Structural characterization of macromolecular assemblies at the atomic level

Lead Research Organisation: Birkbeck College
Department Name: Biological Sciences

Abstract

Developing cures for diseases requires an understanding of how they work. We know, for example, that bacteria must penetrate the membranes of cells to cause infection, but we do not know much about the underlying mechanisms. Which part of the membrane serves as the entry point? Why are some membranes vulnerable while others are not? By answering these kinds of questions, we can take the first steps towards designing drugs capable of blocking these processes. Structural biology aids this project by providing pictures of parts of cells. This is done through the use of experimental techniques such as cryogenic electron microscopy, a method whereby cell components are frozen and then bombarded with electrons, yielding an image of the sample. Though such techniques have led to great discoveries, there are significant limitations to what they can accomplish. The pictures they provide are usually only partial, leaving us with significant gaps in our knowledge. My research aims to overcome this gap. Working with the electron microscopy group at Birkbeck college, I develop computational methods to complement experimental techniques. The project focuses on three diseases-pneumonia, herpes, and Alzheimer‘s-and we use computer modelling to pull together information from a variety of experimental sources to construct clearer and more complete images of the crucial cellular components and processes at work in these diseases.

Technical Summary

One of the central challenges in biology is to gather structural information at the molecular level. Experimental techniques such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy (cryoEM) have so far provided a wealth of structural data on proteins, DNA, and RNA, as well as on their complexes. Despite these successes, many important biological questions remain unanswered, due in part to the limitations of these experimental methods. My research goal is to overcome these limitations and provide more accurate characterizations of macromolecular assemblies at the atomic level through the development a novel hybrid computational method that integrates information from experiments, physical theories, and bioinformatics.
There has been a good deal of important work done that combines low- and intermediate-resolution cryoEM density maps of whole assemblies with atomic-resolution structures of individual components to provide atomic models of the assemblies. My aim in this project is to take the next step forward, building and refining atomic models of assemblies that integrate cryoEM, crystallography, NMR, low-resolution experimental data (chemical cross-linking, site-directed spin labelling, etc.), and protein structure prediction, with the aid of methods from physical and statistical theories. This integrative model will not only provide a superior picture of assemblies, but it will also enable us to observe and simulate dynamic processes so that we can gain a better understanding of how proteins assemble, how their components interact, and what kind of structural rearrangements they undergo.
The specific methodological aims of the project are:
Aim 1: Develop a method of combining computational protein-protein docking, cryoEM density fitting, and other experimental restraints.
Aim 2: Develop a method for modelling new structural folds by direct analysis of subnanometer-resolution cryoEM maps.
Aim 3: Develop a real-space refinement method for modelling conformational changes in atomic structures of assembly components.
Aim 4: Implement and integrate an automated protocol for building atomic models of whole assemblies using cryoEM maps.
Working in collaboration with experimentalists in the School of Crystallography at Birkbeck College (Image Processing Group), the research will focus initially on protein assemblies involved in three biologically and medically important cellular processes, each of which presents significant methodological challenges:
(1) Bacterial Infection (S. pneumoniae toxin pneumolysin)
(2) Viral infection (The connector of Bacillus subtilis bacteriophage SPP1)
(3) Chaperonin-mediated protein folding (E. coli chaperonin folding machine GroEL-GroES).

Publications

10 25 50

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Serysheva II (2008) Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel. in Proceedings of the National Academy of Sciences of the United States of America

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Harvey RJ (2008) The genetics of hyperekplexia: more than startle! in Trends in genetics : TIG

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Connell S (2008) A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation in Nature Structural & Molecular Biology

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Connell SR (2008) A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation. in Nature structural & molecular biology

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Chandramouli P (2008) Structure of the mammalian 80S ribosome at 8.7 A resolution. in Structure (London, England : 1993)

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Topf M (2008) Protein structure fitting and refinement guided by cryo-EM density. in Structure (London, England : 1993)

 
Title Lifting the lid on pilus assembly - journal cover 
Description Cover of eLife 
Type Of Art Image 
Year Produced 2014 
Impact Highlighting our work on pilus assembly (title: "Lifting the lid on pilus assembly") published in Farabella et al 2014 (PMID: 25271373) 
 
Description A direct electron detector for electron cryo-microscopy.
Amount £387,500 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2013 
End 07/2016
 
Description An integrative approach to deciphering the entry process in Herpesviruses
Amount £273,800 (GBP)
Funding ID MR/M019292/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 11/2018
 
Description BC Researcher Exchange Programme (RXP) award
Amount £3,202 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2007 
End 08/2008
 
Description Birkbeck Faculty of Science, Research Grant
Amount £4,980 (GBP)
Organisation Birkbeck, University of London 
Sector Academic/University
Country United Kingdom
Start 01/2007 
End 12/2007
 
Description Bloomsbury Colleges, Phd Studentship
Amount £69,000 (GBP)
Organisation Birkbeck, University of London 
Sector Academic/University
Country United Kingdom
Start 10/2009 
End 09/2012
 
Description Capacity Building Research Studentship
Amount £89,597 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2009 
End 12/2012
 
Description Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM): Supporting the software infrastructure for cryoEM techniques.
Amount £1,177,000 (GBP)
Funding ID MR/N009614/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 02/2016 
End 01/2021
 
Description HFSP - Young Investigator Grant
Amount $300,000 (USD)
Funding ID RGY0079/2009-C 
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 11/2009 
End 06/2013
 
Description Polysomes in Cells
Amount £250,000 (GBP)
Funding ID RPG-2012-519 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description The in situ architecture of the entire Herpes Simplex Virus Type 1
Amount £100,000 (GBP)
Funding ID MRC Centenary Award 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2013
 
Description Timestamping Integrative Approach to Understand Secondary Envelopment of Human Cytomegalovirus
Amount £673,700 (GBP)
Funding ID 209250/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2018 
End 03/2023
 
Description Tools for generating and assessing pseudo-atomic models from 3D electron microscopy maps of macromolecular assemblies
Amount £148,757 (GBP)
Funding ID BB/K01692X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2013 
End 02/2014
 
Description Validation Tools for Cryo-EM
Amount £1,187,077 (GBP)
Funding ID 208398/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 02/2021
 
