scholarly journals Integrative structural modelling of macromolecular complexes using Assembline

2021 ◽  
Author(s):  
Vasileios Rantos ◽  
Kai Karius ◽  
Jan Kosinski
Keyword(s):  
Electron Tomography ◽  
Conformational Space ◽  
Nuclear Pore ◽  
Computer Clusters ◽  
Macromolecular Complexes ◽  
X Ray Crystallography ◽  
Atomic Structures ◽  
Step Procedure ◽  
Combining Data ◽  
Configuration System

Integrative modelling enables structure determination of macromolecular complexes by combining data from multiple experimental sources such as X-ray crystallography, electron microscopy (EM), or crosslinking mass spectrometry (XL-MS). It is particularly useful for complexes not amenable to high-resolution EM-complexes that are flexible, heterogenous, or imaged in cells with cryo-electron tomography. We have recently developed an integrative modelling protocol that allowed us to model multi-megadalton complexes as large as the nuclear pore complex. Here, we describe the Assembline software package, which combines multiple programs and libraries with our own algorithms in a streamlined modelling pipeline. Assembline builds ensembles of models satisfying data from atomic structures or homology models, EM maps and other experimental data, and provides tools for their analysis. Comparing to other methods, Assembline enables efficient sampling of conformational space through a multi-step procedure, provides new modeling restraints, and includes a unique configuration system for setting up the modelling project. Our protocol achieves exhaustive sampling in less than 100 - 1,000 CPU-hours even for complexes in the megadalton range. For larger complexes, resources available in institutional or public computer clusters are needed and sufficient to run the protocol. We also provide step-by-step instructions for preparing the input, running the core modelling steps, and assessing modelling performance at any stage.

Cryoelectron tomography of isolated desmosomes

2008 ◽  
Vol 36 (2) ◽  
pp. 173-179 ◽  
Author(s):  
Gethin Rh. Owen ◽  
Devrim Acehan ◽  
K.D. Derr ◽  
William J. Rice ◽  
David L. Stokes
Keyword(s):  
Electron Tomography ◽  
Mouse Skin ◽  
Freeze Substitution ◽  
Newborn Mouse ◽  
Thin Sections ◽  
X Ray Crystallography ◽  
Atomic Structures ◽  
Mediate Cell ◽  
Physical Principles

Desmosomes are a complex assembly of protein molecules that form at the cell surface and mediate cell–cell adhesion. Much is known about the composition of desmosomes and there is an established consensus for the location of and interactions between constituent proteins within the assembly. Furthermore, X-ray crystallography has determined atomic structures of isolated domains from several constituent proteins. Nevertheless, there is a lack of understanding about the architecture of the intact assembly and the physical principles behind the adhesive strength of desmosomes therefore remain vague. We have used electron tomography to address this problem. In previous work, we investigated the in situ structure of desmosomes from newborn mouse skin preserved by freeze-substitution and imaged in resin-embedded thin sections. In our present work, we have isolated desmosomes from cow snout and imaged them in the frozen unstained state. Although not definitive, the resulting images provide support for the irregular groupings of cadherin molecules seen previously in mouse skin.

Hierarchy of π-Stacking Determines the Conformational Preference of Bis-Squaraines

2020 ◽  
Author(s):  
Abhishek Singh ◽  
Reman K. Singh ◽  
G Naresh Patwari
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Biological Molecules ◽  
Conformational Space ◽  
X Ray Crystallography ◽  
Simple Strategy ◽  
Induced Folding ◽  
Π Stacking ◽  
Varying Length ◽  
Dynamics Simulations

The rational design of conformationally controlled foldable modules can lead to a deeper insight into the conformational space of complex biological molecules where non-covalent interactions such as hydrogen bonding and π-stacking are known to play a pivotal role. Squaramides are known to have excellent hydrogen bonding capabilities and hence, are ideal molecules for designing foldable modules that can mimic the secondary structures of bio-molecules. The π-stacking induced folding of bis-squaraines tethered using aliphatic primary and secondary-diamine linkers of varying length is explored with a simple strategy of invoking small perturbations involving the length linkers and degree of substitution. Solution phase NMR investigations in combination with molecular dynamics simulations suggest that bis-squaraines predominantly exist as extended conformations. Structures elucidated by X-ray crystallography confirmed a variety of folded and extended secondary conformations including hairpin turns and 𝛽-sheets which are determined by the hierarchy of π-stacking relative to N–H···O hydrogen bonds.

