Structural Dynamics II: Conformational Ensembles of Proteins Studied by Cryo-EM and X-ray Scattering

Conference: 2020: 70th ACA Annual Meeting
08/06/2020: 12:00 PM  - 3:00 PM 
Oral Session 


The macromolecules of life are often likened to elaborate machines, with many moving parts that must work collectively to achieve biological function. However, it has proven exceedingly difficult to understand how these machines work from traditional, static "snapshots" of structure alone. Thus, a new field of dynamic structural biology has emerged at the intersection of a diverse and evolving set of techniques. In Part II of this two-part session sponsored by Structural Dynamics, we focus on the analysis of conformational and thermodynamic ensembles by cutting-edge approaches in cryo-electron microscopy (cryo-EM) and solution X-ray scattering. Recent developments in cryo-EM bring us closer to ensemble-like structural depictions that attempt to describe and better understand both compositional and conformational particle heterogeneity. Meanwhile, X-ray scattering allows for an unmatched view of a protein's dynamic behavior in solution, and new avenues of analysis reveal meaningful structural information. This session highlights how cryo-EM and SAXS are uncovering the roles that conformational changes, intrinsic disorder, and structural variation play in protein function.


Opening Remarks

12:00 PM - 12:06 PM 

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility.

12:06 PM - 12:24 PM 
Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that produce diverse secondary metabolites, including antibiotics and other therapeutics. They are arranged as an assembly line of modules where each module incorporates one aminoacyl monomer into the nascent peptide. Little is known about the interplay between modules within multimodular NRPSs. We determined five x-ray crystal structures from the first two modules of linear gramicidin synthetase, including the full core dimodular structure showing trans-module delivery of the peptide intermediate during the condensation reaction. The structures, along with small-angle x-ray scattering data, show that adjacent modules undergo massive conformational rearrangements and that relative module positions are not governed by the catalytic cycle. Using the structures and covariation analysis, we bioengineered a module-swapped dimodular NRPS with improved catalytic abilities. 

View Proposal 330


Janice Reimer, University of California San Diego

Additional Author(s)

Maximilian Eivaskhani, McGill University
Ingrid Harb, McGill University
Alba Guarné, McGill University
Martin Weigt, Sorbonne Université
Martin Schmeing, McGill University

How CRY Regulates the Clock: Structural Studies of a Dynamic Mammalian Circadian Complex

12:24 PM - 12:42 PM 
Circadian oscillators are molecular pathways that confer rhythmic signaling with a period that approximates the day/night cycle. In the mammalian system, the transcription factor CLOCK: BMAL1 forms the core of a transcriptional/translational negative feedback loop that drives the rhythmic expression of large swaths of the genome, including the genes that encode the circadian repressors, CRY1/2 and PER1/2. While repression by CRY and PER is essential to generate circadian rhythms, a mechanistic understanding of how CRY and PER work together to repress CLOCK: BMAL1 has remained elusive, in part through the difficulty in obtaining crystallographic data of circadian protein complexes. Our work combines structural data from single particle cryo-EM and small angle X-ray scattering with computational methods to generate an integrative model of CRY-mediated repression in the clock. We show that CRY1 interacts directly with the PAS-B domain of CLOCK through the secondary pocket of its photolyase homology region (PHR) domain, recruiting PER to the transcription factor and sequestering the transactivation domain of BMAL1 from transcriptional co-activators to repress CLOCK:BMAL1 activity. We also describe how PER2 binding to the PHR domains regulates CRY1/2 in an isoform-specific manner by remodeling a dynamic loop at the secondary pocket in CRY2 that results in CRY1-like affinity for CLOCK:BMAL1, serving as a molecular equalizer. 