Title Flex-EM 
Description a method for fitting and refining atomic models in cryo-electron microscopy maps. 
Type Of Material Improvements to research infrastructure 
Year Produced 2008 
Provided To Others? Yes  
Impact The method (which is freely available via the widely-used modelling program MODELLER) helps in improving the analysis of low-resolution cryo-electron microscopy maps. It has been used by us and other researchers to generate pseudo-atomic models of a number of macromolecular assemblies studied by cryo-electron microscopy, such as various ribosomes (eukaryotic and e-coli), ribosomal factors (EF4, eIF6), chaperonins (GroEL and TRIC/CCT), membrane channels (RyR1), the exosome, the apopotosome, microtubules, metabolic enzymes (Pyruvate carboxylase) and actin. All these studies have been published . The method has been cited 135 times since 2008. The method is currently being implemented in the CCP-EM (Collaborative Computational Project for Electron cryo-Microscopy) package to reach the wider community of users in the UK and worldwide. 
URL http://topf-group.ismb.lon.ac.uk/flex-em/
 
Title Flex-EM for high resolution EM 
Description We improved the Flex-EM method to make it applicable for refinement of high-resolution 3D electron microscopy maps 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact We have used the method to refine a number of new structure, in particular GroEL and microtubule-bound complexes. 
URL http://topf-group.ismb.lon.ac.uk/flex-em/
 
Title RIBFIND 
Description A method for detecting flexibility in protein structures via the clustering of secondary structural elements (SSEs) into rigid bodies. Helps improve flexible fitting into cryoEM maps. 
Type Of Material Improvements to research infrastructure 
Year Produced 2012 
Provided To Others? Yes  
Impact Refining structures in simulated and experimental maps at the 5-15 A resolution range using rigid bodies identified by RIBFIND shows a significant improvement over using individual SSEs as rigid bodies. Furthermore, for some of our test cases we show that at the sub-nanometer resolution range the fits can be further improved by applying a two-stage refinement protocol (using RIBFIND-based refinement followed by an SSE-based refinement). Pandurangan & Topf 2012 (PMID: 22079400) Pandurangan & Topf 2012 (PMID: 22796953) Pandurangan et al. 2014 (PMID: 224333899) 
URL http://ribfind.ismb.lon.ac.uk/
 
Title TEMPy 
Description TEMPy is an object-oriented Python library designed to help the user in the manipulation and analysis of macromolecular assemblies, especially in the context of 3D electron microscopy density maps. It is designed with a set of functionalities that assess the goodness-of-fit between a given atomic model and a density map or between two maps using a variety of different scoring functions. It can also generate various ensembles of alternative fits, which has been shown to access one of the best-fitting models. 
Type Of Material Improvements to research infrastructure 
Year Produced 2015 
Provided To Others? Yes  
Impact Vasishtan and Topf 2011 (PMID: 21296161) Seitsonen et al., 2012 (PMID: 22514349) Pandurangan et al. 2014 (PMID: 24333899) Atherton et al 2014 (PMID: 25209998) Farabella et al 2015 (PMID: 26306092) Lukoyanova et al 2015 (PMID: 25654333) 
URL http://tempy.ismb.lon.ac.uk/
 
Title Validation toold for models in cryoEM maps - TEMPy 
Description We developed new scores for validation of atomic models in cryoEM maps, we also added features for scoring models based on crosslink data obtained from XL-MS 
Type Of Material Improvements to research infrastructure 
Year Produced 2015 
Provided To Others? Yes  
Impact The methods has been used by us an others in the EM community, also in via CCP-EM to validated models in cryoEM maps. Some of the publications describing the use of the software to validate different structures can be found here: http://tempy.ismb.lon.ac.uk/ 
URL http://tempy.ismb.lon.ac.uk/
 
Description A hybrid approach to revealing interaction networks and intermediate structures of herpes viruses 
Organisation London School of Hygiene and Tropical Medicine (LSHTM)
Country United Kingdom 
Sector Academic/University 
PI Contribution Our key goal is to study the interactions and structures of viron subcomplexes during herpesviruses infection cycle. Together with the Alber Lab in USC we have developed an efficient mathematical programming algorithm that simultaneously fits all component structures into a cryoEM density map of a complex at low resolutions (e.g. from tomography - data provided by Prof. Grunewald). The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. Our method generates an assembly configuration in a few seconds, allowing the generation of an ensemble of candidate solutions that can be assessed by an independent scoring function. The method was recently published in Bioinformatics (Zhang et al. 2010) and was recommended and highlighted by the 'Faculty of 1000'. The work was also be presented at the ISMB conference. In addition, we further developed methods for modelling, fitting and refining models in the context of cryoEM maps. First, we developed a web-server for automated homology modelling of assembly components by alternative alignments and fitting into cryoEM maps of their assemblies. The web-server (based on which a paper was published this year in Bioinformatics - Rawi et al. 2010) provides an interactive approach to improving the selection of models based on the quality of their fit into the EM map and enables a large scale modelling (http://choyce.ismb.lon.ac.uk/). Second, we have developed a number of new scoring functions for density fitting, two of which are as good if not better than the currently used score in density fitting (cross correlation). Finally, to improve our flexible fitting program Flex-EM (http://topf-group.ismb.lon.ac.uk/flex-em/), we have developed a method for identifying rigid bodies in proteins structure (RIBFIND). Dr. Grunewald and myself have a joint PhD student who works on modelling of glycoproteins sub-complexes from sub-tomogram averaged maps. She has also developed a protein interaction database and network for HSV-1 (hvint: http://topf-group.ismb.lon.ac.uk/hvint/ ).
Collaborator Contribution Dr. Grunewald has pioneered the application of cryo-electron tomography to isolated pleomorphic viruses revealing their three-dimensional supramolecular organization. His work on the virions of Herpes simplex virus has provided us with new challenges for fitting atomic structures into low-resolution EM maps of large virus assemblies during infection. Prof. Alber's lab has developed a method an efficient mathematical programming algorithm that simultaneously fits all component structures into an assembly electron microscopy density map. The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. Over the past year, the IC group has extended the proteomic techniques employed in the lab and tailored their use towards our goal of elucidating HSV1-host protein interactions. This data is important to be provided as constraint to our modelling of HSV1 proteins during infection using tomograms.
Impact This collaboration is multidisciplinary, involving computational biology, structural biology and cell biology. Zhang et al. (PMID: 20529915) Pandurangan and Topf (PMID: 22079400) Pandurangan and Topf (PMID: 22796953) Maurer et al, and Grunewald (PMID: 23850455 ) Pandurangan et al, and Topf (PMID: 26655474) Farabella et al, and Topf (PMID: 26306092) Zeev-Ben-Mordehai et al (PMID: 27035968) Ashford et al (PMID: 27384951) Joseph et al. (PMID:26988127) Joseph et al. (PMID: 28735107) Joseph et al. (PMID: 28552721)
Start Year 2009
 
