OVERVIEW OF PROGRESS DURING THE LIFETIME OF THE PROJECT
Project objectives and scientific achievements
Introduction: Electron microscopy of biological macromolecules has
become a powerful technique for determining the three dimensional (3D)
structure of proteins and protein complexes. The development of
crystallographic methods and their application to two-dimensional (2D) crystals
produce 3D density maps at 34 Angstrom resolution which can be interpreted by
atomic models (Henderson et al., 1990; Kuhlbrandt et al., 1994; Nogales et al.,
1998). At the same time, electron microscopy of isolated protein molecules and
complexes (single particles) continues to progress to higher resolution and has
yielded 3D maps at 7 resolution in the case of highly symmetrical viruses
(Bottcher et al., 1997; Conway et al., 1997) and 11.5 resolution for the
asymmetrical ribosome (Gabashvili et al., 2000). The overall aim of this
project was to define standards for data interchange, to create a deposition
system and database and to direct efforts to inform the 3D-EM community on
standards and deposition policies.
3D-EM Metadata descriptions. A complete meta description of the 3D-EM experiment has been
developed and presented to the 3D-EM community. The final dictionary of
metadescriptors have been made available on the IIMS web site. A common set of
textual descriptors that will standardise the experimental conditions specific
to 3D-EM has been developed. Areas covered include specimen preparation, EM
imaging and data processing. These descriptors complement the set of other
standard descriptors already contained for X-ray and NMR techniques in the
definition of the EMSD. The data is given in the supplementary material and the
on-line documentation is given in the following table.
An example of the wide-spread acceptance of the
data model is in the PDB where all CRYO-EM entries now use the template created
by MSD group in November 2001 and to date (23/09/2003) 45 EM entries use the
IIMS template out of total of 57 EM entries since start of PDB (1971). The PDB
idcodes since November 2001 are: 1GR5, 1GR6, 1GRU, 1GW7, 1GW8, 1KJU, 1LD4, 1LS2, 1LU3,
1MOF, 1M11, 1M4X, 1M8Q, 1MHS, 1MI6, 1MJ1, 1ML5, 1MVR, 1MVW, 1NO3, 1N6G, 1O18,
1O19, 1O1A, 1O1B, 1O1C, 1O1D, 1O1E, 1O1F, 1O1G, 1OED, 1P6G, 1P85, 1P86, 1P87,
1PDF, 1PDI, 1PDJ, 1PDL, 1PDM, 1PDP, 1PN6, 1PN7, 1PN8, 1UCU.
Example of Published reference to EMDB: see
Rawat, U.B.S., Zavialov, A.V.,
Sengupta, J., Valle, M., Grassucci, R.A., Linde, J., Vestergaard, B.,,
Ehrenberg, M. and Frank, J. (2003). Nature, 421, 87-90. A cryo-electron microscopic study of
ribosome-bound termination factor RF2
The coordinates of the
modified RF2 structure, obtained by fitting individual domains of RF2 (pdb code
1GQE) into the cryo-EM density of RF2(wt), have been deposited in the Protein
Data Bank with accession number 1MI6. The coordinates of the ribosomal
components (1G1X, 1G1Y) interacting with the RF2 domains at the GAC, PTC and DC
aligned to the cryo-EM map of the 70S Escherichia coli ribosome have also been
deposited with accession number 1MVR. The cryo-EM maps of the RC (EMD-1006) and
its interaction with the RF2(GAQ) mutant (EMD-1008) and the wild type
(EMD-1007) have been deposited at the 3D-EM database, EMBL-European Bioinformatics
Institute, Cambridge, UK.
Integrated DataBase: The MSD structure database,
originally built to handle PDB coordinate data is now completely merged with
the EM SQL schema derived from the above EM data definitions. A preliminary
Query/Search interface has been designed and implemented and is available to
the public. (Details are given above).
Deposition System: For deposition, this project was to produce a prototype
deposition interface, however from the outset we undertook to build a fully functional
database and deposition system as we realised that the 3D-EM community would
accept a functioning system rather than a prototype test system. The system has
been accepting depositions since June 2002.
