Microscope Schedule
Logbook
NYSBC Intranet
NYSBC Contacts
Cryo-EM home

NYSBC home
NMR Facility
X-Ray Facility
Protein Production Facility
Electron Crystallography Center

Cryo-EM Site map

Principles & Protocols

Helical Reconstruction using EMIP

This protocol uses software that originated at the Medical Research Council (DeRosier and Moore, 1970 J. Mol. Biol. 52:355), which was further developed by Toyoshima & Unwin for theacetyl choline receptor. A protocol for unbending tubes was later developed by Beroukhim and Unwin (Ultramicroscopy 1997 70:57-81) for higher resolution reconstructions. Many scripts and minor improvements have been implemented by Bill Rice and the graphical interface called EMIP (EM Image Processor) has been developed by David Stokes.

Alternative image processing suites are provided by MRC (image2000) http://www2.mrc-lmb.cam.ac.uk/image2000.html and Scripps (phoelix) http://ami.scripps.edu/software/phoelix/

Another alternative is the real-space method known as Iterative Helical Real Space Reconstruction (IHRSR) developed by Ed Egelman (Ultramicroscopy 2000 85:225-234).

Tube Imaging and Selection

Images of frozen-hydrated tubes suspended over holes should be recorded on film at 1-2 um defocus at 50,000x magnification. To obtain the best results, the carbon film should be baked prior to freezing, which increases its conductivity and strength. Furthermore, the hole should be centered in the electron beam such that carbon film is illuminated at the periphery of the field. This procedure tends to reduce specimen-induced movement (charging?), which is prevalent when unsupported ice is imaged.

Very straight tubes of uniform diameter should be chosen for analysis. Other packages use a spline fitting algorithm for straightening curvy filaments. This approach has not been tested with the current software, but might be ok for low resolution work.

An optical bench is very useful for evaluating defocus, drift and order of the tubes; the presence of carbon film within the image is helpful because it generally produces strong Thon rings from which these parameters can be assessed. A rectangular mask should be used to evaluate diffraction from the tubes themselves. Sharp layers lines that are symmetric about the meridian are sometimes a better predictor than the presence of high resolution reflections.

Good areas should be marked and the very best tubes should be scanned at 7-14 um depending on expected resolution (14 um should be sufficient for 7 A resolution). The long axis of the tube should be carefully aligned with one axis of the scanner (preferably the y-axis). 512 pixels is often sufficient to include the entire width of the tube, whereas the length of the tube can be very large (e.g. 3000 pixels). Selection of the tube orientation depends on the scanner. For very old flat-bed scanners, the long axis was oriented parallel to the scan direction (typically x). The tube was later rotated using a program such as axchgmrc (label has a limit on the dimension). For CCD-based scanners that use "swatch", the long axis is again oriented parallel to the scan: y for the Zeiss Photoscan so there is no need to rotate the image for initial stages of processing. However, for refinement, the tube must be rotated 90deg and the corresponding program (axchgmrc) requires a dimension of either 512 or 1024 pixels, so plan accordingly.

EMIP

This is a GUI written in wxPython that simply collects information from the user and runs existing programs from a variety of different software packages. Error messages and certain results are printed on the console. If the program or script required for any particular step does not exist, is not in your path, is incorrectly configured, or is a version that takes different parameters, EMIP will fail and will probably produce some kind of error message. You should regularly check the directory listing to see what files have been created and to verify that the programs have successfully completed.

• create an alias to the following command in your .cshrc file
  • alias lshead 'ls -lt | head'
• then use lshead to check for the last few files that have been created after each step in the process

See EmIP page for more information and screen shots.

Directory structure

EMIP expects a particular (simple) layout of directories. From a directory of your choice, create a series of directories named for the id of each tube (e.g. ./1791, ./1792-2, ./7796). Copy the scanned image into this directory and start EMIP. You may change directories within EMIP using the File menu.

A little later, you will need another directory at this same level for the first averaged structure (first reference for unbending, e.g. ./ref1).

For unbending, a subdirectory will be created (called refine) within each tube directory (e.g. ./1796/refine)

your processing directory	.
indiv tube directories	7774	1921	1926	7771a	7771b	ref1
refinement subdirectories	refine	refine	refine	refine	refine

File Conversions and Image Display

The file should be converted to MRC integer*2 (16-bit integer) format (mode=1 in MRC header). File conversions are handled on the first page of EMIP ("transform"). Keep in mind that the width of the tube should be 512 of 1024 pixels.