Description A hybrid approach to revealing interaction networks and intermediate structures of herpes viruses 
Organisation Osaka University
Department Department of Biological Sciences
Country Japan 
Sector Academic/University 
PI Contribution Our key goal is to study the interactions and structures of viron subcomplexes during herpesviruses infection cycle. Together with the Alber Lab in USC we have developed an efficient mathematical programming algorithm that simultaneously fits all component structures into a cryoEM density map of a complex at low resolutions (e.g. from tomography - data provided by Prof. Grunewald). The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. Our method generates an assembly configuration in a few seconds, allowing the generation of an ensemble of candidate solutions that can be assessed by an independent scoring function. The method was recently published in Bioinformatics (Zhang et al. 2010) and was recommended and highlighted by the 'Faculty of 1000'. The work was also be presented at the ISMB conference. In addition, we further developed methods for modelling, fitting and refining models in the context of cryoEM maps. First, we developed a web-server for automated homology modelling of assembly components by alternative alignments and fitting into cryoEM maps of their assemblies. The web-server (based on which a paper was published this year in Bioinformatics - Rawi et al. 2010) provides an interactive approach to improving the selection of models based on the quality of their fit into the EM map and enables a large scale modelling (http://choyce.ismb.lon.ac.uk/). Second, we have developed a number of new scoring functions for density fitting, two of which are as good if not better than the currently used score in density fitting (cross correlation). Finally, to improve our flexible fitting program Flex-EM (http://topf-group.ismb.lon.ac.uk/flex-em/), we have developed a method for identifying rigid bodies in proteins structure (RIBFIND). Dr. Grunewald and myself have a joint PhD student who works on modelling of glycoproteins sub-complexes from sub-tomogram averaged maps. She has also developed a protein interaction database and network for HSV-1 (hvint: http://topf-group.ismb.lon.ac.uk/hvint/ ).
Collaborator Contribution Dr. Grunewald has pioneered the application of cryo-electron tomography to isolated pleomorphic viruses revealing their three-dimensional supramolecular organization. His work on the virions of Herpes simplex virus has provided us with new challenges for fitting atomic structures into low-resolution EM maps of large virus assemblies during infection. Prof. Alber's lab has developed a method an efficient mathematical programming algorithm that simultaneously fits all component structures into an assembly electron microscopy density map. The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. Over the past year, the IC group has extended the proteomic techniques employed in the lab and tailored their use towards our goal of elucidating HSV1-host protein interactions. This data is important to be provided as constraint to our modelling of HSV1 proteins during infection using tomograms.
Impact This collaboration is multidisciplinary, involving computational biology, structural biology and cell biology. Zhang et al. (PMID: 20529915) Pandurangan and Topf (PMID: 22079400) Pandurangan and Topf (PMID: 22796953) Maurer et al, and Grunewald (PMID: 23850455 ) Pandurangan et al, and Topf (PMID: 26655474) Farabella et al, and Topf (PMID: 26306092) Zeev-Ben-Mordehai et al (PMID: 27035968) Ashford et al (PMID: 27384951) Joseph et al. (PMID:26988127) Joseph et al. (PMID: 28735107) Joseph et al. (PMID: 28552721)
Start Year 2009
 
Description A hybrid approach to revealing interaction networks and intermediate structures of herpes viruses 
Organisation Princeton University
Country United States 
Sector Academic/University 
PI Contribution Our key goal is to study the interactions and structures of viron subcomplexes during herpesviruses infection cycle. Together with the Alber Lab in USC we have developed an efficient mathematical programming algorithm that simultaneously fits all component structures into a cryoEM density map of a complex at low resolutions (e.g. from tomography - data provided by Prof. Grunewald). The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. Our method generates an assembly configuration in a few seconds, allowing the generation of an ensemble of candidate solutions that can be assessed by an independent scoring function. The method was recently published in Bioinformatics (Zhang et al. 2010) and was recommended and highlighted by the 'Faculty of 1000'. The work was also be presented at the ISMB conference. In addition, we further developed methods for modelling, fitting and refining models in the context of cryoEM maps. First, we developed a web-server for automated homology modelling of assembly components by alternative alignments and fitting into cryoEM maps of their assemblies. The web-server (based on which a paper was published this year in Bioinformatics - Rawi et al. 2010) provides an interactive approach to improving the selection of models based on the quality of their fit into the EM map and enables a large scale modelling (http://choyce.ismb.lon.ac.uk/). Second, we have developed a number of new scoring functions for density fitting, two of which are as good if not better than the currently used score in density fitting (cross correlation). Finally, to improve our flexible fitting program Flex-EM (http://topf-group.ismb.lon.ac.uk/flex-em/), we have developed a method for identifying rigid bodies in proteins structure (RIBFIND). Dr. Grunewald and myself have a joint PhD student who works on modelling of glycoproteins sub-complexes from sub-tomogram averaged maps. She has also developed a protein interaction database and network for HSV-1 (hvint: http://topf-group.ismb.lon.ac.uk/hvint/ ).
Collaborator Contribution Dr. Grunewald has pioneered the application of cryo-electron tomography to isolated pleomorphic viruses revealing their three-dimensional supramolecular organization. His work on the virions of Herpes simplex virus has provided us with new challenges for fitting atomic structures into low-resolution EM maps of large virus assemblies during infection. Prof. Alber's lab has developed a method an efficient mathematical programming algorithm that simultaneously fits all component structures into an assembly electron microscopy density map. The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. Over the past year, the IC group has extended the proteomic techniques employed in the lab and tailored their use towards our goal of elucidating HSV1-host protein interactions. This data is important to be provided as constraint to our modelling of HSV1 proteins during infection using tomograms.
Impact This collaboration is multidisciplinary, involving computational biology, structural biology and cell biology. Zhang et al. (PMID: 20529915) Pandurangan and Topf (PMID: 22079400) Pandurangan and Topf (PMID: 22796953) Maurer et al, and Grunewald (PMID: 23850455 ) Pandurangan et al, and Topf (PMID: 26655474) Farabella et al, and Topf (PMID: 26306092) Zeev-Ben-Mordehai et al (PMID: 27035968) Ashford et al (PMID: 27384951) Joseph et al. (PMID:26988127) Joseph et al. (PMID: 28735107) Joseph et al. (PMID: 28552721)
Start Year 2009
 
