Name: Amyloid and the Cross-Beta Architecture (Video) | JoVE Journal
Uploaded: 2026-02-13T13:15:00.000+00:00
Duration: 9 min 6 s
Description: Learn X-ray fiber diffraction methods to determine amyloid fibril architecture and cross-beta organization in structural biology. This protocol enables detailed molecular modeling of protein misfolding structures.

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5 minutes

The authors would like to acknowledge the help of Dr Pawel Sikorski and Professor Edward Atkins with the development of the CLEARER programme and analysis of patterns. The programme was written by Dr O Sumner Makin. This work has been supported by funding from the Alzheimer&#39;s Research Trust and the BBSRC.

1. Preparation of fibrillar samples for X-ray fiber diffraction
<ol>
	<li>Dissolve protein or peptide of interest in optimal conditions for fibril formation for the selected amyloidogenic peptide and assess fibril formation using electron microscopy or atomic force microscopy. Details of how to prepare samples and conduct imaging are available elsewhere13. Ensure that the fibrils are long, straight, and unbranched as shown in Figure 1. For example, the fibrils shown in Figure 1 were imaged using transmission electron microscopy (TEM). Place a droplet containing fibrils on an electron microscope grid and then negatively stain using uranyl acetate14. 
	NOTE: An even covering and distribution of the fibrils on the grid is desirable.</li>
	<li>Align to produce a bundle of fibrils whose fiber axes are ideally all parallel (Figure 2A).
	<ol>
		<li>Melt candle wax until it is liquid using a hot plate. Using a glass cutter such as a diamond pen, cut 1 mm borosilicate glass capillaries to a length of about 2-3 cm and dip them into the liquid wax briefly. Draw the candle wax up by capillary action (approximately 1-2 mm in length within the capillary). Ensure the wax surface is flat so that the fibril suspension can rest evenly.</li>
		<li>Use plasticine to attach the sealed capillaries horizontally in a Petri dish with a 0.5 mm to 2 mm gap between the wax-filled ends (Figure 2A).</li>
		<li>Using a pipette, carefully suspend a 10 &#181;L droplet of solution between the wax-filled ends of the two capillaries. Ensure that the solution makes contact with both wax ends but does not extend onto the glass.</li>
		<li>Protect the sample from dust by covering the Petri dish. Allow the sample to evaporate overnight, resulting in alignment of the fibrils (see Figure 2A). 
		NOTE: It may take several days for the sample to dry fully, and this will depend on the ambient temperature and humidity, and the viscosity of the sample.</li>
		<li>Seal the plate; incubate at lower temperatures in a refrigerator or incubate using humidity in a sealed box to slow down drying, which may result in better alignment of the sample.</li>
	</ol>
	</li>
	<li>Align amyloid fibrils in a glass capillary (Figure 2B).
	<ol>
		<li>Fix a 1 mL syringe fitted with a small piece of rubber tubing to the wide end to a 0.7 mm diameter, siliconized glass capillary and aspirate up 2-3 cm of fibril suspension.</li>
		<li>Seal one end of the capillary using melted wax and allow the other end to remain open to allow evaporation. Arrange the capillary vertically and allow it to dry over several days or weeks until it has formed a dried disk in the capillary tube (Figure 2B).</li>
	</ol>
	</li>
	<li>Align fibrils as a &#34;mat&#34; or thin film (&#8203;&#8203;Figure 2C).
	<ol>
		<li>Place a high concentration (e.g.,&#62;10 mg/mL) of fibril sample onto a glass slide, parafilm, or piece of Teflon, cover, and allow it to air dry for at least 24 h. This should dry to form a thin, flat film on the surface. Carefully lift this using a scalpel and then attach it to a glass capillary tube using glue or plasticine.</li>
		<li>The mat will have a random arrangement of fibrils (see Figure 2C). Carefully mount the sample on a goniometer head so that the X-ray beam passes through the edge of the sample and parallel to the plane of the film (Figure 2C). 
		NOTE: Diffraction through the face of the film will yield diffraction rings due to random alignment (see Figure 2C).</li>
	</ol>
	</li>
	<li>Examine the sample.
	<ol>
		<li>Investigate how well the sample is aligned by examining it through a cross-polariser under a light microscope. If birefringence is present, it may suggest that there is good alignment in the fibril bundle.</li>
		<li>Attach the capillary fiber sample onto a goniometer head using plasticine. Consider whether the sample is a fiber bundle, mat, or disk when placing it in the X-ray beam.