Validation measurements and
procedures for 3D-EM: It is
essential to use a reliable resolution measure to judge the progress in
resolution made with larger amounts of data and new techniques. Commonly, a
correlation coefficient is used to measure the resolution of a 3D map
calculated from single-particle images. For this purpose, the set of images is
divided into two subsets, each containing one half of the images of the
complete set. The distribution of images between the two sets should be random,
but in practice they are usually divided into odd and even numbered particles.
Two 3D maps are calculated from the subsets and their Fourier transformations,
F1 and F2, are computed. The resolution of the two maps is then estimated by the
Fourier shell correlation (FSC; Harauz & van Heel, 1986),
Accurate measurement of the
resolution of the 3D reconstruction calculated from images of single protein
molecules or complexes is an essential quality assessment for the images
recorded in the electron microscope, as well as for new methods to be developed
to push single-particle methods to near-atomic resolution. Commonly used
measures of resolution, such as the Fourier ring correlation, the spectral
signal-to-noise ratio or the Q factor, can yield unrealistic results. The
Fourier ring correlation is a reliable indicator of a signal present in a 3D
reconstruction only if the alignment of the images in the two half data sets
was performed with two independent reference structures.
community towards achieving better methods and procedures
for 3D-EM structure determination. Partner 2 has work in a new parametric
technique for the determination of the Contrast Transfer Function (CTF) of EM
micrographs (Velazquez-Muriel et al., 2003). Faithful CTF determination is an
essential step prior to CTF correction. Several methods have been proposed in
order to correct the effect of the CTF in a given reconstruction:
Wiener filtering of the reconstructed volume (Frank and
Penczek, 1995)
Regularised steepest-descent technique (Zhu et al. 1997)
Inverse CTF filtering of the reconstructed volume (Stark et
al. 1997)
Incorporation of CTF for each projection and maximum-entropy
reconstruction (Skoglund et al. 1996)
Incorporation of Wiener-like fashion CTF for each projection
and Fourier reconstruction algorithm (Grigorieff 1998)
CTF of projections and weighted Fourier reconstruction
(Ludtke et al. 1999, Ludtke et al. 2001)
As part of the Pilot project for generation of harvest
files for icosahedral reconstruction, the Oxford group have investigated the
software package, iTools. The iTools.parameter file (a single parameter file
that provides all of the parameters used for finding orientations and perfoming
a reconstruction), contains the refined orientation information and the
residuals, this information is being investigated as a means of mapping the
file to the interchange XML for uploading during deposition. The iTools files
allows for the reproduction of a reconstruction together with the fft files
corresponding to the data
The following data sets have been deposited to emdep as part of the prototype
testing and accumulation of data sets for validation trials.
- PRD1 membrane bacteriophage wild type 18
- PRD1 membrane bacteriophage sus1 mutant 13
- PRD1 membrane bacteriophage sus690 mutant 18
- PRD1 membrane bacteriophage P3 shell 11
- PRD1 pseudoatomic models are deposited in PDB, (1GW7, 1GW8,1HB5,1HB7,1HB9)
- Semliki Forest Virus 9
SFV pseudoatomic model is
deposited in PDB (1DYL)
Adenovirus 2 - 25
Reports on extended coordination meetings:
WP8 has involved circulating
meta-data descriptors to a wide community of 3D-EM scientists and in
collaborating with the RCSB in establishing a PDB template for those 3D-EM
structures that may be submitted to the Protein Data Bank. All partners have
made presentations at a deposition/database workshop held at a Conferences and
workshops on Electron Microscopy.
Provide an
update of tables 1 (deliverables), 2 (work packages) and 3 (milestones)
from the technical annex of your
contract, comparing the progress achieved against the planning. Add comments,
if appropriate, indicating significant difficulties or delays encountered, the
activities thus affected and actions taken to remedy them.
(PLEASE see WP list
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