Description A hybrid approach to revealing interaction networks and intermediate structures of herpes viruses 
Organisation University of Oxford
Department Division of Structural Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Our key goal is to study the interactions and structures of viron subcomplexes during herpesviruses infection cycle. Together with the Alber Lab in USC we have developed an efficient mathematical programming algorithm that simultaneously fits all component structures into a cryoEM density map of a complex at low resolutions (e.g. from tomography - data provided by Prof. Grunewald). The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. Our method generates an assembly configuration in a few seconds, allowing the generation of an ensemble of candidate solutions that can be assessed by an independent scoring function. The method was recently published in Bioinformatics (Zhang et al. 2010) and was recommended and highlighted by the 'Faculty of 1000'. The work was also be presented at the ISMB conference. In addition, we further developed methods for modelling, fitting and refining models in the context of cryoEM maps. First, we developed a web-server for automated homology modelling of assembly components by alternative alignments and fitting into cryoEM maps of their assemblies. The web-server (based on which a paper was published this year in Bioinformatics - Rawi et al. 2010) provides an interactive approach to improving the selection of models based on the quality of their fit into the EM map and enables a large scale modelling (http://choyce.ismb.lon.ac.uk/). Second, we have developed a number of new scoring functions for density fitting, two of which are as good if not better than the currently used score in density fitting (cross correlation). Finally, to improve our flexible fitting program Flex-EM (http://topf-group.ismb.lon.ac.uk/flex-em/), we have developed a method for identifying rigid bodies in proteins structure (RIBFIND). Dr. Grunewald and myself have a joint PhD student who works on modelling of glycoproteins sub-complexes from sub-tomogram averaged maps. She has also developed a protein interaction database and network for HSV-1 (hvint: http://topf-group.ismb.lon.ac.uk/hvint/ ).
Collaborator Contribution Dr. Grunewald has pioneered the application of cryo-electron tomography to isolated pleomorphic viruses revealing their three-dimensional supramolecular organization. His work on the virions of Herpes simplex virus has provided us with new challenges for fitting atomic structures into low-resolution EM maps of large virus assemblies during infection. Prof. Alber's lab has developed a method an efficient mathematical programming algorithm that simultaneously fits all component structures into an assembly electron microscopy density map. The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. Over the past year, the IC group has extended the proteomic techniques employed in the lab and tailored their use towards our goal of elucidating HSV1-host protein interactions. This data is important to be provided as constraint to our modelling of HSV1 proteins during infection using tomograms.
Impact This collaboration is multidisciplinary, involving computational biology, structural biology and cell biology. Zhang et al. (PMID: 20529915) Pandurangan and Topf (PMID: 22079400) Pandurangan and Topf (PMID: 22796953) Maurer et al, and Grunewald (PMID: 23850455 ) Pandurangan et al, and Topf (PMID: 26655474) Farabella et al, and Topf (PMID: 26306092) Zeev-Ben-Mordehai et al (PMID: 27035968) Ashford et al (PMID: 27384951) Joseph et al. (PMID:26988127) Joseph et al. (PMID: 28735107) Joseph et al. (PMID: 28552721)
Start Year 2009
 
Description ATP-triggered molecular mechanics of the chaperonin GroEL 
Organisation Birkbeck, University of London
Department Department of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the co-chaperonin GroES, followed by further large movements that eject the substrate polypeptide from a ring of hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. In collaboration with Prof. Helen Saibil, Birkbeck College, we used data from cryo-electron microscopy (EM) to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. For that, we used our flexible fitting program Flex-EM. Based on our refinement we found that the initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other, but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the "power stroke" that ejects substrate into the folding chamber.
Collaborator Contribution The group of Helen Saibil performed the experiments for this work as well as image processing. We currently have a joint PhD student looking at various GroEL maps at multiple resolution to compare the different conformations.
Impact Clare DK et al. 2012 (PMID: 22445172); Joseph et al, 2017 (PMID: 28552721)
Start Year 2010
 
Description Collaboration with CCP-EM 
Organisation Daresbury Laboratory
Country United Kingdom 
Sector Private 
PI Contribution We collaborate on the CCP-EM (Collaborative Computational Project for Electron cryo-Microscopy) project. This project is supported by MRC. We implement some of the software developed in my group via the CCP-EM platform. The idea is to support the users of software for cryo-EM through dissemination of information on available software, and directed training.
Collaborator Contribution Our collaborators (under the supervision of Dr. Martyn Winn) are in the process of making our Flex-EM/RIBFIND software as well as TEMPy available via CCP-EM.
Impact We had a number of productive meetings and workshops. We are working on a number of software packages developed in my group to be implemented in CCP-EM (Flex-EM, RIBFIND, TEMPy). publications: Wood et al. (2015) PMID: 25615866 Joseph et al (2016) PMID: 26988127 Joseph et al (2017) PMID: 28552721 We received a joint MRC Partnership grant together with Martyn Winn (PI) and other 9 Co-Is across the UK. (MR/N009614/1)
Start Year 2012
 
Description Comprehensive molecular structure of the eukaryotic ribosome. 
Organisation Columbia University
Country United States 
Sector Academic/University 
PI Contribution By combining cryo-electron microscopy with RNA and protein homology modeling, we obtained an atomic model of the yeast 80S ribosome with all ribosomal RNA expansion segments and all ribosomal proteins for which a structural homolog can be identified. Mutation or deletion of 80S ribosomal proteins can abrogate maturation of the ribosome, leading to several human diseases. Our modeled enabled researcher in Frank's lab to localize one such protein unique to eukaryotes, rpS19e, whose mutations are associated with Diamond-Blackfan anemia in humans.
Collaborator Contribution Prof. Joachim Frank has been studying the structure of the ribosome for many years using cryo-electron microscopy. Recently, he obtained density maps of the complete yeast 80S ribosome at 8.9 Å. The data provided the basis for the modeling of a large part of eukaryotic ribosome.
Impact Taylor et al. 2009 (PMID: 20004163). The paper was reviewed in: Dinman and Kinzy, Expanding the Ribosomal Universe, Structure (2009), doi:10.1016/j.str.2009.11.003
Start Year 2006
 
