ranging from 0
to Æ60
in steps of 5
–10
, usually (a full exposure
is acquired at a given tilt angle). The finer the angular sampling the
better. A typical dataset will contain $100 to $1000 movies, which
should be spread across the tilt range. Note that in contrast to
electron tomography, where unique samples are imaged in a tilt
series, in the here described approach each 2D crystal is imaged
only once and at one specific tilt angle. However, different 2D
crystals should then be imaged at different fixed tilt angles.
Movies of 2D crystals should be corrected for beam-induced
drift [44] and for beam-induced resolution loss by applying dosedependent temperature factors to each frame before computing
frequency-weighted averages from all frames [45], generating a
drift-corrected average image. Also, defocus and astigmatism have
to be estimated for each image [46, 47]. Using FOCUS, these steps
can be carried out in real time during the microscopy session and
the dataset can be conveniently pruned based on parameters such as
total drift, measured defocus and estimated CTF resolution, among
others [22].
3.2 Conventional 2D
Crystallographic
Reconstruction
Before starting with the single particle approach, it is necessary to
process the data in conventional 2D electron crystallography fashion. For each 2D crystal image in the dataset, the crucial parameters
to be obtained at this stage are:
– The defocus and astigmatism at the center of the image.
– The tilt geometry of the specimen plane, defined by the angles
TLTANG and TLTAXIS.
– The orientation of the 2D crystal on the specimen plane, defined
by the angle TAXA.
– The phase origin (translational shifts).
– The unit cell positions provided by the unbending
algorithm [26].
Ideally, the best possible 3D map should be obtained in this
way by iterative 3D merging and refinement before proceeding,
although an initial, low-resolution map may suffice (see Note 4).
For more information and protocols for the processing of 2D
crystal data, please refer to [17, 19, 21, 43, 48].
3.3 Exporting a 2D
crystal Project
for Single Particle
Analysis
Having performed conventional 2D crystallography data processing, we can proceed to the single particle analysis of 2D crystals.
The single particle scripts are available under the Particles tab on
the top bar of the FOCUS GUI. The first script, Pick & export
particles, is the core of our approach, allowing the user to extract
the particles and export these data in a way that is understood by
single particle programs. The script will operate on the images that
have been selected via the FOCUS library GUI (see Note 5). The
Single Particle Analysis for High-Resolution 2D Electron Crystallography
271
to Æ60
in steps of 5
–10
, usually (a full exposure
is acquired at a given tilt angle). The finer the angular sampling the
better. A typical dataset will contain $100 to $1000 movies, which
should be spread across the tilt range. Note that in contrast to
electron tomography, where unique samples are imaged in a tilt
series, in the here described approach each 2D crystal is imaged
only once and at one specific tilt angle. However, different 2D
crystals should then be imaged at different fixed tilt angles.
Movies of 2D crystals should be corrected for beam-induced
drift [44] and for beam-induced resolution loss by applying dosedependent temperature factors to each frame before computing
frequency-weighted averages from all frames [45], generating a
drift-corrected average image. Also, defocus and astigmatism have
to be estimated for each image [46, 47]. Using FOCUS, these steps
can be carried out in real time during the microscopy session and
the dataset can be conveniently pruned based on parameters such as
total drift, measured defocus and estimated CTF resolution, among
others [22].
3.2 Conventional 2D
Crystallographic
Reconstruction
Before starting with the single particle approach, it is necessary to
process the data in conventional 2D electron crystallography fashion. For each 2D crystal image in the dataset, the crucial parameters
to be obtained at this stage are:
– The defocus and astigmatism at the center of the image.
– The tilt geometry of the specimen plane, defined by the angles
TLTANG and TLTAXIS.
– The orientation of the 2D crystal on the specimen plane, defined
by the angle TAXA.
– The phase origin (translational shifts).
– The unit cell positions provided by the unbending
algorithm [26].
Ideally, the best possible 3D map should be obtained in this
way by iterative 3D merging and refinement before proceeding,
although an initial, low-resolution map may suffice (see Note 4).
For more information and protocols for the processing of 2D
crystal data, please refer to [17, 19, 21, 43, 48].
3.3 Exporting a 2D
crystal Project
for Single Particle
Analysis
Having performed conventional 2D crystallography data processing, we can proceed to the single particle analysis of 2D crystals.
The single particle scripts are available under the Particles tab on
the top bar of the FOCUS GUI. The first script, Pick & export
particles, is the core of our approach, allowing the user to extract
the particles and export these data in a way that is understood by
single particle programs. The script will operate on the images that
have been selected via the FOCUS library GUI (see Note 5). The
Single Particle Analysis for High-Resolution 2D Electron Crystallography
271