View Proposal 317


Colby Sandate, Scripps Research La Jolla, CA 

Additional Author(s)

Jennifer Fribourgh, UC Santa Cruz Santa Cruz, CA 
Alicia Michael, UC Santa Cruz Santa Cruz, CA 
Ashutosh Srivastava, Nagoya University Nagoya
Greg Hura, Molecular Biophysics and Integrated Bioimaging Division (MBIB), Lawrence Berkeley National Laborator Berkeley, CA 
Dina Schneidman-Duhovny, The Hebrew University of Jerusalem Jerusalem
Sarvind Tripathi, UC Santa Cruz Santa Cruz, CA 
Joseph Takahashi, UT Southwestern Dallas, TX 
Gabriel Lander, Scripps Research
Tsuyoshi Hirota, Nagoya University Nagoya
Florence Tama, Nagoya University Nagoya
Carrie Partch, UC Santa Cruz Santa Cruz, CA 

Cryo-EM Reveals Active Site Coordination Within a Multienzyme pre-rRNA Processing Complex

12:42 PM - 1:00 PM 
Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of the cell. Las1 is a recently discovered endoribonuclease that assembles into a multienzyme complex with the Grc3 polynucleotide kinase to orchestrate the targeted removal of a transcribed spacer (ITS2) from precursor ribosomal RNA (pre-rRNA). The essential Las1 endoribonuclease cleaves the ITS2 spacer at a defined site to initiate pre-rRNA processing. The Grc3 polynucleotide kinase subsequently phosphorylates the resulting 5'-hydroxyl RNA to signal for 5'- and 3'-exoribonucleases to degrade the ITS2. Disruption of mammalian Las1-Grc3 has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting its importance in human health; yet, its mechanism of action remains unclear. Here we report that the Las1 endoribonuclease assembles into a higher-order tetrameric complex with its binding partner the Grc3 polynucleotide kinase, which is essential for the activation of its nuclease and kinase functions. To understand how Las1-Grc3 achieves its strict nuclease specificity and coordinates its dual enzymes, we determined a series of high-resolution cryo-EM structures of Las1-Grc3 in multiple conformational states. Structural characterization of Las1-Grc3 reveals its molecular architecture harboring a composite nuclease active site flanked by two discrete RNA kinase sites. Coupled with functional studies, we identify molecular features crucial for RNA specificity and two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish how Las1-Grc3 couples its enzymatic functions to drive ribosome assembly. 

View Proposal 259


Monica Pillon Durham, NC 

Additional Author(s)

Allen Hsu, National Institutes of Environmental Health Sciences
Juno Krahn, National Institutes of Environmental Health Sciences
Jason Williams, National Institutes of Environmental Health Sciences
Kevin Goslen, National Institutes of Environmental Health Sciences
Mack Sobhany, National Institutes of Environmental Health Sciences
Mario Borgnia, National Institutes of Environmental Health Sciences
Robin Stanley, NIEHS/NIH

Coffee Break

1:00 PM - 1:20 PM 

Structural Basis for Strand Transfer Inhibitor Binding to HIV Intasomes

1:20 PM - 1:45 PM 
The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of antiretrovirals, integrase (IN) strand transfer inhibitors (INSTIs). Challenges associated with lentiviral intasome biochemistry have hindered high-resolution structural studies of how INSTIs bind to their native drug target. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of HIV intasomes bound to the latest generation INSTIs. These structures highlight how small changes in the IN active site can have significant implications for drug binding and design and provide mechanistic insights into why a leading INSTI retains efficacy against a broad spectrum of drug resistant variants. The data have implications for expanding effective treatments available for HIV-infected individuals.  