Description EF4-mediated back-translocation on the ribosome. 
Organisation Charité - University of Medicine Berlin
Department Institute of Medical Physics and Biophysics
Country Germany 
Sector Academic/University 
PI Contribution Using our flexible fitting method, we built a model of ribosome-bound EF4 based on the cryo-EM map provided by Prof. Spahn and an EF4 X-ray structure. The model established EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which deviates markedly from the position of a canonical A-tRNA. Our results provided insight into the underlying structural principles governing back-translocation.
Collaborator Contribution Prof. Christian Spahn studies a newly discovered translational GTPase in eubacteria called EF4 (LepA). EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-electron microscopy performed. The corresponding density map provided a basis for further analysis by flexible fitting of EF4, tRNA, ribosomal proteins, and RNA.
Impact - Connell*, Topf et al.* PMID: 19172743 *equal contribution. - The paper was reported in the editor's choice in Science 321:1272, 2008.
Start Year 2007
 
Description Fitting and refining atomic models in cryo-electron microscopy maps. 
Organisation Baylor College of Medicine
Country United States 
Sector Hospitals 
PI Contribution We have developed methods for fitting atomic-resolution structures and models into cryo-electron microscopy maps. Our methods, in particular for flexible fitting (Flex-EM) were applied to some of the assemblies studied in Prof. Chiu's lab (describe above).
Collaborator Contribution Prof. Wah Chiu has provided us with cryo-electron microscopy data on a number of macromolecular assemblies, including: actin, the calcium release channel (ryr1), GroEL, and the eukaryotic chaperonin TRiC/CCT. The data helped us in developing our methods for fitting atomic structures into cryo-EM maps.
Impact Topf et al 2006 (PMID: 16490207) Topf et al 2008 (PMID: 18275820) Serysheva et al 2008 (PMID: 18621707) Booth el al 2008 (PMID: 18536725) Cong et al 2008 (PMID: 18022194)
 
Description Modelling microtubule with microtubule binding proteins 
Organisation Birkbeck, University of London
Department Department of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been generating atomic models of microtubule complexes using 3D cryoEM density maps.
Collaborator Contribution Professor Carolyn Moores studies microtubule organisation and dynamics using cryo electron microscopy methods.
Impact Atherton et al 2014 (PMID: 25209998) Atherton et al 2017 (PMID: 28826477) Atherton et al 2017 (PMID: 28991265) Locke et al 2018 (PMID: 29078367)
Start Year 2014
 
Description Molecular insights into disease mechanisms in glycinergic disorders. 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution We modeled GlyT2 based on recently published crystal structures of related proteins and predicted the consequences of the numerous mutations in this protein. Our analysis revealed insights into mechanisms of hyperekplexia that can be correlated with existing functional data, or used to devise additional functional tests. A joint PhD student has dentifued new human GlyR receptor mutations. A homology model has been built and mutations are analysed both on the GlyT2 and GlyR models.
Collaborator Contribution Prof. Robert Harvey studies defects in glycinergic neurotransmission, which result in a neurological disorder known as hyperekplexia or startle disease. Hyperekplexia affects newborn children and although rare, it can have serious consequences, including brain damage and/or sudden infant death. Hyperekplexia is typically caused by missense and nonsense mutations in GLRA1, which encodes the glycine receptor (GlyR) a1 subunit. A team led by Prof. Harvey recently discovered a second major hyperekplexia gene (SLC6A5; Rees et al., 2006) encoding the presynaptic Na+/Cl- dependent glycine transporter GlyT2. The data was the basis for structural modeling hyperekplexia-associated mutations.
Impact Harvey et al, 2008 (PMID: 18707791) James et al., 2011 (PMID: 22114948) Carta et el., 2012 (PMID: 22700964) Giménez et al., 2012 (PMID: 22753417) Ganser et al, 2013 (PMID: 24029548) James et al, 2013 (PMID: 23238346) Pilorge et al. 2016 (PMID: 26370147) Schaefer et al 2017 (PMID: 28724750) BBC News, Health, 21 July 2012: "She went blue and shook from head to toe." * *our contribution was the modelling the Y705C mutation associated with startle disease, which presents altered H(+) and Zn(2+) dependence of glycine transport, onto our homology model of GlyT2. The model (shown in the BBC article) assisted in proposing that the mutation may affect the function of glycinergic neurotransmission in vivo.
Start Year 2008
 
Description PapC modelling 
Organisation Birkbeck, University of London
Department Department of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili proteins in uropathogenic E. coli, allowing them to infect the human urinary tract and cause disease. To pass through the outer membrane of the E. coli cell, a P pili protein must travel through the translocation domain of PapC. The translocation domain is a large ß-barrel occluded by a middle domain (plug domain), which is only marginally stable, to allow the opening of the barrel. Previous studies suggested that this gating mechanism is controlled by two elements of the translocation domain - a ß-hairpin and an a-helix, but it is not clear how. To investigate the role of these elements in allosteric signal communication, we developed a hybrid computational method combining evolutionary and molecular dynamics studies. Additionally, we used evolutionary analysis, relying on both amino acid conservation and coupling (co-evolution) to highlight particularly important interactions. The data were combined in a large hybrid residue interaction network. Using graph theory approaches, we showed that certain regions of the ß-barrel, located mostly on one side of the barrel, are likely involved in moving the plug domain. This unique computational approach allowed us to detect key amino acids in these distinct regions (residue 'communities') that likely control the position of the plug in the pore, thereby regulating PapC gating and usher's activation. Our results suggest that residues around ß12-ß14 that are beneath the a-helix act as a regulator of ß 12- ß 13 loop (the 'latch') that controls the opening and closing of the ß-barrel. The study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.
Collaborator Contribution The group of Prof. Gabriel Waksman (ISMB, Birkbeck/UCL) solved the structure of the usher. Our predictions were tested by experiments, in collaboration with the Waksman group and two groups in the US - Delcour group at the University of Houston and Thanassi Group at Stony Brook University. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four communities. Our collaborators in Groningen, Prof. Siewert-Jan Marrink, have contributed in training us to use their software for coarse-grained MD simulations.
Impact A multi-disciplinary collaboration. Farabella et al. eLife (PMID: 25271373) The paper was highlighted as a eLife Insight story: http://elifesciences.org/content/3/e04997 The paper was on the cover of eLife.
Start Year 2009
 