Hierarchy of π-Stacking Determines the Conformational Preference of Bis-Squaraines

2020 ◽  
Author(s):  
Abhishek Singh ◽  
Reman K. Singh ◽  
G Naresh Patwari
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Biological Molecules ◽  
Conformational Space ◽  
X Ray Crystallography ◽  
Simple Strategy ◽  
Induced Folding ◽  
Π Stacking ◽  
Varying Length ◽  
Dynamics Simulations

The rational design of conformationally controlled foldable modules can lead to a deeper insight into the conformational space of complex biological molecules where non-covalent interactions such as hydrogen bonding and π-stacking are known to play a pivotal role. Squaramides are known to have excellent hydrogen bonding capabilities and hence, are ideal molecules for designing foldable modules that can mimic the secondary structures of bio-molecules. The π-stacking induced folding of bis-squaraines tethered using aliphatic primary and secondary-diamine linkers of varying length is explored with a simple strategy of invoking small perturbations involving the length linkers and degree of substitution. Solution phase NMR investigations in combination with molecular dynamics simulations suggest that bis-squaraines predominantly exist as extended conformations. Structures elucidated by X-ray crystallography confirmed a variety of folded and extended secondary conformations including hairpin turns and 𝛽-sheets which are determined by the hierarchy of π-stacking relative to N–H···O hydrogen bonds.

Cryo-EM Is a Powerful Tool, But Helical Applications Can Have Pitfalls

Soft Matter ◽  
2021 ◽  
Author(s):  
Edward Egelman ◽  
Fengbin Wang
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Macromolecular Complexes ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Main Technique

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct...

The Cryo-EM Effect: Structural Biology of Neurodegenerative Disease Proteostasis Factors

2021 ◽  
Author(s):  
Benjamin C Creekmore ◽  
Yi-Wei Chang ◽  
Edward B Lee
Keyword(s):  
Neurodegenerative Diseases ◽  
Protein Aggregation ◽  
Neurodegenerative Disease ◽  
Electron Tomography ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
X Ray Crystallography ◽  
New Information ◽  
A Cell ◽  
Regulate Protein

Abstract Neurodegenerative diseases are characterized by the accumulation of misfolded proteins. This protein aggregation suggests that abnormal proteostasis contributes to aging-related neurodegeneration. A better fundamental understanding of proteins that regulate proteostasis may provide insight into the pathophysiology of neurodegenerative disease and may perhaps reveal novel therapeutic opportunities. The 26S proteasome is the key effector of the ubiquitin-proteasome system responsible for degrading polyubiquitinated proteins. However, additional factors, such as valosin-containing protein (VCP/p97/Cdc48) and C9orf72, play a role in regulation and trafficking of substrates through the normal proteostasis systems of a cell. Nonhuman AAA+ ATPases, such as the disaggregase Hsp104, also provide insights into the biochemical processes that regulate protein aggregation. X-ray crystallography and cryo-electron microscopy (cryo-EM) structures not bound to substrate have provided meaningful information about the 26S proteasome, VCP, and Hsp104. However, recent cryo-EM structures bound to substrate have provided new information about the function and mechanism of these proteostasis factors. Cryo-EM and cryo-electron tomography data combined with biochemical data have also increased the understanding of C9orf72 and its role in maintaining proteostasis. These structural insights provide a foundation for understanding proteostasis mechanisms with near-atomic resolution upon which insights can be gleaned regarding the pathophysiology of neurodegenerative diseases.

scholarly journals Nuclear pores constrict upon energy depletion

2020 ◽  
Author(s):  
Christian E Zimmerli ◽  
Matteo Allegretti ◽  
Vasileios Rantos ◽  
Sara K Goetz ◽  
Agnieszka Obarska-Kosinska ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Electron Tomography ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Energy Status ◽  
Energy Depletion ◽  
Time Variations ◽  
Subtomogram Averaging ◽  
Pore Complexes

Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins that form an intricate cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter were reported, but the physiological circumstances and the molecular details remain unknown. Here we combined cryo-electron tomography and subtomogram averaging with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. While actively transporting NPCs adopt a dilated conformation, they strongly constrict upon cellular energy depletion. Fluorescence recovery after photo bleaching experiments show that NPC constriction is concomitant with reduced diffusion and active transport across the nuclear envelope. Our data point to a model where the energy status of cells is linked to the conformation of NPC architecture.

scholarly journals Combining dehydration, construct optimization and improved data collection to solve the crystal structure of a CRM1–RanGTP–SPN1–Nup214 quaternary nuclear export complex

2015 ◽  
Vol 71 (12) ◽  
pp. 1481-1487 ◽  
Author(s):  
Thomas Monecke ◽  
Achim Dickmanns ◽  
Manfred S. Weiss ◽  
Sarah A. Port ◽  
Ralph H. Kehlenbach ◽  
...  
Keyword(s):  
Structure Determination ◽  
Nuclear Export ◽  
Conformational Flexibility ◽  
Small Gtpase ◽  
Nuclear Pore ◽  
Macromolecular Complexes ◽  
Construct Optimization ◽  
Transport Receptors ◽  
Gtpase Ran