Image can be viewed using a number of different programs, all of which have pros and cons. These can be selected from the "transform" page of EMIP. v2 (from EMAN) or imod (Univ. of CO) are all purpose viewers that work with many workstations.
mrcdisp, Ximdisp are MRC viewers that require 8-bit displays.
xdisp and xdispf are from Toyoshima suite and require 8-bit displays. xdispf is good for viewing the Fourier transform of helical tubes (see below).
Easiest way to use 8-bit display is vnc:

• Start server: vncserver -display 8 :10
• Start viewer: vncviewer :10
• Within this virtual desktop, you should be able to run 8-bit display applications

Note that all of these programs can also be run from the command line, independently of EMIP. To do this, you can create a separate 8-bit display. Make a csh command file with the following contents:

#!/bin/csh
# starts an 8-bit display on display 1
#first save current .Xclients setup
cp .Xclients .Xclients.sav
#choose your favorite windows manager
#echo exec /usr/local/bin/fluxbox > $home/.Xclients
echo exec /usr/X11R6/bin/twm > $home/.Xclients
#echo exec /usr/bin/icewm > $home/.Xclients
chmod a+x $home/.Xclients
startx -- :1 -depth 8
#after exiting, restore the original .Xclients file
cp .Xclients.sav .Xclients

Overview of HELICAL1 and HELICAL2 pages

There are two pages for processing helical crystals.

• helical1 is for processing individual tubes.
• helical2 is for averaging tubes together and creating a map.

Note that in addition to tooltips, a "right-click" on many of the buttons will print out a description of the steps taken for that button on the console and will also bring up relevant log files in separate windows for inspection.

You will have to change directories depending on which tube you are processing. This can be done with a pop-up window found under the File menu.

Initial boxing and Fourier transform - FFT, PRJPLT, HFTOUT

On the HELICAL1 page of EMIP, enter the img ID. the "imgID" is used for naming conventions: imgID.fft, imgID.box, imgID.acf, etc. If files exist from a previous session, then most of the fields on this EMIP page will be populated with the corresponding parameters. Otherwise, enter the image filename (e.g. imgID.mrc) and a Title that describes the tube. Enter the pixel size in Angstroms:

• actual dimension of the image pixel (e.g. 15um) / magnification.

Enter parameters for an initial box. This can be done either as xmin/xmax,ymin/ymax or as xcenter/xsize, ycenter/ysize parameter pairs. The center and width can be determined using one of the view programs. To start, a length of 1024 pixels is usually suitable (2500-3000 A). An initial FFT frame size of 1024x2048 is suitable. angle refers to an in-plane rotation of the tube to align its meridional axis with y. Hit FFT to calculate an fft using these parameters.

PRJPLT will produce a 1D plot representing the projection of real-space densities along the meridional axis of the tube. The plot starts from the center of the boxed area and reaches to the outer radius of the tube. The left side of the tube (open squares) is inverted and overlayed with the right side (open circles) of the tube. If the tube is perfectly centered, then the projection from the right side matches that from the left side. The average (filled squares) and the difference (solid line) of the two sides is also plotted. This plot allow one to judge the precise width of the tube and the optimal in-plane rotation angle.

• There is generally a dip in density due to defocus just outside the tube. The radius should be measured at the place where this dip returns to baseline (115 pixels in this example).

• Optimal centering and in-plane rotation generally produces maximal contrast in this projection: i.e. highest peaks and lowest valleys. The two sides match and the difference is therefore minimized.

To view the transform graphically, use xdispf. Right click of the mouse allows you to adjust the contrast of the transform (type new min/max values on the console). q will quit the program. Left click of the mouse allows you to evaluate position and intensity of the layer lines. Recall that you need 8-bit display for xdispf. This can be run as a standalone program on a separate display.

To evaluate phases and location of the layer lines, use HFTOUT. The cutoff and threshhold control the contrast of the output and may need to be adjusted. The output consists of 2 pages: the first represents the amplitudes and the second represents the phases. The equator runs up the right-hand margin of the page. The meridian runs horizontally along the middle of the page.

