The overall goal of this procedure is to show how to define the structure of factor VIII heli organized on lipid nanotubes by cryo-electron microscopy. This is accomplished by first organizing helic purified factor VIII on lipid nanotubes and collecting cryo-electron microscopy data at liquid nitrogen temperature and low electron doses. Next, the factor VIII membrane bound structure is calculated by combining single particles and helical 3D reconstruction algorithms.
Then electron tomography data is collected and the 3D reconstruction is generated. Ultimately, the calculated factor VIII lipid nano tube structures are validated by segmenting the 3D volumes and comparing them to the tomography reconstructions. The demonstrated method can help answer key question in the field of structural biology and blood coagulation, such as what is the membrane bound organization of factor VIII and how it relates to its function in coagulation.
The GM 2100 lab six is equipped with a TE con operating system consisting of a computer connected to the electron microscope. A screen with windows reading the vacuum system, illumination system, HT lens currents and two panels left and right placed on either side of the column, the beam shift, XY knobs and the multifunction or deaf stick knobs are on both panels. On the left panel is the illumination or brightness knob.
The right panel houses the magnification and focus knobs and the three buttons for imaging mag one, mag two and low mag, and one for diffraction mode. For this protocol, acquire data in mag one at alpha two using generic settings. Set the minimum dose illumination conditions or MDS required for cryo EM data acquisition with the F1 through F six buttons located on the top row of the right panel.
Use F1 to raise or lower the screen F two for search mode and F three to focus. Choose F four for photo mode F five to switch MDS off and on, and F six for beam blank, which is used to shield the specimen from radiation damage by deflecting the beam. After preparing cryo EM samples, according to the text protocol, place the cryo holder into the cryo station and with liquid nitrogen, fill the doer of the holder and the cryo station.
When the temperature reaches negative 192 degrees celsius, open the shutter on the holder tip, place the previously frozen cryo EM grid into the designated place and use a ring clamp to secure it. Next, insert the cryo holder into the electron microscope and use liquid nitrogen to refill the doer of the cryo holder and the anti ator chamber. After the holder has stabilized for 30 to 60 minutes, turn on the filament.
When the filament is saturated, press F six off and open the shutter on the holder. To view the grid, press low mag alpha one and locate areas on the grid with thin ice. Then switch to mag one alpha two to set minimum dose or MDS mode and acquire cryo EM data at low electron doses so as not to damage the specimen.
Choose F two to set search mode and set the magnification at 40, 000 x. Use the brightness knob to enlarge the beam to the minimal electron dose of about 0.04 electrons per angstrom squared times seconds. Then press diff to switch to diffraction mode with the mag cam length knob.
Set the camera length to 120 centimeters. Then locate areas on the grid that have lipid nanotubes in the holes of the carbon film within the preselected areas. Next, press F four to set photo mode at 40, 000 x magnification and use the brightness knob to set the illumination at doses of 16 to 25 electrons per angstrom square times seconds.
Then press the standard focus button to set the focus conditions, adjusting the Z height of the specimen with the Z up down buttons Set defocus between negative 1.5 and negative 2.5 micrometers. Align search and photo mode by drawing a square in search mode in the live view window on the digital camera monitor corresponding to the area to be imaged in photo mode, press F three to set the focus mode at 100, 000 x magnification. Then focus the illumination to cover the CCD chip and off axis to not irradiate the area to be imaged.
In photo mode, adjust the defocus between negative 1.5 and negative 2.5 micrometers correct for astigmatism of the image if needed. In search mode, select the F eight L and T to be imaged by acquiring live images in the live view window on the digital camera monitor center, the F eight L and T in the square drawn on the live view window. Switch to photo mode and click the acquire button on the digital micrograph camera monitor.
To record a digital image on the CCD camera, press CTRL F to obtain a fast Fourier transform to check the quality and the defocus of the acquired image. After using the free scientific imaging processing softwares Eman two and I-H-R-S-R to perform 2D image analysis and helical reconstruction according to the text protocol. To visualize and segment the F eight L and T volumes open the final 3D reconstructions in UCSF cera select tools, volume data segment map, and in the segment map tab, select volume and click segment to segment the volume data.
Then press control shift and click group to group segments corresponding to one unit cell. To emphasize the structural features, choose actions color to color the segments by unit cell and helix. To collect the electron tomography data, the negatively stained electron microscopy grid is transferred into a single tilt holder before transferring the holder into the electron microscope tilt.
Series of ally organized porcine factor VIII on lipid nanotubes were collected with the serial EM software. According to the text protocol, the Tom was reconstructed in IM and visualized in 3D mod to decrease the size of the to use imad bin Val tobin it by four, then use the IM mod rotate vol command to select the proper angle to rotate the lipid nano tube along the Y axis and rotate the full tommo with the IM mod clip resize command crop, the selected lipid nano tube along the Y axis use 3D mod to open the cropped sub tommo. Finally click on the Tommo Graham to visualize the arrangement of factor VII molecules along the slices in the Z axis and along the Y axis in the sub tomo volume.
As shown here, recombinant human and porcine F eights were successfully organized hally on negatively charged single bilayer lts resembling the activated platelet surface. This figure shows that the helical order of the membrane bound human and porcine F eights and the lipid bilayer in the best control LNT and F eight LNT 2D class averages are well-defined and reveals the variations in the helical organization between the two proteins. These panels represent the initial 3D reconstructions for the human and porcine F eight L and T that were carried out with 1000 representative particles from the final human and porcine F eight L and T data sets here, the foer shell correlation plots for human and porcine F eight LNT indicate a resolution of 20.5 angstroms at a Fourier shell correlation equal to 0.5 in this figure.
The final volumes show eight human F eight and 10 porcine F eight membrane bound molecules organized around the helical axis. Each human F eight molecule is translated 41.2 angstroms and rotated 42.0 degrees from the previous one, and each porcine F eight molecule is translated 35.9 angstroms and rotated 35.2 degrees from the previous molecule corresponding to the helical parameters of the final 3D reconstructions. After watching this video, you should have a good understanding of how to acquire cryo-electron microscopy data of membrane brown protein ally organized on lipid nanotubes, and calculate a 3D structure close to physiological condition.
This video, it's a first step toward a high resolution membrane-bound structure of a clotting protein, which can be applied to other proteins which function in a lipid environment.
