1: Preparation to run TYGRESS
1.1 To use TYGRESS on the example data set, download and unzip the TYGRESS source code and example data set package (www.tygress.org), and then follow Procedure step 2 (“Preparation of TYGRESS input files” below).
Note：TYGRESS is also available on Code Ocean as a compute capsule (https://doi.org/10.24433/CO.2034333.v1). Please read the readme file on the Code Ocean TYGRESS page for more details.
1.2 To use your own data set, please finish the data collection, tomogram reconstruction, and subtomogram averaging before following Procedure 2, below.
1.2.1 Data collection. During data collection, two data sets need to be acquired for each region of interest. (1) First, acquire a 2D image at 0 degree using an electron dose typical for conventional SP-cryo-EM (HD image), which is used for final TYGRESS reconstruction; (2), immediately after collecting the HD image, collect a traditional low-dose tilt series (LD images) of the same region, which is used for retrieving the position information for each particle of interest after tomogram reconstruction and subtomogram averaging. Note: to minimize the effects of radiation damage in the final reconstruction, the HD image must be recorded prior to the LD images.
1.2.2 Motion correction. If movie stacks were taken during the data collection, run the whole-frame motion correction using IMOD scripts.
1.2.3 Alignment of the combined tilt series. Each HD image needs to be inserted into its corresponding tilt series at the angle where the HD image was taken using the “newstack” command in the IMOD package, resulting in a “combined tilt series”. For example, an HD image recorded at 0 degrees should be inserted just before the LD image at 0 degrees. After tilt series alignment using the IMOD package, the combined HD and LD images ensure a common reference frame for the later steps in the TYGRESS procedure.
1.2.4 Tomogram reconstruction. After alignment, only the LD images are used to generate the tomogram using the IMOD package.
1.2.5 Subtomogram averaging. The subtomogram averaging can be performed using the PEET package in IMOD. Run PEET averaging for each tilt series and all of the particles of interest, respectively.
2: Preparation TYGRESS input files.
2.1 Prepare two TYGRESS input text files.
2.1.1 There is one file named as “TYGRESS_input.txt” in the TYRESS source code folder (as shown below in the example data set), which contains all the parameters to run TYGRESS, such as the work directory, file path, 3D reconstruction parameters, etc. Update these parameters as needed to reflect the location and organization of your files. If you are following along with the example dataset, modify the following:
· fnDataDir= (copy the full path name for the example data folder that you downloaded from this tutorial)
· fncode= (copy the full path name for the TYGRESS source code folder that you downloaded from this tutorial)
· fnref= (copy the full path name for the .mrc file within the example folder)
Example of a “TYGRESS_input.txt” file
=========================================================================
Part I Parameters for running TYGRESS
fnDataDir = /project/cellbiology/Nicastro_lab/nusr/shang/linux/Tygress/example/ % project data directory
fncode = /home2/s165838/Tygress/Tcoden/Tcode/ % TYGRESS code directory
fnWorkDir = combined_PEET/ % combined peet directory
fnDef = ctffind/DefocusList.txt % defocus file
fnref = /home2/s165838/Tygress/ref.mrc % reference masked after PEET average
fnCombinedHD = HD_particles/ % high dose 2D particle directory
fnmod = combined_mod_csv/ % combined mod & csv directory
fncsv = Iter1_particles/ % updated peet run for whole dataset
csvDirname = Itr1/ % csv directory
csvname_h = _Itr1_MOTL_Tom % csv root name after PEET of individual tilt series
csvname_t = _Iter5.csv % csv tail name after PEET of individual tilt series
modname_h = A1.mod.FOIE. % mod root name after PEET of individual tilt series
modname_t = .txt.mod.txt.Ax_Pred_.txt.mod % mod tail name after PEET of individual tilt series
fnPEETRoot = example % root name for PEET running
particleSize = 180 % particle size for PEET running
fnDist = N % file for correct distortion (gif camera), 'N' for no correction
tail_st = _fused.st % entire tilt date set
tail_hd = _HD.st % file name use for save HD micrograph
amplitude_contrast = 0.14 % thin ice sample use 0.07 and thick ice use 0.14
axoRepeat =180 % pixel size for particle (used for remove edged particles) in high dose micrograph
Mag1 = 22500 % micrograph magnification
PickSize = 300 % size for pick 2D high dose particle
sizex = 200 % size for final out put structure
root_csv = _MOTL_Tom % csv root name after run entire PEET
tail_csv = _Iter2.csv % csv tail name after run entire PEET