Description PapC modelling 
Organisation Stony Brook University
Country United States 
Sector Academic/University 
PI Contribution PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili proteins in uropathogenic E. coli, allowing them to infect the human urinary tract and cause disease. To pass through the outer membrane of the E. coli cell, a P pili protein must travel through the translocation domain of PapC. The translocation domain is a large ß-barrel occluded by a middle domain (plug domain), which is only marginally stable, to allow the opening of the barrel. Previous studies suggested that this gating mechanism is controlled by two elements of the translocation domain - a ß-hairpin and an a-helix, but it is not clear how. To investigate the role of these elements in allosteric signal communication, we developed a hybrid computational method combining evolutionary and molecular dynamics studies. Additionally, we used evolutionary analysis, relying on both amino acid conservation and coupling (co-evolution) to highlight particularly important interactions. The data were combined in a large hybrid residue interaction network. Using graph theory approaches, we showed that certain regions of the ß-barrel, located mostly on one side of the barrel, are likely involved in moving the plug domain. This unique computational approach allowed us to detect key amino acids in these distinct regions (residue 'communities') that likely control the position of the plug in the pore, thereby regulating PapC gating and usher's activation. Our results suggest that residues around ß12-ß14 that are beneath the a-helix act as a regulator of ß 12- ß 13 loop (the 'latch') that controls the opening and closing of the ß-barrel. The study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.
Collaborator Contribution The group of Prof. Gabriel Waksman (ISMB, Birkbeck/UCL) solved the structure of the usher. Our predictions were tested by experiments, in collaboration with the Waksman group and two groups in the US - Delcour group at the University of Houston and Thanassi Group at Stony Brook University. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four communities. Our collaborators in Groningen, Prof. Siewert-Jan Marrink, have contributed in training us to use their software for coarse-grained MD simulations.
Impact A multi-disciplinary collaboration. Farabella et al. eLife (PMID: 25271373) The paper was highlighted as a eLife Insight story: http://elifesciences.org/content/3/e04997 The paper was on the cover of eLife.
Start Year 2009
 
Description PapC modelling 
Organisation University College London
Department Structural Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili proteins in uropathogenic E. coli, allowing them to infect the human urinary tract and cause disease. To pass through the outer membrane of the E. coli cell, a P pili protein must travel through the translocation domain of PapC. The translocation domain is a large ß-barrel occluded by a middle domain (plug domain), which is only marginally stable, to allow the opening of the barrel. Previous studies suggested that this gating mechanism is controlled by two elements of the translocation domain - a ß-hairpin and an a-helix, but it is not clear how. To investigate the role of these elements in allosteric signal communication, we developed a hybrid computational method combining evolutionary and molecular dynamics studies. Additionally, we used evolutionary analysis, relying on both amino acid conservation and coupling (co-evolution) to highlight particularly important interactions. The data were combined in a large hybrid residue interaction network. Using graph theory approaches, we showed that certain regions of the ß-barrel, located mostly on one side of the barrel, are likely involved in moving the plug domain. This unique computational approach allowed us to detect key amino acids in these distinct regions (residue 'communities') that likely control the position of the plug in the pore, thereby regulating PapC gating and usher's activation. Our results suggest that residues around ß12-ß14 that are beneath the a-helix act as a regulator of ß 12- ß 13 loop (the 'latch') that controls the opening and closing of the ß-barrel. The study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.
Collaborator Contribution The group of Prof. Gabriel Waksman (ISMB, Birkbeck/UCL) solved the structure of the usher. Our predictions were tested by experiments, in collaboration with the Waksman group and two groups in the US - Delcour group at the University of Houston and Thanassi Group at Stony Brook University. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four communities. Our collaborators in Groningen, Prof. Siewert-Jan Marrink, have contributed in training us to use their software for coarse-grained MD simulations.
Impact A multi-disciplinary collaboration. Farabella et al. eLife (PMID: 25271373) The paper was highlighted as a eLife Insight story: http://elifesciences.org/content/3/e04997 The paper was on the cover of eLife.
Start Year 2009
 
Description PapC modelling 
Organisation University of Groningen
Department Groningen Biomolecular Sciences and Biotechnology Institute (GBB)
Country Netherlands 
Sector Academic/University 
PI Contribution PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili proteins in uropathogenic E. coli, allowing them to infect the human urinary tract and cause disease. To pass through the outer membrane of the E. coli cell, a P pili protein must travel through the translocation domain of PapC. The translocation domain is a large ß-barrel occluded by a middle domain (plug domain), which is only marginally stable, to allow the opening of the barrel. Previous studies suggested that this gating mechanism is controlled by two elements of the translocation domain - a ß-hairpin and an a-helix, but it is not clear how. To investigate the role of these elements in allosteric signal communication, we developed a hybrid computational method combining evolutionary and molecular dynamics studies. Additionally, we used evolutionary analysis, relying on both amino acid conservation and coupling (co-evolution) to highlight particularly important interactions. The data were combined in a large hybrid residue interaction network. Using graph theory approaches, we showed that certain regions of the ß-barrel, located mostly on one side of the barrel, are likely involved in moving the plug domain. This unique computational approach allowed us to detect key amino acids in these distinct regions (residue 'communities') that likely control the position of the plug in the pore, thereby regulating PapC gating and usher's activation. Our results suggest that residues around ß12-ß14 that are beneath the a-helix act as a regulator of ß 12- ß 13 loop (the 'latch') that controls the opening and closing of the ß-barrel. The study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.
Collaborator Contribution The group of Prof. Gabriel Waksman (ISMB, Birkbeck/UCL) solved the structure of the usher. Our predictions were tested by experiments, in collaboration with the Waksman group and two groups in the US - Delcour group at the University of Houston and Thanassi Group at Stony Brook University. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four communities. Our collaborators in Groningen, Prof. Siewert-Jan Marrink, have contributed in training us to use their software for coarse-grained MD simulations.
Impact A multi-disciplinary collaboration. Farabella et al. eLife (PMID: 25271373) The paper was highlighted as a eLife Insight story: http://elifesciences.org/content/3/e04997 The paper was on the cover of eLife.
Start Year 2009
 