		<ol>
			<li>Note that the sample rotation axis will be around the fiber axis for a fiber bundle (Figure 2A), while in disks the fibers tend to align across the diameter of the tube (Figure 2B). A mat will have two distinct directions (parallel to the plane of the film and perpendicular to it) (Figure 2C).</li>
		</ol>
		</li>
		<li>Collect a diffraction pattern (as shown in Figure 3A) using in-house X-ray equipment (CuK&#945; for protein crystallography) or using synchrotron radiation. Exposure times will vary depending on the intensity of the X-ray beam; adjust this depending on recommended collection times for the diffraction source.</li>
	</ol>
	</li>
</ol>
2. Data analysis
<ol>
	<li>Use of CLEARER for data analysis15. 
	NOTE: The X-ray fiber diffraction pattern analysis program is available free to academic users from the corresponding author. Other methods for diffraction analysis are available16,17 (www.esrf.fr/computing/expg/subgroups/data_analysis/FIT2D)18.&#160;Italics within parentheses indicate menu commands.
	<ol>
		<li>Convert the diffraction pattern to a .tiff file and upload the file into CLEARER (Figure 4).</li>
		<li>Input the diffraction settings (Diffraction Pattern: Diffraction settings), including the pixel size in the TIFF image, the X-ray source, and the sample to detector distance.</li>
		<li>Ensure that the diffraction pattern is oriented so that the meridian is vertical (Image Processing: Rotate). Use the Centering module (Diffraction Pattern: Centre Image) to centre the image. Define the two main axes (meridian and equator):- This module compares intensity along these two axes of the pattern. Once centering is completed, save the image and ensure that it is used for subsequent steps.</li>
		<li>Specify a suitable angle (set along the two main axes [meridian and equator]) and output a graph showing diffraction signal position against intensity using the Radially Average module (Diffraction Pattern: Radially Average) (Figure 4A).</li>
		<li>Measure and automatically output the diffraction signal positions using the Peak Find module (Diffraction Pattern: Radially Average: Peak Find) (Figure 4B). Ensure that all diffraction signals are being detected accurately by adjusting the peak search width while making sure that noise is not being detected.</li>
		<li>Check the signal positions output by Peak Find by using the Zoom &#38; Measure module (Diffraction Pattern: Zoom &#38; Measure). Verify all signal positions are listed and are assigned to the correct axis.</li>
		<li>Explore unit cell dimensions (b and c) that will generate the observed diffraction signals using the Unit Cell Optimization module (Diffraction Pattern: Unit Cell Optimization) (Figure 5A). Examine the strong signals found on the equator to generate an initial guess to input into the unit cell input table and specify the min and max [hkl] indices over which CLEARER searches for potential cells (a dimension is meridional and defaults to 4.76 &#197;).</li>
		<li>Calculate potential unit cell dimensions and compare the predicted diffraction signal positions with observed reflections. Unit cells are scored depending on the degree of agreement between the predicted and experimental signals. The top twenty matches are then displayed. Consider the peptide precursor length and other information from electron microscopy, AFM, or solid state nuclear magnetic resonance (ssNMR) to help select the best possible unit cells to fit the data and the sample.</li>
	</ol>
	</li>
</ol>
3. Structural modeling and testing the model using CLEARER
<ol>
	<li>Construct molecular models (PDB format). 
	&#8203;NOTE: Utilize protein modelling programmes such as Pymol (DeLano)19 or Insight II (Accelerys) and others, to generate PDB coordinates of the precursor peptide in &#946;-strand conformation and in a &#946;-sheet arrangement. This section describes a simple process used for an orthorhombic unit cell (all 90&#176; angles). Non-orthogonal organizations are possible where more expert knowledge of diffraction is required. These can be explored in CLEARER for more complex unit cells.
	<ol>
		<li>Build three-dimensional arrangements of the peptides in a &#946;-sheet arrangement to fit into the determined unit cell(s). Ensure this model contains all symmetry-related objects within the cell.</li>
		<li>Load the PDB model into the Structure Chain Generator module (Diffraction Simulation: Structures: Structure Chain Generator) to check the constructed structure coordinates. Ensure that the fiber axis and beam axis are correctly assigned. Input the determined unit cell dimensions. Check whether clashing occurs for the selected cell dimensions (Figure 5B).</li>
		<li>Upload PDB model coordinates into the Fiber Diffraction Simulation module (Diffraction Simulation: Fiber Diffraction Simulation).