• Adjust the cutoff/threshhold until the layer lines are clearly visible above the background on the amplitudes page (e.g. '5' and '6' along the layer lines and lesser symbols for the background).

• Examine the amplitudes for the equator and the layer lines. They should run parallel to the x pixels axis (vertical on the page). If not, adjust the angle until the amplitudes follow the scanning raster. The equator should remain along a single pixel. Use the sharpest layer lines that go to the highest radius to evaluate this angle. Also refer to PRJPLT output for finest adjustment.

• On the first page, find the first 2 or three peaks along the equator. On the second page, find the phase at those peaks. There is a letter code for the phases, which is indicated along the top margin of the second page. A=0, B=10, C=20 . . . S=180, T=190 . . . 9=350. These uppercase letters correspond to locations on the first page that have amplitudes of '5' or above. For locations with smaller amplitudes, there is a similar code with lowercase letters: a=0, b=10 . . . s=180, t=190, etc.

• When the tube is centered, the phases along the equator will all be 0 or 180 deg. When off-center, the phases will start gradually get farther and farther from these ideal values. Not the current values, move the center of the box xctr by 1 pixel. Recalculate the FFT and reexamine the corresponding transform using HFTOUT. Thus, adjust the xcenter until the phases at peaks along the entire length of the equator are close to 0 or 180 deg. Your tube is now close to center.

hftout amplitudes
hftout phases

Index the helical symmetry - llindex

This can be the most difficult and frustrating part of helical reconstruction. Please read and understand the nl plot as discussed in the following papers:

The first step is to identify the locations of layerlines from HFTOUT output and to record the following 3 parameters:

• their location along the meridional direction (l)
• the location at which the initial peak in the radial starts to rise (R)
• the relationship of phases under the first peak from either side of the meridian
  • ideally, these phases should either be equal or vary by 180 deg
  • if the tube has out-of-plane tilt, then this phase relationship will be perturbed
  • this perturbation increases with l, so only low lying layer lines should be trusted
  • if the phases are equal, then the Bessel order for that layer line is even
  • if the phases differ by 180deg, then the Bessel order is odd

The next step is to assign "Miller indices" (h,k) to each of the layer lines.

• Pick two strong, low-order layer lines and assign them as 1,0 and 0,1 layer lines respectively.
• Based on this choice, all other layer lines can now be assigned their respective h,k
• All you need to look at are the l values:
  • l(1,1) = l(1,0) + l(0,1)
  • l(2,1) = 2 * l(1,0) + l(0,1)
  • l(1,-1) = l(1,0) - l(0,1)

Now run the program llindex. You will need to guess at the correct indexing scheme by entering trial Bessel orders for the 1,0 and 0,1 layer lines. Then enter the values you have measured for R for each of the layerlines. llindex will calculate the expected radius of the tube in real space from each layer line value according the the following equation:

• 2piRr ~= n+2 ~= 1.1n + 0.9 - where r=real space radius of the tube

If you have selected the correct indexing scheme, then the estimates for r from all the layer lines should be consistent with one another and consistent with the plot from PRJPLT

• keep in mind that out-of-plane tilt will cause high-order layer lines to move closer to the meridian. This is especially marked for layer lines that are close to the meridian (i.e. small Bessel order).

Determine the repeat distance - ACF

The repeat distance along the tube is determined by an auto-correlation function. You need to define a small reference area from the bottom of the tube and a larger "test" area composing the rest of the tube. These two areas cannot overlap. The reference area should be slightly smaller than the width of the tube. The test area should be 38 pixels wider (for clean output). In the corresponding boxes, enter the center of each area followed by the size of each area. The length of the reference area is usually chosen as 140 pixels, but this can be changed depending on the application.

The output consists of a large table of normalized cross-correlation coefficients as the reference area is moved across the test area. The highest value (100) is not necessarily the best repeat distance to use. Start by scanning down the table and recording the location of all peaks >80.

These peaks should coincide with the axis of the tube (i.e., if the tube has an in-plane rotation of 1 deg, then these peaks should reflect that rotation and gradually move towards one side). For each maximum, the repeat distance is calculated by subtracting the value at the left-hand margin (the y-coord for the test image) from the y-center of the reference image. Make a table of peak strength vs. repeat distance. Look for any trends:

• is there a second order or third order peak present
• are there subsidiary peaks

Choose a couple of plausible repeat distances to evaluate. Optimal repeat distance is ~1024 pixels. A very short repeat distance will generate a lot of overlapping layer lines. A very long repeat distance will make you more susceptible to distortions and bending along the tube.

