The Molecular Foundry - Imaging and Manipulation of Nanostructures - Staff

Gang Ren

Staff Scientist, Imaging and Manipulations of Nanostructures Facility

Research Interests

The research objective of this laboratory is mainly focusing on developing the individual particle electron microscopic (IPET) technique for intermediate-resolution structure determination of an individual protein. By particle-by-particle structure determination using IPET, this lab is for understanding the protein structure, fluctuation, function and mechanism of highly dynamic and heterogeneous proteins. The development of the IPET technique is performed from two aspects: i) High-resolution electron tomography imaging of an individual protein; ii) high-resolution electron tomography reconstruction algorithm.

Potential Impact: The dynamic nature and structural heterogeneity is essential for protein functions. However, structure determination of dynamic protein is frustrated by current techniques, such as X-ray, NMR and single-particle electron microscopy. This lab invented individual particle electron tomography (IPET) technique can be a fundamental solution to be used to determine the structural dynamics at an intermediate resolution (1-2 nm). IPET is a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single ''snapshot'' molecule structure determination. Thus, IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.

Research Areas: The research in this lab falls into three main categories: i) developing high-resolution IPET technique; ii) structural studying the dynamic proteins, such as human IgG antibody and human "good-cholesterol" molecule (HDL, in a total mass of 120kDa-200kDa); iii) studying protein function and mechanism at atomic resolution level by molecule dynamic simulation based on EM structures as constrains.

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Current Projects

Toward a high-resolution structure of an individual protein
The long-term goal of this lab is to develop a robust method to determine a 3D image of an individual protein at a resolution that sufficient to answer biological stories. This lab recently reported a 14Å-resolution 3D image of an individual IgG antibody from negative-staining electron tomography (NS-ET) and a 36Å-resolution 3D image of an individual HDL particle from cryo-electron tomography (cryoET) proved the concept of that an individual protein 3D map at an intermediate resolution is possible to be achieved by this lab invented method - individual particle electron tomography (IPET) (PLoS ONE, 2012, 7(1):30249).

The strategy in improvement of electron tomography is from two aspects, high-quality (includes high-resolution and high-contrast) image acquirement and high-resolution reconstruction algorithm. For high-quality imageing, this lab developed an optimized negative-staining protocol (OpNS) for high-contrast imaging (JLR, 2010, 51(5):1228-36; and JLR, 2011, 52(1):175-84), invented cryo-positive-staining (cryoPS) protocol (NBC, 2012, 8(4): 342-349) for high-resolution and high-contrast imaging, and improved cryo-electron microscopy (cryoEM) protocol for high-resolution imaging (JBC, 2010, 24;285(52):41161-71) For high-resolution reconstruction, this lab developed IPET method via a focused electron tomography reconstruction (FETR) for high-resolution reconstructing a high-resolution 3D map from high-noise images of an individual protein.

The structural dynamics of human IgG antibody

The human IgG antibody (molecular weight: ~150 kDa) is well known as naturally dynamics and fluctuation in solution. X-ray diffraction provides detail structure of IgG antibody (1iGT) that locked in their lattice positions. Thus, the structure lost the information about its dynamic and fluctuation. To uncover the dynamics and fluctuation of IgG antibody, we use IPET to track and window a targeted single-instance of an antibody particle from each tilt micrograph after CTF correction by TOMOCTF, and then FETR reconstructed the images of this particle into a 3D density map at 14 Å resolution (PLoS ONE, 2012, 7(1):30249). The 3D density map contained rich structural details, including the shape of each domain, and even the holes inside each domain. The 3D reconstruction from single-instance antibody that is free of conformational dynamics and heterogeneity can be treated as a "snapshot" of the dynamic structure of antibody. By comparing these "snapshot" IgG antibody structures from different antibody particles, this method could allow us to study antibody dynamics. For example, by aligning two docked PDB files by aligning their F_c domain, the F_ab domains are different from each other in location and orientation, suggesting the equilibrium fluctuation and structural dynamic character of IgG antibody.

