The overall goal of this procedure is to provide a method to obtain a three-dimensional structure of heli assembled molecules using cryo-electron microscopy. The protocol begins with cryo EM specimen preparation using a rapid dilution and backside blotting method to transiently reduce the salt concentration when preparing the frozen hydrated EM grid. This process ensures protein stability while reducing background noise and improving the signal to noise ratio during the cryo EM low dose data collection that follows subsequent to data collection.
Helical indexing of diffraction patterns is completed. Image processing is then performed, followed by real space 3D reconstruction, resulting in the final density map of the HIV one capsid or ca tube. Generally, individual new to this method will struggle because there are several critical points for optimal data collection and analysis.
For example, in the case of HIV one capsid tubular assemblies that only form in one molar source solution rapidly reducing salt concentration without affecting the structure. It's critical to obtain high quality cryo EM images. Given a tube image, it is very difficult to learn what to do to index a critical tube and which software could be considered to do 3D reconstruction.
This video provides a detailed and straightforward approach to obtain a 3D reconstruction of physical objects and could be used as a reference for your experiment. I will be demonstrating the procedure with Dr.Puja. We are both postdocs from Dr.Laboratory To prepare the HIV one capsid protein assemblies for cryo em begin by glow, discharging the carbon side of 200 mesh copper grids under 25 milliamps for 25 seconds.
Use a nebulizer to bring the humidity to 80%in the environmental chamber, which is a homemade manual gravity plunger. Meanwhile, cool liquid ethane in a vitro bott plunger doer with liquid nitrogen mount the plunge freezing doer onto the manual gravity plunger. Next, use forceps to clamp the pincers closed onto the grid edge.
Apply 2.5 microliters of a preassembled protein solution onto the carbon side of the grid. Then load the forceps onto the plunger with the carbon side of the grid facing away. Add three microliters of low salt dilution buffer to the backside of the grid.
Immediately blot the grid with a piece of filter paper on the same side. Keep the whole back surface of the grid in close contact with the filter paper for approximately six seconds. After removing the filter paper, immediately plunge the grid into liquid ethane.
Finally, remove the forceps from the plunger and quickly transfer the grid into a grid storage box. Begin cryo-electron microscopy by loading the frozen hydrated grid into an FEI polar G two electron microscope. Operating at 200 kilovolts and equipped with a gatin 4K by 4K CCD camera.
Under low dose search mode, add a magnification of about 200 x. Screen the whole grid for areas with suitable ice. Save the positions of these areas in a stage file.
Recall the saved positions and further screen these areas. Add a magnification of 3, 900 x in low dose search mode. Select the areas with uniform thin layer ice for data collection, which are characterized by containing long tubes suspended over a hole.
Save all the areas in a second stage file. Switch to exposure mode. Add a magnification of 59, 000 x and inset a 100 micrometer objective aperture.
Then adjust the objective stigmatism and beam intensity for a dose of approximately 15 electrons per angstrom squared per exposure. Return to the low dose search mode and move to a saved position. Identify and center a good tube.
Using the CCD camera switch to the focus mode, adjust the focus and set a defocus value normally between 0.5 and 2.5 micrometers. Then switch to the exposure mode and set an exposure time of 0.3 to 0.5 seconds for a dose of 15 electrons per angstrom squared. Collect the images on a plate camera, allowing the films to settle for 10 seconds before an exposure is taken.
Following collection of images, move to the next saved position and repeat this process to collect more images. Develop the films in full strength D 19 for 12 minutes. Then digitize the images using a Nikon Super cool scan 9, 000 ED scanner at a pixel size of 6.35 micrometers.
A helical object can be indexed by two parameters. The Bessel order N and layer line number L in the forer transform of a helical object's surface lattice. Each layer line is characterized by N and L and corresponds to a set of lines on the surface lattice as denoted by H and K indices.
To begin helical indexing, use the Iman's program Helix boxer to box out a relatively long and straight tube with uniform diameter and save the image in MRC format. Then measure the radius of the tube using Iman's program boxer. Determine the HEL repeat distance using a cross correlation based program such as I-M-G-C-C-F in the MRC package.