To evaluate a particular repeat distance, alter the y size of the box used for the FFT (yctr/siz) to match the repeat distance. Examine the output of HFTOUT and note the strength of the signal along the layer lines, particular high order layer lines. Try increasing or decreasing the repeat distance by 1, 2, or 3 pixels to look for subtle differences (only necessary for well ordered tubes). Note the location of layer lines and watch out for overlap (i.e. does the -1,1 layer line overlap with the 2,0 layer line). Ultimately, you will choose the repeat distance that gives you the best signal-to-noise ratio along the layer lines and produces minimal layer line overlap.

The choice of repeat distance is somewhat arbitrary. There is no correct repeat distance. There may be many approximate repeat distances that will produce comparable results. Choose the one that is most convenient for subsequent processing and that produces a good signal-to-noise ratio.

Refine center and determine out-of-plane tilt - SRCH

Enter (n,l) for both the 1,0 and 0,1 layerlines. Also, identify the strong (good) layer lines that you want to use for this refinement. These are the layer lines that are visible in the output from hftout. Enter their height (l).

The output of SRCH (on the console) will indicate an out of plane tilt, an x shift (should be less than 1 pixel), and a residual (<30 deg is acceptable). The tilt and the xshift are multiplied by 100. The residual is multiplied by 1000. The following example corresponds to 1.73deg tilt, 0.4 pixels shift, 18.32deg residual:

Final srch residual:
173 40 18320
Actual out-of-plane tilt of the tube,
accounting for scale factor: 0.9 deg

The actual out-of-plane tilt takes into account the anisotropic stretching of the tube along the helical axis and is only for informational purposes.

If the x shift is larger than 1 pixel, change the xctr of the box and calculate a new transform using FFT button.

The details of the SRCH button are as follows. It involves running a number of programs and is derived from hlxsymsearch.pl.

• nltabl creates a table of layer lines based on 1,0 and 0,1 assignments
  • maxn and Rcutoff affect the output of nltabl.
  • Rcutoff should be chosen to coincide with the 1st zero of the CTF (at most)
  • output files are ID.id and imgID.id and imgID.tbl. Feel free to view these important files.
• lnl retrieves specific information about layer lines from imgID.id,
  • only those layer lines matching the good ll specified in the GUI layerlines are used
  • these goodll are stored in a file call imgID.goodll
• hlxs_thresh is run interatively to determine an amplitude threshhold for selecting peaks for refinement
  • hlxs_thresh plots the layer line data and picks off points from the strongest peaks along the selected layer lines
  • the phases from these points are used to refine the xshift and out-of-plane tilt
  • output file is hlxsall.log. Feel free to look at it as it plots the data along the layer lines
  • hlxsall.log can be used to evaluate the starting position of the layer line data for indexing purposes
• srch uses the few points with highest signal-to-noise ratio to determine xshift and out-of-plane tilt (omega)
  • this is run in two steps. 1st with a coarse search then with fine search
  • log files are in srch1.log and srch2.log, respectively
  • the resulting values for xshift and omega are entered automatically into the GUI. They should not normally be changed.

Extract layer lines and calculate statistics for the tube - STATS

The Amplitude threshhold is used to select data included in the statistical evaluation. This threshhold corresponds to the percentage of the maximum amplitude that is considered. Two statistical measures are run:

• near/far phase residual compares phases across the meridian after applying corrections for xshift and out-of-plane tilt. This is the only measure you can use unless you have p2 symmetry. 90 deg corresponds to random data.
• twofold phase residual compares the phases of the near/far average relative to those expected for twofold symmetry (i.e. 0 or 180 deg). 45 deg corresponds to random data.