A computer animation demonstrates the flexible dynamics — the moving parts — of human IgG antibody. 3-D images of two individual antibody particles (gray) were generated using EM tomography with IPET. The demonstration shows how the same molecular chains (red, orange, and green noodle-like models) of antibody particle #1 can fit precisely into particle #2, which was found under the microscope in an entirely different pose.

The structural dynamics of human high-density lipoprotein (HDL, "good-cholesterol")

Human high-density lipoprotein (HDL, so-called "good cholesterol"), a highly dynamic protein conveys excess cholesterol from peripheral tissues to the liver and steroidogenic organs for clearance during reverse cholesterol transport. HDL particles in vivo vary in size, shape, components, and biological functions. A fraction, nascent HDL is a critical intermediate between lipid-poor apoA-I and mature spherical HDL during HDL assembly. Nascent HDL contains 120-240 phospholipids and 2-3 amphipathic apolipoprotein A-I (apoA-I, MW: 28kDa) molecules (in total MW of 140-240 kDa). Although nascent HDL is small protein that is super challenging for imaging by cryoEM and cryoET and even challenging for 3D reconstruction, we have successfully imaged nascent HDL embedded in vitreous ice by cryoET (JBC, 2010, 24;285(52):41161-71) and then reconstructed at 36 Å resolution 3D map (PLoS ONE, 2012, 7(1):30249), suggesting our IPET method is a robust and powerful approach. The 3D reconstruction from a single-instance HDL particle that is free of conformational dynamics and heterogeneity can be treated as a "snapshot" of the dynamic structure of protein. By comparing these "snapshot" HDL structures, this method could allow the study of HDL structural dynamics.

3-D images from a single particle (A) a series of images of an ApoA-1 protein particle, taken from different angles as indicated. A succession of four computer enhancements (projections) clarifies the signal. In the right column is the 3-D image compiled from the clarified data. B) is a close-up of the reconstructed 3-D image. C) Analysis shows how the particle structure is formed by three ApoA-1 proteins (red, green, blue noodle-like models)
The function and mechanism of human cholesteryl ester transfer protein (CETP)

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using our reported OpNS and cryoPS for imaging single-particle approach for 3D reconstruction and molecular dynamics simulation for understanding the mechanism, we discovered that: i) CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein; ii) the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

Past Research

Structure of low-density lipoprotein (LDL, "bad-cholesterol" ) by conventional single-particle cryoEM.
The heterogeneous nature of lipoproteins is a major difficulty in obtaining 3D reconstructions at intermediate resolution (~2 nm) by single-particle analysis of cryo-EM. Since we were not able to improve the homogeneity of lipoprotein particles by biochemical methods, we developed several programs for further selecting lipoprotein particles with a homogeneous size for 3D reconstruction and refinement that we term "computational size-exclusion gel-filtration" algorithms. Using this method, we determined the structure of human plasma LDL alone and bound to the LDL receptor, by single particle cryo-EM. To obtain a high-quality 3D map, we selected a homogenous population that constituted only 10-20% of the total data set. These 3D reconstructions of LDL alone and LDL bound to LDL receptor yielded interesting results that include the juxtaposed-stack model of cholesteryl ester cores, the spatial arrangement of the five domains of apoB-100, and the location of the β-propeller domain of the LDL receptor on the LDL surface.
Atomic resolution structure of Aquaporin-1 transmembrame protein by cryo-electron crystallography

Many protein/soft materials preferred to form in two-dimensional crystal (2-D array) instead of 3-D crystal that is required by X-ray structure determination. For atomic resolution structure determination from the 2D crystal, we experienced in structure determination of 2D crystal of aquaporin 1 (AQP1) water channel at 3.7Å resolution by cryo-electron crystallography (PNAS, 2001, 98(4): 1398-403). The structure is first atomic structure determined preserved in vitrified buffer instead of any additive (such as glucose or trehalose). The paper has been referenced by the Advanced Information for the 2003 Nobel Prize in Chemistry.
Elastic and inelastic electron scattering factors

