**SIMPLE 2.0** is not a standalone suite for single-particle reconstruction, but complements available developments. It is assumed that the windowed single-particle images represent 2D projections, and therefore, that the projection slice theorem applies (Bracewell, 1956). This requires correction of the micrographs due to the contrast transfer function (CTF) of the electron microscope and particle windowing using other software. “CTF-corrected images” refers to images corrected using the simple heuristic of binary phase flipping, adopted in numerous image-processing packages. CTF-correction by phase flipping only corrects the resolution-dependent CTF phase inversions, disregarding the damping of Fourier amplitudes with increasing resolution. Phase-flipped images are noisier than images corrected with more accurate approaches, such as the reciprocal space adaptive Wiener filter (Penczek, 2010). We have been primarily concerned with the problem of reconstructing accurate low-resolution maps *ab initio *from very noisy images. More sophisticated approaches for dealing with the CTF will be implemented in the future. In addition, the windowed projections should be roughly centered in the box. Many program suites are available for dealing with these and other matters (Frank et al., 1996; Hohn et al., 2007; Ludtke et al., 1999; Sorzano et al., 2004). In EMAN, the envelope component of the CTF, describing the fall-off of the signal with resolution, is parameterized and then used in the class averaging procedure in the refinement. Some implementations only phase-corrects the micrographs, ignoring the envelope function initially. Instead, the final refined maps are B-factor sharpened. SIMPLE has so far been use in combination with the latter approach, assuming the same resolution fall-off for all micrographs. The SIMPLE workflow is divided into four phases:

1) CTF correction and particle windowing

We usually execute phase (1) by parameterizing the CTF with Ctffid3 (Mindell and Grigorieff, 2003), doing CTF phase correction of the micrographs in Spider (Frank et al., 1996), and using EMAN’s (Ludtke et al., 1999) program BOXER for particle windowing. EMAN’s graphical user interface is used for quick inspection of volumes and class averages. UCSF Chimera is used for more detailed analysis of the reconstructed volumes (Pettersen et al., 2004). Phases (2)-(4) are executed with **SIMPLE 2.0**.

2) *ab initio* reconstruction method** PRIME**

**PRIME** is a powerfull *ab inito* reconstruction method that can:

***) **** in a single step, generate an ** **initial 3D map directly from the noisy images**

***) overcome model bias introduced by an erroneous initial map**

***) get the PRIME paper here**

Example: asymmetric reconstruction of 1000 images of the eukaryotic ribosome

Dowload the data:

**STKrib.spi** - stack of single particle images with SNR=0.05

nptcls=1000

box=120

smpd=3.54

**ribosome_rec.spi **- the correct map to compare with your result, remembrer that your map can have a different hand, so you might need to mirror it

**1) generate a random reconstruction (a blob) - simple_rndrec**

**>> simple_rndrec** stk=STKrib.spi box=120 smpd=3.54 nthr=16

where nthr is the number of threads

**output:**

cmdline.txt - a file with your input parameters

rndoris.txt - random Euler angles

startvol_state1.spi - your blob

**2) Execute simple_prime** - should converge in 7-10 rounds

run in the background with nohup &

**>> **nohup** **stk=STKrib.spi box=120 smpd=3.54 vol1=startvol_state1.spi dynlp=yes oritab=rndoris.txt ring2=50 nthr=16 > OUTPUT &

**output:**

recvol_state_iter_X.spi - reconstructions from different rounds, the last one is your final map

prime_docX.txt - alignment file (Euler angles and translations)

**If you use the trs parameter:** you can shift the stack with:

>>>**simple_stackops **stk=STKrib.spi box=120 smpd=3.54 outstk=STKshifted.spi oritab=merged.txt shalgn=yes

3) Heterogeneity analysis (coming soon)

4) Refinement (coming soon)