Description PapC modelling 
Organisation University of Houston
Country United States 
Sector Academic/University 
PI Contribution PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili proteins in uropathogenic E. coli, allowing them to infect the human urinary tract and cause disease. To pass through the outer membrane of the E. coli cell, a P pili protein must travel through the translocation domain of PapC. The translocation domain is a large ß-barrel occluded by a middle domain (plug domain), which is only marginally stable, to allow the opening of the barrel. Previous studies suggested that this gating mechanism is controlled by two elements of the translocation domain - a ß-hairpin and an a-helix, but it is not clear how. To investigate the role of these elements in allosteric signal communication, we developed a hybrid computational method combining evolutionary and molecular dynamics studies. Additionally, we used evolutionary analysis, relying on both amino acid conservation and coupling (co-evolution) to highlight particularly important interactions. The data were combined in a large hybrid residue interaction network. Using graph theory approaches, we showed that certain regions of the ß-barrel, located mostly on one side of the barrel, are likely involved in moving the plug domain. This unique computational approach allowed us to detect key amino acids in these distinct regions (residue 'communities') that likely control the position of the plug in the pore, thereby regulating PapC gating and usher's activation. Our results suggest that residues around ß12-ß14 that are beneath the a-helix act as a regulator of ß 12- ß 13 loop (the 'latch') that controls the opening and closing of the ß-barrel. The study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.
Collaborator Contribution The group of Prof. Gabriel Waksman (ISMB, Birkbeck/UCL) solved the structure of the usher. Our predictions were tested by experiments, in collaboration with the Waksman group and two groups in the US - Delcour group at the University of Houston and Thanassi Group at Stony Brook University. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four communities. Our collaborators in Groningen, Prof. Siewert-Jan Marrink, have contributed in training us to use their software for coarse-grained MD simulations.
Impact A multi-disciplinary collaboration. Farabella et al. eLife (PMID: 25271373) The paper was highlighted as a eLife Insight story: http://elifesciences.org/content/3/e04997 The paper was on the cover of eLife.
Start Year 2009
 
Description Polysomes in Cells 
Organisation University of Oxford
Department Division of Structural Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution 3D image analysis of polysomes in cells.
Collaborator Contribution Cryo-electron tomography of polysomes performed in Kay Gruenewald's group, Oxford
Impact The collaboration combines experimental work on polysomes in human cells with pattern recognition methods. We work together with Gruenewald lab to improve sub-tomogram averaging.
Start Year 2012
 
Description RNA channelling by the eukaryotic exosome. 
Organisation Birkbeck, University of London
Department Department of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Our contribution to the work was to model the entire exosome and fit it into the EM map. We used homology modelling and flexible fitting and provided for the first time a pseudo-atomic model for complete yeast exosome in an RNA-bound and unbound states.
Collaborator Contribution The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. The structure of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states was studies by Prof. Helen Saibil and Dr. Esben Lorentzen. The structure, together with the density fitting (performed by us) revealed that in the RNA-bound structures a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.
Impact Malet H, et al (PMID: 21072061)
Start Year 2008
 
Description Structure and function of glycine receptors. 
Organisation University College London
Department Centre for Clinical Pharmacology & Therapeutics
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed homology modeling of the glycine receptor and in conjunction with the electrophysiology studies performed in Prof. Smart's lab we provided a new view for the activation of a Cys-loop receptor where, following agonist binding, the hydrophobic core and interfacial loops reorganize in a concerted fashion to induce downstream gating. Currently we are working together with Prof. Smart and Prof. Harvey (UCK School of Pharmacy) to explain new mutations on the receptor, and perform some docking calculations to identify the glycine binding pocket.
Collaborator Contribution Prof. Trevor Smart and Dr Paul Miller study Cys-loop ligand-gated ion channels, which mediate rapid neurotransmission throughout the central nervous system. Their investigation of the glycine receptor (GlyR) has led to identification of a scaffold of hydrophobic residues enabling allosteric communication between glycine-agonist binding loops A and D, and the Zn2+-inhibition site. Mutating these hydrophobic residues disrupted Zn2+ inhibition, generating novel Zn2+-activated receptors and spontaneous channel activity. Electrophysiology, together with our further modelling, revealed that these phenomena are caused by disruption to three residues on the '-' loop face of the Zn2+-inhibition site, and to D84 and D86, on a neighbouring b3 strand, forming a Zn2+-activation site.
Impact Miller et al, 2008 (PMID: 18806798)
Start Year 2008
 
Description Structure and function of glycine receptors. 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed homology modeling of the glycine receptor and in conjunction with the electrophysiology studies performed in Prof. Smart's lab we provided a new view for the activation of a Cys-loop receptor where, following agonist binding, the hydrophobic core and interfacial loops reorganize in a concerted fashion to induce downstream gating. Currently we are working together with Prof. Smart and Prof. Harvey (UCK School of Pharmacy) to explain new mutations on the receptor, and perform some docking calculations to identify the glycine binding pocket.
Collaborator Contribution Prof. Trevor Smart and Dr Paul Miller study Cys-loop ligand-gated ion channels, which mediate rapid neurotransmission throughout the central nervous system. Their investigation of the glycine receptor (GlyR) has led to identification of a scaffold of hydrophobic residues enabling allosteric communication between glycine-agonist binding loops A and D, and the Zn2+-inhibition site. Mutating these hydrophobic residues disrupted Zn2+ inhibition, generating novel Zn2+-activated receptors and spontaneous channel activity. Electrophysiology, together with our further modelling, revealed that these phenomena are caused by disruption to three residues on the '-' loop face of the Zn2+-inhibition site, and to D84 and D86, on a neighbouring b3 strand, forming a Zn2+-activation site.
Impact Miller et al, 2008 (PMID: 18806798)
Start Year 2008
 
Description Structure of the mammalian 80S ribosome at 8.7 A resolution. 
Organisation Boston University
Country United States 
Sector Academic/University 
PI Contribution Together with Prof. Akey's group, we modeled the ribosome by docking homology models of subunit rRNAs and conserved proteins into the density map. We found unclaimed density for ~20 novel proteins. The model provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation.
Collaborator Contribution Prof Christopher Akey determined the structure of the mammalian ribosome at 8.7A° resolution by electron cryo microscopy and single-particle methods. The data was the basis for analysis using homology modeling and fitting to provide the first atomic model of the mammalian ribosome.
Impact Chandramouli et al., 2008. (PMID:18400176)
 
Description The structure of pore-forming proteins 
Organisation Birkbeck, University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed EM fitting to model conformational changes during pore formation by the perforin-related protein pleurotolysin and suilysin (a bacterial cholesterol-dependent cytolysin).
Collaborator Contribution Generated an 11 Å resolution cryo-Electron Microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), and solved the crystal structures of both components (the lipid binding PlyA protein and the pore forming MACPF component PlyB). The data revealed a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA.
Impact publications: Leung et al. 2014 (PMID: 25457051) Lukoyanova et al 2015 (PMID: 25654333) 5 February 2015: cover story in New Scientist: "Mushroom kills with cookie cutter trick"
Start Year 2013
 
