Rigid body matching/fitting in intermediate-low resolution density - PowerPoint Presentation

1. Rigid body matching/fitting in intermediate-low resolution density Agnel Praveen JosephSTFC, Harwell

2. Sample classigicationTaxonomyPfam domains (fitted models)Uniprot ID (text search/Pfam link)Alignment scores, transformation matrix Web-serverDatabaseUser’s query map/ New EntryUser’s query PDB/ New EntryData organisation & annotation(Work along with the data submission pipeline at EMDB/PDBe and as a development for CCP-EM)

3. MethodsDensity based 6D search : Fast Translational Matching (COLORES) : Fast Fourier Transform (FFT) driven translational search at each rotation (Chacon and Wriggers, 2002). Cross correlation scores with or without Laplacian filters, are used for matchingAuthor test cases: Simulated maps down to 34Å resolution were used, and correct alignments were obtained down to 25Å resolution using Laplacian correlation filter. Components of microtubule were also assembled successfully in a 20Å experimental map.Fast Rotational Matching (ADP-EM,) : Spherical Harmonics accelerated rotational search at each translation (Garzon et al. 2007, Kovacs et al. 2003). Cross correlation scores with or without Laplacian filters, are used for matchingAuthor test cases: Simulated maps of down to 30Å were used for testing and accurate results were obtained even at 30Å resolution, using higher harmonic bandwidth values. Fitting components on a 23Å experimental map was also successful.Random Sampling (CHIMERA,ModEM): Randomly sample rotation and translation space (Goddard et al. 2007, Topf et al.2005). Overlap and correlation based scores are used for matching.http://3dem.ucsd.edu/SI_Table1_ver2.pdfVilla and Lasker, Finding the right fit: chiseling structures out of cryo-electron microscopy mapsCurr Opin Struct Biol. 2014

4. Reduced representation : Gaussian Mixture Model (GMFIT): Linear combination of N 3D gaussian density (Kawabata, 2008). A gaussian overlap metric is used to score the alignment.Author test cases: With the right choice of the number of GDFs, correct alignments were obtained using simulated maps down to 30Å resolution. Tests on a 23.5Å experimental map also gave ‘near-native’ alignment with respect to a reference. FoldEM : Local density gradients are described as orthogonal feature vectors which are rotationally invariant (represented as vectors covering a set of neighboring grid points: Local Region Descriptors). Graphs are constructed with these descriptors as nodes and a graph-matching technique is applied to detect the maximal sub-graph. The size of the common sub-graph is returned as the score, along with an atom inclusion score from Chimera (Pettersen et al., 2004).Author test cases: Tests were carried out with simulated EM maps down to 20Å resolution and correct fits were obtained with resolutions down to 15Å. Comparison of 11Å ribosome (experimental) maps in two conformational states also gave a good alignment. Methods

5. GMfitNumber of 3D gaussians used to approximate the density/model depends on the number of local features/components. Both map and model needs to be converted into gaussian mixture models prior to matching EMD-1056, E-coli 70S, 9ÅEMD-2017, E-coli 30S, 13.5ÅAlignment of gaussian mixtures

6. ADP-EMSpherical harmonics are an infinite set of harmonic functions defined on the sphere. The basis functions are indexed according to two integer constants, the order, l, and the degree, m. As the number of coefficients increases, higher frequency signals can be approximated more accurately. The two arguments l and m break the family of polynomials into bands of functionsOption of laplacian filter for gradient detection

7. Atomic structure modeling and processing If experimentatly determined structures are not available: Fold recognition and Homology modelingRemove flexible loops. Separate compact domains connected by a hinge/flexible loop.Check if sub-complexes can be modelled 2631/4umm : 11.6Å cryo-EM structure of palindromic DNA bound USP/EcR nuclear receptorMaletta et al. The palindromic DNA-bound USP/EcR nuclear receptor adopts an asymmetric organization with allosteric domain positioning. Nat comm 2014