		<ol>
			<li>Input the fiber axis direction, beam orientation, unit cell dimensions selected from step 3.1.2, X-ray source and detector settings, and click simulate. For an orthorhombic unit cell simulation, the fiber axis is defined by the Cartesian axis related to the orientation of the fiber axis in the model used.</li>
			<li>Ensure that the beam axes are perpendicular to fiber axis, i.e., on one of the other two possible axes. Save the simulation settings as .html files (File: Save As). These can be uploaded and edited later to iteratively explore the optimal model structure.</li>
			<li>Save the resulting simulated pattern in a raw format, which can be uploaded later for further analysis, or as a .tiff (this format has additional contrast information discarded) (Simulation: Save Viewable Image as TIFF). Then, compare these to the experimental pattern.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Compare simulated diffraction patterns with experimental patterns for presentation.
	<ol>
		<li>Use steps 2.1.5-2.1.6 to measure simulated diffraction signals and compare with those measured from experimental patterns (Figure 6). Record these in a table.</li>
		<li>Use a module called Layers (Diffraction Pattern: Layers) to overlay simulated and experimental patterns to compare signal positions ( Figure 6C).</li>
		<li>Compare signal positions and relative intensities between the experimental and simulated patterns by arranging a quadrant of the simulated pattern alongside the experimental pattern (as shown in Figure 6B)8,20,21.</li>
		<li>Evaluate the validity of a suggested unit cell and model, and then iteratively alter the model and cell to improve the similarity between the simulated pattern and experimental diffraction pattern.</li>
		<li>Extensively compare the results of different models and cells using the procedure outlined in step 3.1 to test several alternatives. Ensure that information gathered from other techniques, such as TEM, AFM, and NMR, is considered to constrain modeling and to arrive at the best possible structural representation that fits the data8,21,22,23.</li>
	</ol>
	</li>
</ol>

<ol><li>Shirahama, T., Cohen, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+resolution+electron+microscopic+analysis+of+the+amyloid+fibril%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">High resolution electron microscopic analysis of the amyloid fibril.</a> J Cell Biol. 33, 679-706 (1967).</li>
<li>Buxbaum, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyloid+nomenclature+2024%3A+Update%2C+novel+proteins%2C+and+recommendations+by+the+International+Society+of+Amyloidosis+%28ISA%29+Nomenclature+Committee%5BTitle%5D)+AND+%22Amyloid%22%5BJournal%5D)">Amyloid nomenclature 2024: Update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee.</a> Amyloid. 31 (4), 249-256 (2024).</li>
<li>Dobson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structural+basis+of+protein+folding+and+its+links+with+human+disease%5BTitle%5D)+AND+%22Philos+Trans+R+Soc+Lond+B+Biol+Sci%22%5BJournal%5D)">The structural basis of protein folding and its links with human disease.</a> Philos Trans R Soc Lond B Biol Sci. 356 (1406), 133-145 (2001).</li>
<li>Sunde, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Common+core+structure+of+amyloid+fibrils+by+synchrotron+X-ray+diffraction%5BTitle%5D)+AND+%22J.+Mol.+Biol%22%5BJournal%5D)">Common core structure of amyloid fibrils by synchrotron X-ray diffraction.</a> J. Mol. Biol. 273, 729-739 (1997).</li>
<li>Astbury, W. T., Woods, H. J., Bragg, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+studies+of+the+structure+of+hair%2C+wool%2C+and+related+fibers.+II.+The+molecular+structure+and+elastic+properties+of+hair+keratin%5BTitle%5D)+AND+%22Philos+Trans+R+Soc+Lond+A%22%5BJournal%5D)">X-ray studies of the structure of hair, wool, and related fibers. II. The molecular structure and elastic properties of hair keratin.</a> Philos Trans R Soc Lond A. 232, 333-394 (1934).</li>
<li>Geddes, A. J., Parker, K. D., Atkins, E. D., Beighton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%26%2334%3BCross-beta%26%2334%3B+conformation+in+proteins%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">&#34;Cross-beta&#34; conformation in proteins.</a> J Mol Biol. 32 (2), 343-358 (1968).</li>
<li>Sawaya, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic+structures+of+amyloid+cross-beta+spines+reveal+varied+steric+zippers%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Atomic structures of amyloid cross-beta spines reveal varied steric zippers.</a> Nature. 447 (7143), 453-457 (2007).</li>