High conformational flexibility is an intrinsic and indispensable property of nuclear transport receptors, which makes crystallization and structure determination of macromolecular complexes containing exportins or importins particularly challenging. Here, the crystallization and structure determination of a quaternary nuclear export complex consisting of the exportin CRM1, the small GTPase Ran in its GTP-bound form, the export cargo SPN1 and an FG repeat-containing fragment of the nuclear pore complex component nucleoporin Nup214 fused to maltose-binding protein is reported. Optimization of constructs, seeding and the development of a sophisticated protocol including successive PEG-mediated crystal dehydration as well as additional post-mounting steps were essential to obtain well diffracting crystals.

scholarly journals Proteasomes tether to two distinct sites at the nuclear pore complex

2017 ◽  
Vol 114 (52) ◽  
pp. 13726-13731 ◽  
Author(s):  
Sahradha Albert ◽  
Miroslava Schaffer ◽  
Florian Beck ◽  
Shyamal Mosalaganti ◽  
Shoh Asano ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Electron Tomography ◽  
Nuclear Pore ◽  
Cellular Environment ◽  
Subtomogram Averaging ◽  
Substrate Processing ◽  
Cellular Components ◽  
Transport Of Macromolecules

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.

scholarly journals Multidrug Resistance in Mammals and Fungi—From MDR to PDR: A Rocky Road from Atomic Structures to Transport Mechanisms

2021 ◽  
Vol 22 (9) ◽  
pp. 4806
Author(s):  
Narakorn Khunweeraphong ◽  
Karl Kuchler
Keyword(s):  
Multidrug Resistance ◽  
High Resolution ◽  
Fungal Pathogens ◽  
Molecular Mechanisms ◽  
Transport Systems ◽  
Parasitic Infections ◽  
New Era ◽  
X Ray Crystallography ◽  
Atomic Structures ◽  
Catalytic Cycles

Multidrug resistance (MDR) can be a serious complication for the treatment of cancer as well as for microbial and parasitic infections. Dysregulated overexpression of several members of the ATP-binding cassette transporter families have been intimately linked to MDR phenomena. Three paradigm ABC transporter members, ABCB1 (P-gp), ABCC1 (MRP1) and ABCG2 (BCRP) appear to act as brothers in arms in promoting or causing MDR in a variety of therapeutic cancer settings. However, their molecular mechanisms of action, the basis for their broad and overlapping substrate selectivity, remains ill-posed. The rapidly increasing numbers of high-resolution atomic structures from X-ray crystallography or cryo-EM of mammalian ABC multidrug transporters initiated a new era towards a better understanding of structure–function relationships, and for the dynamics and mechanisms driving their transport cycles. In addition, the atomic structures offered new evolutionary perspectives in cases where transport systems have been structurally conserved from bacteria to humans, including the pleiotropic drug resistance (PDR) family in fungal pathogens for which high resolution structures are as yet unavailable. In this review, we will focus the discussion on comparative mechanisms of mammalian ABCG and fungal PDR transporters, owing to their close evolutionary relationships. In fact, the atomic structures of ABCG2 offer excellent models for a better understanding of fungal PDR transporters. Based on comparative structural models of ABCG transporters and fungal PDRs, we propose closely related or even conserved catalytic cycles, thus offering new therapeutic perspectives for preventing MDR in infectious disease settings.

scholarly journals The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome

2013 ◽  
Vol 24 (24) ◽  
pp. 3920-3938 ◽  
Author(s):  
Mario Niepel ◽  
Kelly R. Molloy ◽  
Rosemary Williams ◽  
Julia C. Farr ◽  
Anne C. Meinema ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Regulation Of Gene Expression ◽  
Nuclear Pore ◽  
Docking Site ◽  
Discrete Structure ◽  
Transport Channel ◽  
Macromolecular Complexes ◽  
Nuclear Stability

The basket of the nuclear pore complex (NPC) is generally depicted as a discrete structure of eight protein filaments that protrude into the nucleoplasm and converge in a ring distal to the NPC. We show that the yeast proteins Mlp1p and Mlp2p are necessary components of the nuclear basket and that they also embed the NPC within a dynamic protein network, whose extended interactome includes the spindle organizer, silencing factors, the proteasome, and key components of messenger ribonucleoproteins (mRNPs). Ultrastructural observations indicate that the basket reduces chromatin crowding around the central transporter of the NPC and might function as a docking site for mRNP during nuclear export. In addition, we show that the Mlps contribute to NPC positioning, nuclear stability, and nuclear envelope morphology. Our results suggest that the Mlps are multifunctional proteins linking the nuclear transport channel to multiple macromolecular complexes involved in the regulation of gene expression and chromatin maintenance.

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