An example from a moderately ordered tubular crystal (which appears on the console). Note that the two-fold statistics are broken down into resolution ranges. The % data included in each resolution range depends on the amplitude threshhold. Values of phi,z are used to adjust the phase origin to coincide with a two-fold axis. More information can be obtained from log files hlxresidual.log and twofold_imgID.log :

Near/far phase residual:
average residual among 8 layer lines from hlxs: 43.04

Overall twofold residual:
--50.00(A) --40.00(A) --30.00(A) --25.00(A) --20.00(A)
14.46(130) 18.25(126) 18.11(332) 35.72(139) 0.00( 0)
( 94.20%) ( 87.50%) ( 90.96%) ( 75.54%) ( 0.00%)
( 138) ( 144) ( 365) ( 184) ( 0)
18.943 degrees at (phi= 7.932, z=-10.400)

Again, this is a complex of individual programs involving the following

• hlxmk to create a control file for extracting layer lines using info from imgID.id and from results of srch
  • takes data from imgID.tbl to create a control file for hlxfl
• hlxfl to extract layer lines and create imgID.nea and imgID.far files (for near and far layer line data)
  • control file is imgID.hlx. output file is imgID.nea and imgID.far
• nfavg to average near and far sides to create imgID.avg layer line data file
• hlxresidual.com to calculate near/far phase residual (control file is imgID.halfit)
• hlx2fld to calculate two-fold phase residual (control file is twofold_imgID.cnt)

Average data from multiple tubes - create an initial averaged dataset

The next step in processing a tube is unbending. However, in order to detect the distortions of an individual tube, a reference data set is requires. The initial reference data set could potentially come from a single tube and if you choose to do this, then the imgID.avg file created by the STATS button. A better idea is to average data from several tubes together, to produce a reference with better signal-to-noise ratio. To do this, use the second EMIP page entitled helical2.

• create a directory (e.g. ref) to do this averaging. This should be at the same level as the individual tube directories which were names according to their id. If the directory structure is correct, then EMIP will be able to find the files for the individual tubes and move them when necessary to the ref directory.
• move into this directory (under the File menu you can Change Dir)

With this page, the layer line files from all the tubes (imgID.avg) are adjusted to the same phase origin and then averaged. The first step is to designate a reference tube. This should be your best tube (best phase residuals). Enter information for that tube into the first line in the HELICAL2 window of EMIP. If the tube has two-fold symmetry, then the phase origin should be adjusted to coincide with one of the two-fold origins (determined in the previous step by the hlx2fld program run as part of the STATS button. If the tube has p1 symmetry, then the phase origin is arbitrary and does not need to be adjusted.

• enter the id and filename of the reference file. The other fields should be automatically populated if the corresponding files are found
• run ADJUST PHASE ORIGIN to adjust the phase origin of the reference file. This will also place an entry in the Tube list
  • if you have a p1 tube and do not want to change the phase origin, just enter "0 0" in the phase origin phi/z field
  • you must hit the ADJUST PHASE ORIGIN button to enter the reference tube info into the Tube list

Layer line data from each of the other tubes must be "reindexed" to match the indexing of the reference file. Although all the files must have the same helical symmetry (i.e., the same Bessel order for 1,0 and 0,1 layer lines), the height of these layer lines (l) will be variable. Reindexing simply changes the value of l for these layer lines to match the reference. In doing that, the repeat distance is artificially set to be the same as the reference tube also - so it is as if all tubes have exactly the same selection rule. This artificial procedure can produce slight distortions in the individual tubes and may ultimately limit resolution if the underlying unit cell of individual tubes is variable.

• enter the ID and filename of each "test" tube. The n,l values should be automatically entered if the file is found in the expected location (i.e. ../imgID/)
• run AddTube to create imgID.ren file (based on input file: ../imgID/imgID.avg)
  • for this initial dataset, you want to find a common phase origin relative to the reference. To do this, select the fit to ref checkbox.
  • when selected, the fit to ref checkbox will cause HLXFIT to be compare tstID.ren with refID.avg. The corresponding values for phi, z, rscale will be entered in Tube list along with the fitting residual. That residual should be 50-60 deg.
  • If the residual is too high, limit the extent of the layer lines being used (goodll and Rcutoff on helical1 page)
• if two-fold symmetry is present, then you may want to use the phase origin determined by HLX2FLD instead of the one determined by HLXFIT. This is generally recommended. The two phase origins should be similar.
  • To do this, hit 2FLD-TST button, which will report the twofold origin for tstID.ren.
  • you must run HLXFIT prior to HLX2FLD in order to ensure that you use the same two-fold origin as the reference file. Just make sure the Fit to ref checkbox is selected when you hit AddTube
  • If satisfied with the two-fold origin, then manually substitute the corresponding origin (reported on the console) in the appropriate place in Tube list, thus replacing the values initially determined by HLXFIT.

