Tools for Generating and Assessing Pseudo-atomic Models from 3D Electron Microscopy Maps of Macromolecular Assemblies.

Lead Research Organisation: Birkbeck College
Department Name: Biological Sciences

Abstract

Cellular processes are governed by the complex coordination and dynamics of biological macromolecules called proteins and nucleic acids. These macromolecules do not act in isolation but form assemblies. Understanding the structure of these macromolecular assemblies can often teach us how they function, which is important for the basic understanding of the cell as well as for developing cures for disease.

Structural biology aids this project by providing "pictures" of macromolecular assemblies. 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. These images can be combined to give us a low-resolution 3-dimensional picture of the assembly structure. Though this technique has led to great discoveries, there are limitations to what it can accomplish due to the low-resolution of the picture, leaving us with significant gaps in our knowledge.

Our research aims to overcome this gap by developing computational methods that pull together information from a variety of experimental sources to construct clearer and more complete pictures of the assembly structures imaged by electron microscopy. Like a jigsaw puzzle, we will fit atomic structures of proteins (known from other experiments) together, within the low-resolution picture, creating a refined "pseudo-atomic" picture of the entire assembly.

Technical Summary

Many cellular processes are governed by the complex coordination and dynamics of macromolecular assemblies. A detailed description of the structures of these assemblies can be extremely useful in understanding these processes. In the last decade, 3D electron microscopy (EM) techniques have become essential in achieving this goal, with an exponentially increasing number of structures solved. Although 3D EM allows us to determine the structural organisation of assemblies not amenable to other methods, the resolution range of the resulting density maps usually reveals the overall molecular shape but does not allow for an atomic description. For this reason, 3D EM maps are almost always further interpreted by fitting in them atomic structures of assembly components determined using X-ray crystallography, NMR, and atomic models from protein structure prediction methods.

We recently introduced a testing procedure for determining the quality of component fits and a number of scores that could be used for that purpose. We propose to build upon this work and create a computational tool for scoring and assessment of fits into 3D EM density maps. We also aim to develop a tool for simultaneous fitting of multiple components into low-resolution 3D EM maps of large assemblies. Our tools will be combined into a fully automated open-source software package, which will be made publicly available, especially to serve the EM community and related structural and computational biology fields. Ultimately, the software will be applied to provide pseudo-atomic models of many macromolecular assemblies, involved in a variety of cellular functions.

Planned Impact

The computational tools for model generation and assessment from 3D electron microscopy (EM) data proposed in this project will primarily be of benefit to the increasingly growing EM community in the UK and internationally due to recent development in the field and a dramatic increase in data generation on structures of macromolecular assemblies. These methodologies and tools will be of general interest to bioscience researchers via systems level analysis of biological processes, as highlighted as one of BBSRC principal strategic aims ("Developing and embedding a 'systems' approach to biosciences in order to advance fundamental understanding of complex biological processes"). Additionally, since the assemblies expected to be analysed by the proposed tools are involved in many cellular functions (including those related to infection and disease), researchers in other fields of life science and medical research are also likely to benefit.

More specifically, the project could also have a great impact on human health since the resulting methods will be applied to 3D EM data on Type 4 secretion systems, which have been shown to be involved in antibiotic resistance and infection. A detailed description of the interactions within the entire system will be of high interest to the pharmaceutical industry, potentially allowing development of inhibitory drugs that target protein-protein interactions. Additionally, the software will contribute to the understanding of the mechanism of membrane pore forming proteins that are important virulence factors in diseases such as pneumonia and meningitis. Thus, results may have impact on the economic competitiveness of the United Kingdom in pharmaceutical research.

Publications

10 25 50
 
Description We have developed a software tool to help researchers better analyse data on structures of macromolecular machines studied by a technique called cryo-electron microscopy (cryoEM). Our software is now used by researchers in the field to fit and validate atomic structure in cryoEM maps It is also used in the following projects:
a. Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM) from STFC.
b. The Electron Microscopy Databank (EMDB) at EBI.
c. The Critical Assessment of protein Structure Prediction (CASP) from the Protein Structure Prediction Centre.
Exploitation Route Our software helps researchers to provide better atomistic models for the systems they study by cryoEM thereby helping them to understand the underlying biological mechanisms. It is particularly useful to validate atomic models with cryoEM density maps at different resolutions. Validation of atomic models is one of the most important problems the cryoEM field is currently facing (due to the so-called "resolution-revolution"). We are actively developing approaches to address this problem and our TEMPy tool is an excellent platform to implement our developments. The software is open-source and can be easily adopted by the community. It can also by accessed via the user-friendly CCP-EM platform. The TEMPy paper (Farabella et al 2015, PMID: 26306092) has been cited 27 times since 2015.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://tempy.ismb.lon.ac.uk/
 
Description A direct electron detector for electron cryo-microscopy.
Amount £387,500 (GBP)
Funding ID 101488/Z/13/Z 
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 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 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 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 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 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 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 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 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 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 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 Instruct Workshop: Computational tools for combining atomic and volume data 
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 Course hosted by CCP-EM at Diamond Light Source and Research Complex at Harwell in April 2015. Funded by Instruct, CCP4 and CCP-EM.
~20 pupils attended. The aim was to teach computational tools for combining atomic and volume data
Year(s) Of Engagement Activity 2015
URL http://www.ccpem.ac.uk/training/instruct_apr15.php
 
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