Then calculate the foyer. Transform with a new box length that is an integral of the repeat distance. Next, choose two principle layer lines that define two basic surface lattice vectors one zero and zero one in units of FFT pixel.
Determine the layer line numbers as well as the radii of maximum amplitude for the two principle layer lines in the Fourier transform. Given the layer line numbers and the values of Bessel orders for the two principle layer lines, the rotation between subunits and the axial rise of the one star helix can be obtained using the selection rule as described in the written procedure. These two real numbers describe the screw symmetry of the tube.
3D reconstruction begins with particle segmentation using the Iman program.Boxer. After opening a micrograph containing helic particles, cut a particle into overlapping segments in the control panel of boxer. Choose helix mode and set the parameters for boxing.
The size of the box should be larger than the diameter of the particle and the value for OLA should be about 90%of the box size. After left, clicking on either end of the particle boxer will automatically generate a series of particle boxes along the helix length, save the boxed segments as well as their coordinates. The next step is to perform initial 3D reconstruction using the iterative helical real space reconstruction method I-H-R-S-R prior to processing with I-H-R-S-R.
Invert the contrast of the cryo-EM images and apply LOWPASS filtering. Then open the graphical interface of the I-H-R-S-R program by typing generator. Provide the graphical interface with all the information for the boxed particles stack, including the name and path of the stack, number of images in the stack and the values for symmetry parameters.
Click the finish button to create the reconstruction script. B 25 SP.I use a solid or hollow cylinder as an initial reference and allow the procedure to cycle until there are no changes in the defined screw symmetry, which usually occurs after a few cycles. A right helic symmetry should give a stably converged reconstruction.
The reconstruction generated in the last cycle will be used as an initial reference for further refinement. Finally, perform 3D reconstruction with iterative refinement using the 3D density map generated by I-H-R-S-R as an initial reference for additional refinement during the refinement. The helical symmetry is fixed at the rotation between subunits and axial rise, which are determined from the I-H-R-S-R procedure.
Next, determine the defocus and astigmatism present in the micrograph using programs CTF find three and CTF tilt using spider programs termed FT and mu multiply particle segments by the contrast transfer function. Abbreviated CTF perform projection matching by comparing projections of the reference volumes with the CTF corrected images using multi reference alignment. The variation in the out of plain tilt angle is limited to plus or minus 10 degrees and sampled in one degree steps introduce constraints such as high correlation coefficients in plain angles near zero degrees or 180 degrees and limited X shifts.
For the alignment parameters of each segment include in the reconstruction only those segments that satisfy the constraints. After each iterative refinement cycle, A 3D reconstruction is generated using back projection and divided by some over the CTF squared impose the helical symmetry to generate a symmetries volume. The iterative refinement is terminated when no further improvement in the resolution of the new 3D reconstruction occurs.
A single HIV one capsid assembly, a 92 e tube, was boxed out and its Fourier transform is calculated for helic indexing the two radius, the layer line numbers and the radii of maximum amplitude for layer lines one zero and zero one were determined. End values of negative 12 and 11 were then calculated for one zero and zero one respectively with a repeat distance of 5195.48 angstroms. The screw symmetry of the tube was determined as delta Z equals 6.81 angstroms and delta five equals 328.88 degrees delta Z and delta five were refined to 7.13 angstroms and 328.86 degrees using I-H-R-S-R and an initial reconstruction was generated after 10 iterative cycles.
The final reconstruction after iterative refinement improved the density map significantly from the initial model calculated with I-H-R-S-R. The density map of capsid assembly tubes is displayed as three orthogonal slices parallel to the tube axis and close to the surface, perpendicular to the tube axis and parallel to and through the tube axis. The displayed structure results from surface rendering of the 3D density map contoured at 1.8 Sigma enclosing 100%volume.
After watching this video, you should have a very good understanding of how to obtain a 3D structure of ally assembled molecule using crow electron microscopy.