Process all the tubes in this way and checking the data in Tube list for accuracy. This data will be used to create a control file for hlxadd and thus to create a global average. Enter a name for the output file and hit HLXADDALL button.

• To check the two fold statistics for the average file, hit the 2FLD-AVG button. The statistics are reported on the console and should indicate a substantial improvement in resolution relative to the individual tubes.
• The control file is twofold_xxx.cnt. You may edit this to extend the resolution range at the end of this file. Log file is twofold.log.

CTF determination

PLTCTFX

• this first runs sctoravgft using the Sectors to Calc parameter to determine the number of sectors in which to divide up the FFT
  • sctravgft creates _imgID.ctf
• enter the relevant information about kV, Cs, fraction of amplitude contrast (see Toyoshima papers on this topic: 1988 Ultramicroscopy 25:279 and 1993 Ultramicroscopy 48:165).
• sample size corresponds to the pixel size and magnification should be calibrated for your microscope
• min/max defocus helps constrain PLTCTFX to get the correct solution.
• PLTCTFX requires a definition file containing the above EM parameters (pltctf.def). Uncheck the box if you wish EMIP to create this file.
• You may be able to get a result by simply hitting NEXT for the several windows presented by PLTCTFX. However, better results may obtained by selecting data along each of the sectors. Do this by clicking the start position along the axis followed by Start R. Then click the ending position along the axis followed by End R. Do this sequentially for each sector. Subsequent windows will indicate the region of the layer line that has been selected.
• The result is obtained after running CTFFIT. If you don't like the result, just try again.
  • PLTCTFX creates a postscript record of the process if you choose HARCOPY (ctf.ps). Also, CTFFIT creates a control file imgIDctf.cnt and a log file ctffit.log.

CTFFIND3

• CTFFIND3 divides the image up into tiles. Enter the size of those tiles
• Res Min and Res Max correspond to high pass and low pass filters respectively
• Step Size is for fitting the ctf to the resulting power spectrum. Smaller steps should provide more accurate fitting
• hit View Output to see result graphically and printed out on the console. Check log file for more details.

• Return to the helical1 page.
• change directory to a given tube (imgID)
• enter the imgID into the first field - the data in all the other fields should be repopulated

Prepare directory for unbending

Hit UNBENDPREP button to create the refine directory and copy/create all the relevant files.

• will run axchgmrc to rotate the tube 90 deg. If the tube dimension is not 512 or 1024, this program will fail. You can either trim/pad the image using label from the command line.
• will reindex the reference tube to match the tube to be unbent
• will make a file for ctfplot containing the defocus parameters
• will use box2box.com to create a box file with various tube alignment parameters
• runs setup_hlxdat.pl to finish setup of the refine directory

UNBEND the tube

• this step is a very complicated series of steps described in the paper by Beroukhim and Unwin (Ultramicroscopy 1997 70:57-81) and in the document http://cryoem.nysbc.org/tubenotes.txt
• the process is controlled by a csh script called hlx_unbend.com and creates a log file hlx_unbend.log, results files __*.rr and imgIDa.curves
• flowchart describing the unbending process: unwin-refine-outline.pdf

Correct CTF for the unbent tube CTFPLOT, NFADD

• CTFPLOT actually multiplies the amplitides by the CTF and inverts the phases in the first, third, fifth maxima.
• CTFPLOT also adds a column of values corresponding to the CTF² at each position along the layer line
• output files are prepended with "ctf". e.g. ctfimgIDac300.nea, ctfimgIDac300.far
• control file is ctfplot.cnt, log file is ctfplot.log and a series of plot files ctfplot?.plt which are generally ignored

Next, hit NFADD to average near and far data sets. This will create ctfimgIDac300.avg.