We have developed a robust algorithm and computer program for the parameterization of elastic and absorptive electron atomic scattering factors (Acta Cryst., 1996, A53: 257-276; and A52: 456-470). The algorithm is based on a combined modified simulated-annealing and least-squares method, and the computer program works well for fitting both elastic and absorptive atomic scattering factors with five Gaussians. As an application of this program, the elastic electron atomic scattering factors have been parameterized for all neutral atoms and for s up to 6 Å-1. For other application, Debye-Waller factors and absorptive scattering factors are given of 44 elemental crystals over the temperature range from 1 to 1000 K or to the melting temperature. These data can be used to estimate the Debye-Waller factor at any temperature using the analytical Debye expression of the phonon density of states. Since 2006, these data have been collected by the International Tables For Crystallography. Volume C (Chapter 4.3: Electron Diffraction by Colliex, C., J. M. Cowley, S. L. Dudarev, M. Fink, J. Gjonnes, R. Hilderbrandt, A. Howie, D. F. Lynch, L. M. Peng, G. Ren, A. W. Ross, V. H. Smith Jr, J. C. H. Spence, J. W. Steeds, J. Wang, M. J. Whelan and B. B. Zvyagin, International Tables For Crystallography. Volume C: Mathematical, physical and chemical tables, Edited by E.Prince, Fourth Edition, Published by Kluwer Academic Publishers, 2006, pp259-429)

Selected Publications

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein. Zhang, L., F. Yan, S. Zhang, D. Lei, M. A. Charles, G. Cavigiolio, M. Oda, R. M. Krauss, K. H. Weisgraber, K.A. Rye, H.J. Pownall, X. Qiu & G. Ren*. Nature Chemical Biology, (2012), 8(4): 342-349.
IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure. Zhang, L. and G. Ren*. PLoS ONE, (2012), 7(1):30249
The Morphology and Structure of Lipoprotein Revealed by Optimized Negative Staining. Lei Zhang, James Song, Giorgio Cavigiolio, Brian Y. Ishida, Shengli Zhang, John P. Kane, Karl H. Weisgraber, Michael N. Oda, Kerry-Anne Rye, Henry J. Pownall, and Gang Ren*, J. Lipid Research, (2011) 52(1):175-84.
Model of Human Low Density Lipoprotein and Bound Receptor Based on Cryo-EM. Gang Ren*, Gabby Rudenko, Steven J. Ludtke, Johann Deisenhofer, Wah Chiu*, Henry J. Pownall*. Proc. Natl. Acad. Sci. USA. (2010) 107: 1059-1064.
An Optimized Negative-staining Protocol of Electron Microscopy for apoE4.POPC Lipoprotein. Zhang, L., J. Song, Y. Newhouse, S. Zhang, K.H. Weisgraber, and G. Ren*, J. Lipids Research, (2010), 51(5):1228-36.

(* corresponding authors)

Education

B.A., Theoretical Physics, Lanzhou University, China, 1986-1990

M.S., Theoretical Physics (General relativity and gauge theory), Lanzhou University, China, 1990-1993, Advisor: Prof. Yi-shi Duan. Thesis: "Theory and Application of 2+1 Dimensional Topological Current"

Ph.D. Material Physics (Electron microscopy), Univ. of Science and Technology Beijing, and Beijing Laboratory of Electron Microscopy, Chinese Academy of Science, Beijing, China, 1993-1997, Advisor: Profs. Lian-mao Peng (2012 chair of IUCr -Commission of Electron crystallography) and Kehsin Kuo, Thesis: "Quantitative Electron Diffraction Theory and Application"

Previous Positions

Postdoctoral Fellows (American Heart Association and University of California), Cell Biology, the Scripps Research Institute, La Jolla, CA, 1997-2003, Advisors: Profs. Alok K. Mitra and Mark Yeager

Staff Scientist (non-PI), National Center for Macromolecular Imaging, Baylor College of Medicine, Houston, TX, 2004-2006, Director: Prof. Wah Chiu

Keck Fellow (Principal Investigator), Dept. of Biophysics and Biochemistry, University of California, San Francisco, CA, 2006-2010