Amyloid and the Cross-Beta Architecture

Our research uses X-ray fiber diffraction to investigate the cross-beta amyloid fibril structure and its molecular organization. This protocol uses X-ray fiber diffraction to confirm cross-beta patterns and elucidate fibril architectures. This protocol enables structural analysis of amyloid fibrils from confirming cross-beta organization to elucidating further molecular details.

To begin, align the protein or peptide fibrils to produce a bundle whose fiber axes are ideally all parallel. Melt candle wax with a hot plate until it liquefies. Using a glass cutter, cut 1 millimeter borosilicate glass capillaries to a length of about 2-3 centimeters.

Dip the cut capillaries briefly into the liquid wax. Then draw the candle wax up by capillary action to approximately 1-2 millimeters within the capillary. Now use plasticine to attach the sealed capillaries horizontally inside a Petri dish.

Maintain a gap of 0.5-2 millimeters between the wax-filled ends of the capillaries. With a pipette, carefully suspend a 10-microliter droplet of fibril solution between the wax-filled ends of the two capillaries. Cover the Petri dish to protect the sample from dust and allow to evaporate overnight.

Then seal the plate to help maintain a controlled drying environment. To align the amyloid fibrils in a glass capillary, fix a 1-milliliter syringe with rubber tubing to the wide end of a 0.7-millimeter diameter siliconized glass capillary. Aspirate 2-3 centimeters of fibril suspension into the capillary.

Seal one end of the capillary using melted wax and leave the other end open to allow evaporation. Arrange the capillary vertically and allow it to dry until a dried disk is formed. To align fibrils as a mat or thin film, place a high concentration fibril sample onto a glass slide and cover before air drying for 24 hours.

Carefully mount the fibril bundle stock on a goniometer head and orient it so that the X-ray beam passes through the bundle. If using a fibril mat sample, orient it so that the X-ray beam passes through the edge of the mat. Examine the sample under a light microscope equipped with a cross polarizer to observe birefringence.

Position the sample in the X-ray beam based on whether it is a fiber bundle, mat or disk. Collect a diffraction pattern using in-house X-ray equipment or synchrotron radiation. To perform data analysis, upload the file into the CLEARER diffraction pattern analysis software.

Navigate to Diffraction Pattern and Diffraction Setting to input diffraction settings, including pixel size, X-ray source and sample to detector distance. Click on Image Processing and Rotate to rotate the diffraction image so that the meridian is vertical. Use the centering module by navigating to Diffraction Pattern and clicking on Center Image to center the image.

Define the meridian and equator axes. Save the centered image for subsequent analysis. Next, specify an appropriate angle along the meridian and equator.

Click Diffraction Pattern and then Radially Average module to generate a graph of diffraction signal position. versus intensity. Navigate to the Peak Find module from the Diffraction Pattern menu to measure and automatically output the diffraction signal.

Adjust the peak search width to detect true signals without noise. Use the zoom and measure option to verify diffraction signal position's output using peak find. Explore potential unit cell dimensions using the Unit Cell Optimization module.

Input initial estimates based on strong equatorial signals and define the minimum and maximum indices for cell search. Calculate potential unit cell dimensions and compare the predicted diffraction signal positions with observed reflections. Select the unit cell that best fits the data and the sample.

To construct molecular models, build 3D arrangements of the peptides in a beta-sheet configuration that fits the determined unit cell. Ensure all symmetry-related components are included. Next, click on Diffraction Simulation followed by Structures and Structure Chain Generator and load the PDB model.

Assign the fiber axis and beam axis, input unit cell dimensions and then check for steric clashes. Navigate to Diffraction Simulation and click Fiber Diffraction Simulation to upload the PDB coordinates into the Fiber Diffraction Simulation module. Input the fiber axis direction, beam orientation, unit cell dimensions, X-ray source and detector settings, then click Simulate to run the simulation.

Ensure the beam axis is perpendicular to the fiber axis. Then navigate to File and click on Save As to save the simulation settings as html files for later editing. Save the simulated diffraction pattern in RAW format or as a TIFF file.

Compare the simulated pattern with the experimental diffraction pattern. Measure simulated diffraction signals using the same analysis workflow. Record and compare these values with experimental data.

Electron micrographs showed a high density of amyloid fibrils on the grid suitable for preparation of an X-ray fiber sample. Stretch frame aligned fibers produced a characteristic cross-beta diffraction pattern with a meridianal reflection at 4.76 angstroms and equatorial reflections arising from chain length, sheet spacing and protofilament size. An idealized cross beta diffraction pattern demonstrated that meridianal and equatorial reflections correspond to repetitive spacings along the fiber axes.

A well-centered diffraction pattern was symmetric with equatorial and meridianal peaks overlapping in position. Diffraction peaks were automatically detected and listed using peak finding analysis. The detected diffraction peaks were used to identify plausible unit cell dimensions.

A structural model was prepared using the identified unit cell and visualized prior to diffraction simulation. Simulated diffraction data generated from the proposed structural model showed reasonable agreement with the experimental diffraction pattern in both reflection positions and relative intensities. Overlaying simulated and experimental diffraction patterns further demonstrated alignment of corresponding reflections.

Proper sample alignment is critical because it determines the quality of the diffraction data and the information obtainable from it. This work enables atomic resolution structural modeling and integration with other structural techniques, such as AFM, to enable detailed molecular structures.