View Proposal 199


Dmitry Lyumkis, The Salk institute for Biological Studies San Diego, CA 

CryoDRGN: a tool for reconstructing highly heterogeneous structural ensembles from cryo-electron micrographs

1:45 PM - 2:10 PM 
Massive macromolecular machines such as the ribosome and spliceosome perform essential cellular functions by undergoing dramatic structural changes. Some such complexes exhibit seemingly continuous conformational changes, whereas others transition between disparate states in a highly cooperative manner such that conformational intermediates are sparsely populated. To understand the extent of complex heterogeneity, the degree of cooperativity in their conformational changes, and to estimate ensembles of such structures, we have developed a neural-network based single-particle analysis framework. This approach, named cryoDRGN, maps individual particles to a low-dimensional latent space, which we find arranges structurally related particles in close proximity. Direct inspection of such a structurally ordered latent space provides insights into the degree of structural heterogeneity in the dataset, provides estimates of particle abundance in each state, and relates the observed states to one another. Additionally, we demonstrate that three-dimensional structures can be sampled from this latent space, allowing users to visualize conformational trajectories sampled along the data manifold. In this talk, I detail our method and apply it to exemplar simulated and experimental datasets to illustrate its utility in analyzing both continuously and discretely heterogeneous complexes. We find that these neural networks accurately estimate the conformational landscape of simulated datasets, reveal significant discrete and continuous conformational heterogeneity in experimentally derived particle stacks, and accurately produce ensembles of medium-resolution density maps, which can be used to understand how the complex's structural changes drive its function. 

View Proposal 300


Joey Davis, MIT Cambridge, MA 

Additional Author(s)

Ellen Zhong, MIT Cambridge, MA 
Bonnie Berger, MIT Cambridge, MA 
Tristan Bepler, MIT Cambridge, MA 

Hyper-Molecules: High Dimensional Maps of Molecular Conformations

2:10 PM - 2:35 PM 
One of the open problems in cryo-EM is mapping complex heterogeneity, such as continuous heterogeneity. We begin our discussion with the question what does it mean to recover a heterogeneous structure, compared to a homogeneous structure or several distinct structures? We introduce "hyper-molecules," a mathematical formulation which captures the continuum of states and the relationships between them, and a Bayesian framework for recovering these "hyper-molecules." We present preliminary implementations and results, which demonstrates how the heterogeneous structures can be recovered from synthetic and real data, and discuss some of the practical challenges and solutions. We discuss next steps in this work on a scalable framework which would map complex heterogeneous structures, and would optionally allow researchers to explicitly encode prior knowledge, such as general physical properties, and existing knowledge about the specific structure, when such knowledge is available, in order to resolve complex structures which could otherwise require unrealistic amounts of data. 

View Proposal 238


Roy Lederman

Malaria parasite translocon structure and mechanism of effector export

2:35 PM - 3:00 PM 
The putative Plasmodium translocon of exported proteins (PTEX) is essential for transport of malarial effector proteins across a parasite-encasing vacuolar membrane into host erythrocytes, but the mechanism of this process remains unknown. Here we show that PTEX is a bona fide translocon by determining structures of the PTEX core complex at near-atomic resolution using cryo-electron microscopy. We isolated the endogenous PTEX core complex containing EXP2, PTEX150 and HSP101 from Plasmodium falciparum in the 'engaged' and 'resetting' states of endogenous cargo translocation using epitope tags inserted using the CRISPR–Cas9 system. In the structures, EXP2 and PTEX150 interdigitate to form a static, funnel-shaped pseudo-seven-fold-symmetric protein-conducting channel spanning the vacuolar membrane. The spiral-shaped AAA+ HSP101 hexamer is tethered above this funnel, and undergoes pronounced compaction that allows three of six tyrosine-bearing pore loops lining the HSP101 channel to dissociate from the cargo, resetting the translocon for the next threading cycle. Our work reveals the mechanism of P. falciparum effector export, and will inform structure-based design of drugs targeting this unique translocon. 

View Proposal 180


Chi-Min Ho, Columbia University Irving Medical Center New York, NY 

Additional Author(s)

Josh R. Beck, Iowa State University Ames, IA 
Mason Lai, UCLA Los Angeles, CA 
Yanxiang Cui, UCLA Los Angeles, CA 
Daniel E. Goldberg, Washington University in St. Louis St. Louis, MO 
Pascal F. Egea, UCLA Los Angeles, CA 
Hong Zhou, UCLA Los Angeles, CA