• log file is nfadd.log

Average unbent data from several tubes

• enter the ref file ID and filename
• change the phase origin phi/z to "0 0"
• hit Adjust Ref Origin to add this reference file to the Tube list
• although hlxfit is run, it doesn't actually create anything useful at this stage. The main purpose of this step is to add the entry to Tube list

• enter the first tst file ID and filename
• uncheck the fit to ref checkbox and hit AddTube
• change the phi,z,rscale values in the Tube list to 0 0 1
• this will run hreindex to alter the indexing and create the file *.ren and then add an entry to Tube list
  • log file is hreindex imgID.log

When finished adding all the tubes to the Tube list, enter filename for avg and hit HLXADDALL

• HLXADDALL uses hlxadd to sum the amplitudes, perform amplitude-weighted averaging of the phases, and sum up the CTF² values at each radial position along the layer lines
• control file is hlxadd.all, log file is hlxadd_all.log

You may check the two fold statistics of the resulting, averaged file using 2FLD-AVG button

• as before, the control file for the hlx2fld program is twofold_avgfile.cnt and the log file is twofold.log
• this twofold is done with amplitude threshhold of 0.1. Edit the control file if you want to change this parameter

If you wish to do another round of unbending, then you may repeat the entire process with this new average file (not twofold enforced)

• two rounds of unbending are generally sufficient as judged by a lack of further improvement in resolution as judged by two-fold statistics

CTF correction and map calculation, DIVCTF, HLXFB, HXSEC

To correct the CTF, the summed amplitudes are divided by the sum of the CTF². This is done by DIVCTF button. One potential problem is that CTF² can be close to zero due to the oscillatory behavior of the CTF. This is particularly problematic as the equator approaches the origin, where the value of the CTF becomes vanishingly small. This may also be the case at the nodes of the CTF, but by averaging several tubes together, the fact that they have different defocus values tends to fill in the nodes. At the origin, the CTF always goes to zero.

• ctf min is a parameter that restricts the minimum CTF² that is used to correct the amplitudes. Actually, the values stored in the averaged layer line file (bigG file) represent the sum of the CTF² from all the tubes. So if you add together 10 tubes, the max value of CTF² is 10 and 0.3 actually corresponds to 0.03 for an individual tube. Choose a value that makes you comfortable. Remember that where the CTF is very low, the signal-to-noise ratio is also large and you do not want to allow noisy data to dominate your reconstruction.
• equator corr is a parameter that controls the CTF correction along the equator. The equator is important because it determines the mean radial density distribution of the reconstruction. Values along the equator that are closer to the origin than equator corr are given full CTF correction, regardless of the value of ctf min. This is problematic if there are values of 0.000 for CTF² in the layer line file. In that case, you get INF in the CTF corrected file and it cannot be used in subsequent steps. The file is ascii and you can examine it using more. You only need to look at the first screen full of data, which corresponds to the region along the equator that is close to the origin.
• The program is divctfbeq, control file is divctf.com and log file is divctf.log

If desired, run HLX2FLD button to create a two-fold constrained file for your data. You can also look at the two-fold statistics, which will be somewhat different than for HLXADDALL output file due to amplitude weighting that is affected by CTF correction.

• uses hlx2fld program, twofold_limfile.cnt for control file and twofold.log for logfile

Use HLXFB button to calculate little g's.

• r limits are the min/max radius of little g in real space as well as their sampling interval. These

• output filename is same as input filename but with extension .lgS, where S is the sampling interval
• program is hlxfb, control file is hlxfb.cnt, logfile is hlxfb.log

Use HXSEC to calculate the 3D map. This map consists of sections normal to the helical axis and tangential to the wall of the tube, i.e. longitudinal sections. Other programs exist for cutting cross-sections (hpsec) and cylindrical sections (hclnd), but you have to figure out how to use those yourself.

• xlim, philim, zlim control the extent of the map.
• scale controls the density scale. For helical

• output filename is same as input filename, but with extension .xS, where S is the sampling interval for HLXFB
• The resulting map can be viewed by chimera (see below) or any other program that can read MRC format files (e.g. IMOD).
• program is hx, control file is hxsec.cnt.

Use IMGPLT to create a contour plot from the 3D map. This produces an output file (imgplt.ps).

• If the scale is too big, the map becomes

• intervals should be 4 numbers (e.g., 20 40 60 10):
  • 1st three numbers are the density threshhold for dotted, solid, bold contours.
  • 4th number is the interval between contours.
  • ticks checkbox controls whether the map has vernier scale drawn around it. This feature increases the file