# Single Particle Analysis Preparation

## 1 Directory layout and parameter files

Regardless of the SPA strategy followed, it is recommended that a layout similar to that shown in Table 1 be adopted for processing in Bsoft. In the case of independent data sets ("golden standard"), the best is to split the full data set into two or more major directories and process each completely separately.

The parameter files (typically STAR files) embed the image paths relative to its location. So it is best not to move the parameter files once they have been created. If you need the parameter file in a different location, recreate it by using a program such as bmg and write the new file in the desired place. The intent is that this will modify the image paths so they remain correctly specified in the parameter file.

Table 1: Suggested directories for SPA in Bsoft
Directory | Purpose
--- | ---
mg | Raw micrographs
mg_b2 | Micrographs binned two-fold
part | Particle images extracted from the micrographs
ctf | CTF-corrected particle images
ref | Initial reference map(s) for orientation-finding
run1 | First run of determining particle orientations with the resultant reconstruction(s)
run2 | Second run of determining particle orientations with the resultant reconstruction(s)
... | ...

Put all the micrographs in the mg directory.

All programs handling parameter files (such as bmg, bpartsel, borient, etc.) can read multiple files and concatenate them into one large internal parameter database. The whole internal database is then written out into one parameter file by specifying the "-output" option. If the user requires individual parameter files for each micrograph, the program bmg has a "-split" option to generate one parameter file per micrograph. Some of the programs also allow the user to set the path for files, which becomes very important to ensure a smooth and easy workflow.

## 2 Handling different types of micrographs

The concept of a micrograph used in Bsoft is the equivalent of taking a single 2D image on a photographic film and scanning it in a digitization device. Single images taken on CCD or direct cameras qualify as simple micrographs. Dose-fractionation (also called movie mode) results in a series of 2D images (frames) where the aligned average is considered equivalent to a micrograph. The initial processing of the micrographs therefore depends on how they were acquired. The best advice is to keep the individual micrographs and their derivatives separate and use a script to automate all the initial processing up to fitting the CTF parameters (see later).

### 2.1 Gain-correction

In a CCD or direct detector, the gain is typically automatically corrected and this step can be skipped. However, sometimes gain correction is not done to speed up acquisition. In this case, the correction is done by multiplying the raw micrograph with the gain image:

bop -verb 7 -sam 0.537 -mult 1,0 klh.tif klh_gc.mrc

### 2.2 Binning

Micrographs are typically taken at very small pixel sizes, resulting in slow processing times. This can be sped up by binning the data:

bint -v 7 -bin 2 klh_gc.mrc klh_gc_b2.mrc

### 2.3 Generating parameter files

Once the initial image manipulations have been done, the next step is to start constructing a proper database where all the processing parameters will be stored. In Bsoft these are typically STAR files, although XML is also supported. One or more micrograph images can be specified as well as several parameters such as the pixel size and acelleration voltage.

Extracting frame (dose fractionated) information:

bmg -verb 7 -extract frame -Pixel 0.537 -Volt 300 -Amp 0.07 -Cs 2.7 -out klh.star klh_*.mrc

Extracting micrograph (single image) information:

bmg -verb 7 -extract mg -Pixel 0.537 -Volt 300 -Amp 0.07 -Cs 2.7 -out klh.star klh_*.mrc

This will generate the first parameter file with references to the micrograph files (if single images) or micrograph frames (if taken on a direct detector with dose fractionation).

## 3 Aligning frames

This step can be skipped if single micrographs were used for the previous step. The frames are first aligned to the reference frame indicated, progressively building up an average to improve subsequent frame alignment. This is then followed by several iterations of alignment to the average until the shifts decrease below a threshold:

bseries -verb 1 -frames -align 5 -resol 20,500 -shift 100 -bin 2 -average t.pif -out klh_aln.star klh.star

## 4 CTF parameter fitting

The most efficient way is to run an automated CTF fitting first on each micrograph, generating an output parameter file for each:

bctf -verb 1 -action prepfit -Range 0.5,3.5 -envtype 4 -frames -tile 512,512,1 -sam 0.537 -resol 5,20 -Volt 300 -Amp 0.07 -Cs 2.7 -out klh_aln_ctf.star klh_aln.star

The resultant fits need to be checked and refined in bshow.

## 5 Examining micrographs and CTF fits

All the output STAR files from the previous steps should be combined in one large parameter file:

bmg -verb 7 -out klh_all.star *ctf.star

The combined parameter file can be opened in bshow:

bshow klh_all.star

This will present the user with a list of image files referenced in the parameter file. At this stage the CTF fits should be examined to make sure they are the best possible. Therefore, choose a power spectrum from the list of images (indicated by a "ps" in the file specification). Then choose the "Micrograph/Fit CTF" option and follow instructions as described here.

## 6 Automated script

prepfit).