :Rob Shih Hsin Huang ( 黃士炘 ) Coach Professor: Dr. Wei Hau Chuang (Institute of Chemistry) Sit in Professor: Dr. Mei I Su (Institute of Biochemistry) Chair Professor: Dr. Joseph Jen Tse Huang (Institute of Chemistry) 2011 / 04 / 21

2 Scheme Introduction of Cryo Electron Microscopy (mechanism, advantages, drawbacks, sample preparation etc.) Concepts of single particle reconstruction Time resolved Cryo Electron Microscopy Application I : Observing trna movement Application II: 30S ribosome biogenesis

3 Introduction of Cryo Electron Microscopy

4 Introduction of Cryo Electron Microscopy (Cryo EM) Transmission electron microscope (TEM) (Detect transmitted electrons) Scanning electron microscope (SEM) (Detect secondary electrons ) Reflection electron microscope (REM) (Detect elastically scattered electrons) Cryo = Low Temperature Images from : Wikipedia

5 Pros and cons of three structural determination method nowadays X ray Advantages: Atomic resolution, No limitations of protein sizes for research Drawbacks: Challenge in crystallization, Snap shots in static, Large amount of sample required NMR Advantages: Atomic resolution, Similar to native status (solution phase), Dynamic and time resolved information Drawbacks: Hard to get complex structure information, Size limitation of sample Cryo EM Advantages: Native sample, less amount of sample required, Suitable for large or complex proteins, Dynamic and time resolved information Drawbacks: Expensive, Near atomic resolution, Low S/N ratio, Challenge of cryosample preparation

6 (Free electron laser scattering,fels) ACTA BIOPHYSICA SINICA 2010 Vol.26 No

7 Macromolecular structure determination methods by cryo EM 2D crystals Helical assemblies (1D crystals) Tomography (cryo ET) Single particle reconstruction Tomography Nat Methods Nov;6(11): EMDataBank.org: unified data resource for CryoEM, Nucleic Acids Research, 2010, 1 9

8 Plunge freezing machine Cryo Electron Microscopy sample preparation ACTA BIOPHYSICA SINICA Vol.26 No.7 Jul Electron Microscopy Methods and protocols 2nd edition, John Kuo, 2007

9 What cryo sample means? Vitreous ice layer 1. Glass like layer 2. Freeze native sample (c.f. dehydration) 3. Prevent from radiation damage Annu.Rev.Biophys.Biomol.Struct :303 19

10 Cryo Electron Microscopy sample preparation Images from : Wikipedia Electron Microscopy Methods and protocols 2nd edition, John Kuo, 2007

11 Introduction of Single Particle Reconstruction

12 Purpose Rebuilding single particle 3D structures from 2D image projections

13 Image acquisition Particle selection (Boxing) Acta. Cryst. (2000).D56, Electron Microscopy Methods and protocols 2nd edition, John Kuo, 2007

14 Class averaging Acta. Cryst. (2000).D56, Similar images are classified into subgroups on the basis of the coefficients of these components. Similar average of each subgroup gives an image with a greatly improved signal to noise ratio.

15 Euler angle determination (angular reconstitution) Quarterly Reviews of Biophysics 33,4(2000),pp The angular reconstitution technique is based on the common line projection theorem stating that two different 2D projections of the same 3D objectal ways have 1D line projection in common.

16 3D reconstruction from 2D projections

17 Summary of single particle reconstruction Numbers of images Programs Distribution of angles Quarterly Reviews of Biophysics 33,4(2000),pp

18 Time resolved Electron Cryo microscopy

19 Purpose X ray crystallographic techniques are less useful for studying pathway intermediates because this process involves large changes in the conformation of the constituents in short time. How to solve it?

20 Aim Image from : ACTA BIOPHYSICA SINICA 2010 Vol.26 No Image from : Wikipedia

21 Aim Using cryo electron microscopy and computational sorting of the cryo EM images present a movie of the sequence of events and the structural nature of the conformational changes.

22 Case I : Max Planck Institute, Germany

23 Elongation state Recycle Translation Ribosome of the bacterium Escherichia coli Translation is a dynamic process: both mrna and trnas move along the ribosome a process called translocation as amino acids are added to the nascent polypeptide chain. Images from: nobelprize.org

24 Introduction Kinetic analysis by cryo EM generally has too low a time resolution to follow the dynamics of EF G catalyzed forward translocation. But in the absence of EF G, spontaneous translocation occurs at a much lower rate in both the forward and backward (retro translocation) directions. So Fischer and co workers set out to investigate the kinetics of spontaneous retro translocation.

25 Introduction Translocation; one way, energy required Retero translocation; bi direction, spontaneous, no energy involved NATURE, 466(15), July 2010

26 But It would be a time consuming computing process if we add one more (time) parameter in single particle reconstruction procedure.

27 How to solve it? Graphic Processing Unit = GPU (NVidia) Video Processing Unit = VPU (ATI) GPU (VPU) is a specialized circuit designed to rapidly manipulate and alter memory in such a way so as to accelerate the building of images in a frame buffer intended for output to a display. GPUs are used in embedded systems, mobile phones, personal computers, workstations, and game consoles. from : wikipedia from : wikipedia

28 Fast computational sorting of the cryo EM images Images from : 48 CPUs PC cluster 15,000 images 7days 200 GPUs server 1000,000 images 14hrs (800x fast)

29 2 million single particle images captured in 28 hours 50 distinct three dimensional reconstructions ~9 to 20 A resolution 5 time interval in 3 temperature conditions

30 top (upper row) solvent side of the 30S subunit

31 Schematic of trna movement through the ribosome Pre Post Green: deactyl trna (first trna) purple: fmet Val trna (second trna) (b) Time course of retrotranslocation determined From cryo EM images. (d) Equilibrium constants for the transitions

32 50S subunit 30S subunit 50S subunit "Handover" of the deacylated trna from protein L5 to the L1 stalk. 30S subunit The anticlockwise 30S body rotation from pre1 to pre5 and the switch back to the non rotated conformation upon transition to post1

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35 Discussion As Fischer and colleagues point out, the main question now is whether EF G catalysed forward translocation can also be understood in terms of the ribosome as a Brownian machine. Brownian ratchet

36 Summery of Case I Fischer and colleagues seminal cryo EM work has revealed important and intriguing aspects of the interplay between rapidly interconverting ribosomal conformations.

37 Case II : Scripps Institute, USA

38 Clin Infect Dis. (2004) 38 (Supplement 4): S322 S327 Ribosome of the bacterium Escherichia coli The small subunit,30s,is made of 16S rrna(1,542nt) and 21 ribosomal proteins (r proteins)

39 Pulse chase monitored by quantitative mass spectrometry (PC/QMS) is a powerful technique for studying the assembly of macromolecular complexes Nature 438, (1 December 2005) Methods 49(2009)

40 Nature 438, (1 December 2005)

41 1 Million images

42 Structural determination of assembly intermediates Nature 438, (1 December 2005)

43 Assembly intermediate population flux overtime

44 Nomura assembly map on a time axis by PC/QMS Summary A parallel mechanism for 30S subunit assembly performed by DSP

45 Summary of Case II The DSP method used for this work is a novel approach for understanding structure and dynamics that can be applied to a wide range of large biologically important complexes.

46 Thanks for your attention!!!