Automatic CTF correction for single particles based upon multivariate statistical analysis of individual power spectra.

Three-dimensional electron cryomicroscopy of randomly oriented single particles is a method that is suitable for the determination of three-dimensional structures of macromolecular complexes at molecular resolution. However, the electron-microscopical projection images are modulated by a contrast transfer function (CTF) that prevents the calculation of three-dimensional reconstructions of biological complexes at high resolution from uncorrected images. We describe here an automated method for the accurate determination and correction of the CTF parameters defocus, twofold astigmatism and amplitude-contrast proportion from single-particle images. At the same time, the method allows the frequency-dependent signal decrease (B factor) and the non-convoluted background signal to be estimated. The method involves the classification of the power spectra of single-particle images into groups with similar CTF parameters; this is done by multivariate statistical analysis (MSA) and hierarchically ascending classification (HAC). Averaging over several power spectra generates class averages with enhanced signal-to-noise ratios. The correct CTF parameters can be deduced from these class averages by applying an iterative correlation procedure with theoretical CTF functions; they are then used to correct the raw images. Furthermore, the method enables the tilt axis of the sample holder to be determined and allows the elimination of individual poor-quality images that show high drift or charging effects.

[1]  H. Zeichhardt,et al.  Determination of the location of proteins L14, L17, L18, L19, L22 and L23 on the surface of the 50S ribosomal subunit of Escherichia coli by immune electron microscopy , 2004, Molecular and General Genetics MGG.

[2]  E. Stadtman,et al.  Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. , 1968, Biochemistry.

[3]  W Chiu,et al.  CTF determination of images of ice-embedded single particles using a graphics interface. , 1996, Journal of structural biology.

[4]  A. Morineau,et al.  Multivariate descriptive statistical analysis , 1984 .

[5]  A. Klug,et al.  Measurement and compensation of defocusing and aberrations by Fourier processing of electron micrographs , 1971 .

[6]  J. Frank,et al.  Correspondence analysis of aligned images of biological particles. , 1982, Journal of molecular biology.

[7]  J. Frank,et al.  Three-dimensional reconstruction with contrast transfer function correction from energy-filtered cryoelectron micrographs: procedure and application to the 70S Escherichia coli ribosome. , 1997, Journal of structural biology.

[8]  B. Böttcher,et al.  Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy , 1997, Nature.

[9]  Gregory K. Miller,et al.  Elements of Applied Stochastic Processes , 1972 .

[10]  M van Heel,et al.  A new generation of the IMAGIC image processing system. , 1996, Journal of structural biology.

[11]  B. Kastner.  Purification and Electron Microscopy of Spliceosomal snRNPs , 1998 .

[12]  N. Unwin,et al.  Contrast transfer for frozen-hydrated specimens: determination from pairs of defocused images. , 1988, Ultramicroscopy.

[13]  O. Scherzer.  The Theoretical Resolution Limit of the Electron Microscope , 1949 .

[14]  J. Schenkel.  RNP Particles, Splicing and Autoimmune Diseases , 1998, Springer Lab Manual.

[15]  Koji Yonekura,et al.  Contrast transfer for frozen-hydrated specimens II. Amplitude contrast at very low frequencies , 1993 .

[16]  J. Conway,et al.  Methods for reconstructing density maps of "single" particles from cryoelectron micrographs to subnanometer resolution. , 1999, Journal of structural biology.

[17]  Expected contribution of the field-emission gun to high-resolution transmission electron microscopy , 1994 .

[18]  Marin van Heel,et al.  Multivariate statistical classification of noisy images (randomly oriented biological macromolecules) , 1984 .

[19]  K. Downing,et al.  Assessment of resolution in biological electron crystallography. , 1992, Ultramicroscopy.

[20]  Holger Stark,et al.  Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle , 2001, Nature.

[21]  Joachim Frank,et al.  On the correction of the contrast transfer function in biological electron microscopy , 1995 .

[22]  W Chiu,et al.  EMAN: semiautomated software for high-resolution single-particle reconstructions. , 1999, Journal of structural biology.

[23]  E. Hewat,et al.  Characterization of the performance of a 200-kV field emission gun for cryo-electron microscopy of biological molecules. , 2002, Journal of structural biology.

[24]  M. Heel,et al.  Single-particle electron cryo-microscopy: towards atomic resolution , 2000, Quarterly Reviews of Biophysics.

[25]  Errol C. Caby.  Elements of Applied Stochastic Processes , 2003, Technometrics.

[26]  M. Radermacher,et al.  The structure of the V(1)-ATPase determined by three-dimensional electron microscopy of single particles. , 2001, Journal of structural biology.

[27]  Joachim Frank,et al.  An assay for local quality in cryo-electron micrographs of single particles. , 2002, Ultramicroscopy.

[28]  José-Jesús Fernández,et al.  A spectral estimation approach to contrast transfer function detection in electron microscopy , 1997 .

[29]  J. Dubochet,et al.  Cryo-electron microscopy of viruses , 1984, Nature.

[30]  R. Wade,et al.  Electron microscope transfer functions for partially coherent axial illumination and chromatic defocus spread , 1977 .

[31]  W Chiu,et al.  Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination. , 2001, Journal of structural biology.

[32]  Hiroyuki Sasabe,et al.  A set of computer programs for determining defocus and astigmatism in electron images , 1996 .

[33]  F. Thon.  Notizen: Zur Defokussierungsabhängigkeit des Phasenkontrastes bei der elektronenmikroskopischen Abbildung , 1966 .