8. A case: 1534/2y7c/2y7h18Å EcoK1 methyltransferase with a bound antirestriction proteinT7 phage antirestriction protein ocrROSETTA(http://robetta.bakerlab.org/) ab-inito model HsdS-ocr complex modeled using guided multi-body docking by HADDOCK (http://haddock.science.uu.nl/services/HADDOCK2.2/haddock.php) HsdMHsdS

9. Volume processing Shift background peak to zeroSome scores to measure fit quality are sensitive to scale of data values e.g: overlap and correlation (not about mean) metrics in Chimera use sum of products of density values.Select a contour thresholdUsually subjective. Can be calculated from molecular weight of sample.Calculations on a subset from EMDB shows a peak at 2 sigmaA higher contour may be considered for local weak density regions:Contour level suggested by authors (EMD-5287, 26Å).

10. Search spaceProbe/target size ratio should be considered unless you are using an exhaustive search method. Target map should be segmented to increase probe/target(segment) size ratio.Gmfit: Random sampling (-I R), segmentation (-I SF) and symmetry based search (-I Y) options are available.adp_em: masking search (-s 2, default): the translational space is limited to positions on which the dimension of the probe (atomic structure) roughly fits inside the experimental EM map. radial search (-s 1): more uniform and useful for structures with holes.exhaustive search (-s 0)Chimera parameter : search N (number of random configurations)

11. Translation and rotation SamplingDepends on resolution/grid spacing – coarse sampling for low resolution maps Size of probe : finer rotation sampling for a larger probe adp_em : higher bandwidth - finer rotational sampling and more accurate harmonic description. 16 default. Bandwidth values correspond to 360/(2*B) degrees of rotation. translational sampling in Å: -t (one/two voxel steps)

12. Try different methods: An example EMD-2631For intermediate/low resolution fitting:Multiple methods might have to be triedDifferent solutions might have to be checked (for those that are structurally stable, functionally relevant and supported by experiments)Multiple scores might be required to re-rank the solutions : surface/envelope matching scores are especially useful at low resolutions. TUTORIAL

13. /scratch/ccpem_tutorial/apj_tutorial_22_04/examples/2631module load chimeramodule load adp_emmodule load gmfitmodule load gmconvert

14. Volume matching pipeline(ccp-EM/EMDB-PDBe)

15. Current version under developmentDatabase of EMDB volume alignments and PDB model – EMDB volume comparisons (fitting)Web service and software for volume-volume and model-volume alignmentsWeb service to search user model/map in EMDB for similar volumes, volumes from certain taxa and sample categories.Web service and software for 3D analysis of alignments, map feature representations (gaussian mixtures, points), calculate difference maps.Annotation (sequence/interactions/taxonomy) of entries in EMDB

16. Volume pre-processingFeature pointsContoured densityGaussian mixtureDustingSegmentation424.8Å424.8Å

17. Scores (TEMPy)TEMPy (Farabella et al.) scores used for re-ranking hits obtained.Global scores are influenced by non uniform noise, interpolation and padding effects. Local scores used - maps need to be contoured prior to calculations. If the resolution of the map is very low to be informative (density variation) – Envelope/Surface based scores may be useful. Local cross-correlation, Local mutual information, surface distance score and Overlap score are used for re-ranking solutions

18. ExamplesE-coli 70S ribosome: 9Å vs E-coli 30S ribosome: 13.5ÅMethanococcus maripaludis Mm-cpn chaperone: 4.9Å vs E-coli GroEL/GroES (mut) chaperone: 9.2ÅHuman Echovirus 12: 16Å vsHuman Coxsackievirus A21: 8Å

19. Surface definitions based on a given contour: Partial surface overlap detection22.0Å Dengue virus9Å 70S E coli vs 6.6Å 80S yeastSurface feature extractionSurface point definitions

20. ReleaseTest version of volume matching software is available at: http://www.ccpem.ac.uk/download.phpCurrently full functionalities are not included: multiple scores, user map input, more methods to be added (adp_em, shapeEM)Fully functional version should be released in the next few months.Web service (EMDB/PDBe) will be also available in the next few months will all functionalities

21. Martyn WinnMaya TopfArdan Patwardhan Ingvar LagerstedtThank You