SizeRef = 180 % size of reference volume
sx = 4 % shift range of coordinates, nm
sa = 2 % shift range of orientation, degree
parallel_f = 1 % set 1 for run job in parallel job
Ncpu = 5 % cup number for parallel job
tail_rec = _fused_exHD.rec % tail part of rec file
pixel_size = 5.562 % angstrom per pixel for raw particle
kV = 300 % voltage
Cs = 2 % value of spherical aberration coefficient of objective in mm
use_ctf = 1 % set 1 for ctf correction
out_mrc = 96nm % output reconstruction file
imageSize = 2048 % entire raw image size K*K
sizescale = 0.6
n_neighbor = 2 % number of neighbored particles which used for background reference
dis_neighbor = 9 % distance (# pixels) used for selecting nearby particles for background reference
Part II Parameter file for reconstruction, visit FREALIGN webpage for more details
data_input 96nm
raw_images1 96nm
thresh_reconst 90.0
thresh_refine 90.0
pixel_size 5.562
dstep_frealign 13
WGH 0.14
kV1 300.0
Cs 2.0
RI 0.0
RO 3000.0
PBC 2.0
BOFF 75.0
ASYM 0
DANG 1
ITMAX 10
MODE 1
XSTD 0.0
RBFACT 0
FPART F
FMAG F
FDEF F
FASTIG F
IEWALD 0
dfsig 100
FBEAUT F
FCREF F
res_reconstruction 3.0
res_low_refinement 800.0
res_refinement 3.0
start_process 1
end_process 1
first_particle 1
last_particle 471
increment 10
=========================================================================
Note：When modifying the “TYGRESS_input.txt” file, please use the full path name to the directories for 'fnDataDir', 'fncode', and 'fnref', and only use the relative file folder names for the other parameters when needed.
2.1.2 The file named “DataList.txt” in the TYGRESS source code folder (as shown below in the example data set) has three columns containing the “data set main name” + “data set sub name” + “total .mod number”, which you should change as needed.
Example of a “DataList.txt” file
=========================================================================
WT_20120914 S1 9
WT_20120914 S2 9
WT_20120914 S3 9
WT_20120914 S4 9
WT_20120914 S5 9
=========================================================================
2.2 Modify the “ctffind.csh” file in the TYGRESS source code folder, which will be used to determine CTF. Please see the example in the “ctffind.csh” file for details. You may at least need to change the path of the “ctffind3.exe” file. For more details about how to modify this file, please visit (http://grigoriefflab.janelia.org/sites/default/files/readme_ctf.txt).
2.3 Modify the path of your FREALIGN installation in the “mreconstruct_noMask.com” file. Please see the example “mreconstruct_noMask.com” file for details.
3: Run TYGRESS
Open the Matlab interface, and set the TYGRESS source code folder as the work path. Then, in Matlab, open the TYGRESS_GUI (Figure 1). Click the “Tygress parameter file” button, input the “TYGRESS_input.txt” file, click the “Data list” button, and input the “DataList.txt” file. Click “Yes” on the corresponding step to have TYGRESS run that step (Figure 1). All steps should be run in order if it is your first time processing your TYGRESS data. (Note: You can process the steps one by one, or you can run all selected steps together.)
Final 3D reconstruction is performed with FREALIGN, and all output is saved under the “Reconstruction” folder. For more information about FREALIGN, please visit (http://grigoriefflab.janelia.org/frealign).
4: Directory tree of TYGRESS (Figure 2)
Please note:
· .mod(1) and .csv(1) are the parameters after running PEET for individual tilt series
· .mod(2) and .csv(2) are the parameters for running PEET for the entire data, and .csv(3) is the output
· .csv(3) and mod(2) are used for TYGRESS alignment and to generate the .par file
· .mrc(1) is the 2D particle data set and .mrc(2) is the final output for the 3D structure
· .txt(1) is the parameter for defocus and .txt(2) coordinates parameters of each particle
· .m is the code for running TYGRESS
· Project data folder contains all tilt series data; each tilt series is processed (alignment and reconstruction in IMOD) in its own particles folder, including particles_Predict and Iter folders (alignment results from individual PEET runs).
· Combined_PEET folder includes combined alignment for all 3D subtomogram particles in PEET (Iter1_particles); Combined_mod_csv contains the file for running PEET for the entire dataset; 2D HD particles are picked in each data set under the HD_particles folder. The defocus detection for HD images is put in the ctffind folder.
· TYGRESS code folder stores all codes needed for running TYGRESS. The ‘m’extension indicates matlab scripts, which can be only executed in Matlab. The structure is output as a .mrc file.
· Reconstruction folder stores all output results after reconstruction with FREALIGN.