Description The structure of pore-forming proteins 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution We performed EM fitting to model conformational changes during pore formation by the perforin-related protein pleurotolysin and suilysin (a bacterial cholesterol-dependent cytolysin).
Collaborator Contribution Generated an 11 Å resolution cryo-Electron Microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), and solved the crystal structures of both components (the lipid binding PlyA protein and the pore forming MACPF component PlyB). The data revealed a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA.
Impact publications: Leung et al. 2014 (PMID: 25457051) Lukoyanova et al 2015 (PMID: 25654333) 5 February 2015: cover story in New Scientist: "Mushroom kills with cookie cutter trick"
Start Year 2013
 
Description The structure of the apoptosome. 
Organisation Boston University
Country United States 
Sector Academic/University 
PI Contribution Our contribution to the work was to model the pseudo-atomic structures of both the human Drosophila apoptosomes by using the EM maps at 9.5 Å and 6.9 Å resolution, respectively. We recently showed an induced-fit mechanism for cytochrome c binding to regulatory ß-propellers of the apoptosome, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange.
Collaborator Contribution In the intrinsic death pathway, cytochrome c binds to Apaf-1 and triggers apoptosome assembly in the presence of dATP. This platform binds procaspase-9, which activates procaspase-3 and other downstream procaspases. To understand this process, Prof. Akey determined a structure of the human apoptosome at 12.8Å resolution and created a model of this heptameric platform, which contains 49 domains and 7 cytochrome c molecules. Currently, we are determining structures of the Drosophila Apaf-1 related killer (Dark) and of other signaling platforms that mediate inflammatory responses to bacteria.
Impact Yuan et al. 2010 (PMID: 20462491) Yuan et al. 2011 (PMID: 21220123) Yuan et al. 2013 (PMID: 23521171)
Start Year 2008
 
Description cryoEM studies on coxsackievirus A7 virus 
Organisation University of Helsinki
Department Institute of Biotechnology
Country Finland 
Sector Academic/University 
PI Contribution Performed flexible fitting using the subnanoresolution structure of cav7 and the crystal structure (at different conformation)
Collaborator Contribution The structures of CAV7 (full and empty capsids) were solved at subunanometrer resolution in the lab of Prof. Sarah Butcher using cryo-EM. A methodology paper (led by us) is on the way (currently under revision) introducing new ways of assessing flexible fitting
Impact Seitsonen et al 2012 (PMID: 22514349) Pandurangan et al 2014 (PMID: 24333899)
Start Year 2011
 
Title Flex-EM 
Description Flexible fitting of atomic structures into electron microscopy density maps 
Type Of Technology Software 
Year Produced 2008 
Impact The method (which is freely available via the widely-used modelling program MODELLER) helps in improving the analysis of low-resolution cryo-electron microscopy maps. It has been used by us and other researchers to generate pseudo-atomic models of a number of macromolecular assemblies studied by cryo-electron microscopy, such as various ribosomes (eukaryotic and e-coli), ribosomal factors (EF4, eIF6), chaperonins (GroEL and TRIC/CCT), membrane channels (RyR1), the exosome, the apopotosome, microtubules, metabolic enzymes (Pyruvate carboxylase) and actin. All these studies have been published . The method has been cited 135 times since 2008. The method is currently being implemented in the CCP-EM (Collaborative Computational Project for Electron cryo-Microscopy) package to reach the wider community of users in the UK and worldwide. 
URL http://topf-group.ismb.lon.ac.uk/flex-em/
 
Title RIBFIND 
Description Software to for finding rigid bodies in protein structures 
Type Of Technology Software 
Year Produced 2012 
Open Source License? Yes  
Impact The software has been used in the EM community for the purpose of EM flexible density fitting as well as in general analysis of protein structure (cited 19 times since 2012). Pandurangan & Topf 2012 (PMID: 22079400) Pandurangan & Topf 2012 (PMID: 22796953) Pandurangan et al. 2014 (PMID: 224333899) 
URL http://ribfind.ismb.lon.ac.uk
 
Title TEMPy 
Description model validation in EM maps. 
Type Of Technology Software 
Year Produced 2015 
Open Source License? Yes  
Impact Vasishtan and Topf 2011 (PMID: 21296161) Seitsonen et al., 2012 (PMID: 22514349) Pandurangan et al. 2014 (PMID: 24333899) Atherton et al 2014 (PMID: 25209998) Farabella et al 2015 (PMID: 26306092) Lukoyanova et al 2015 (PMID: 25654333) 
URL http://tempy.ismb.lon.ac.uk/
 
Description CCP-EM Developers Meeting 2016 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact CCP-EM Developers Meeting
Year(s) Of Engagement Activity 2016
 
Description CCP4 (CCP-EM) Developers Meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact new initiatives for development as part of CCP4 and CCP-EM projects. (CCP-EM is supported by MRC).

CCP-EM is starting to be used by the EM community in the UK.
Year(s) Of Engagement Activity 2013
URL http://www.ccpem.ac.uk/
 
Description EMBO course on integrative modelling of biomolecular interactions 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact EMBO course on integrative modelling of biomolecular interactions
Year(s) Of Engagement Activity 2016
URL http://events.embo.org/16-biomol-interact/
 
Description EMBO practical Course on Image Processing for Cryo EM 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Contributed to the design of the EMBO course on Image Processing for Cryo-electron Microscopy and presented on the course (2007,2009,2011, 2013, 2015).

The students were trained in using my software.
Year(s) Of Engagement Activity 2007,2009,2011,2013,2015
 
Description EMBO practical course in computational structural biology 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The students learned about my method for fitting models into EM maps.

Engagement with students, Dissemination of the methids,
Year(s) Of Engagement Activity 2012
 
Description EMBO practical course on cryoEM and image processing 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact EMBO practical course on cryoEM and image processing
Year(s) Of Engagement Activity 2016
URL https://www.embl.de/training/events/2016/CRY16-01/
 
Description ISMB talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Institute of Structural and Molecular Biology graduate symposium (2012)

engagement with students, dissemination of my work (software)
Year(s) Of Engagement Activity 2012
 
Description public talk in Birkbeck Science week 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact general talk about the role of computational modelling in Structural Biology
Year(s) Of Engagement Activity 2016
URL http://www.bbk.ac.uk/science/about-us/events/science-week/science-week-2016-highlights