<li>Makin, O. S., Atkins, E., Sikorski, P., Johansson, J., Serpell, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+basis+for+amyloid+fibril+formation+and+stability%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Molecular basis for amyloid fibril formation and stability.</a> Proc Natl Acad Sci U S A. 102 (2), 315-320 (2005).</li>
<li>Eanes, E. D., Glenner, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+diffraction+studies+on+amyloid+filaments%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">X-ray diffraction studies on amyloid filaments.</a> J Histochem Cytochem. 16 (11), 673-677 (1968).</li>
<li>Kirschner, D. A., Abraham, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+diffraction+from+intraneuronal+paired+helical+filaments+and+extraneuronal+amyloid+fibers+in+Alzheimer+disease+indicates+cross-beta+conformation%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">X-ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross-beta conformation.</a> Proc Natl Acad Sci U S A. 83 (2), 503-507 (1986).</li>
<li>Ke, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Half+a+century+of+amyloids%3A+Past%2C+present+and+future%5BTitle%5D)+AND+%22Chem+Soc+Rev%22%5BJournal%5D)">Half a century of amyloids: Past, present and future.</a> Chem Soc Rev. 49 (15), 5473-5509 (2020).</li>
<li>Sipe, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nomenclature+2014%3A+Amyloid+fibril+proteins+and+clinical+classification+of+the+amyloidosis%5BTitle%5D)+AND+%22Amyloid%22%5BJournal%5D)">Nomenclature 2014: Amyloid fibril proteins and clinical classification of the amyloidosis.</a> Amyloid. 21 (4), 221-224 (2014).</li>
<li>Morris, K. L., Serpell, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+fiber+diffraction+studies+of+amyloid+fibrils%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">X-ray fiber diffraction studies of amyloid fibrils.</a> Methods Mol Biol. 849, 121-135 (2012).</li>
<li>Al-Hilaly, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tau+%28297-391%29+forms+filaments+that+structurally+mimic+the+core+of+paired+helical+filaments+in+alzheimer%27s+disease+brain%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Tau (297-391) forms filaments that structurally mimic the core of paired helical filaments in alzheimer's disease brain.</a> FEBS Lett. 594 (5), 944-950 (2020).</li>
<li>Makin, O. S., Sikorski, P., Serpell, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clearer%3A+A+new+tool+for+the+analysis+of+X-ray+fiber+diffraction+patterns+and+diffraction+simulation+from+atomic+structural+models%5BTitle%5D)+AND+%22Appl+Cryst%22%5BJournal%5D)">Clearer: A new tool for the analysis of X-ray fiber diffraction patterns and diffraction simulation from atomic structural models.</a> Appl Cryst. 40 (5), 966-972 (2007).</li>
<li>Winn, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+overview+of+the+CCP4+project+in+protein+crystallography%3A+An+example+of+a+collaborative+project%5BTitle%5D)+AND+%22J+Synchrotron+Radiat%22%5BJournal%5D)">An overview of the CCP4 project in protein crystallography: An example of a collaborative project.</a> J Synchrotron Radiat. 10 (Pt 1), 23-25 (2003).</li>
<li>Squire, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+CCP13+software+and+the+strategy+behind+further+developments%3A+Stripping+and+modelling+of+fiber+diffraction+data%5BTitle%5D)+AND+%22Fibre+Diffr+Rev%22%5BJournal%5D)">New CCP13 software and the strategy behind further developments: Stripping and modelling of fiber diffraction data.</a> Fibre Diffr Rev. 11, 7-19 (2003).</li>
<li>Makin, O. S., Serpell, L. C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMakin%2C+OS+%22Amyloid+Proteins%3A+Methods+and+Protocols%22">Amyloid Proteins: Methods and Protocols</a>. , Humana Press. Totowa, NJ. (2005).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22The+pymol+molecular+graphics+system%22">The pymol molecular graphics system</a>. , DeLano Scientific. San Carlos, USA. (2002).</li>
<li>Jahn, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+common+architecture+of+cross-beta+amyloid%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">The common architecture of cross-beta amyloid.</a> J Mol Biol. 395 (4), 717-727 (2009).</li>
<li>Madine, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+insights+into+the+polymorphism+of+amyloid-like+fibrils+formed+by+region+20-29+of+amylin+revealed+by+solid-state+NMR+and+X-ray+fiber+diffraction%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Structural insights into the polymorphism of amyloid-like fibrils formed by region 20-29 of amylin revealed by solid-state NMR and X-ray fiber diffraction.</a> J Am Chem Soc. 130 (45), 14990-15001 (2008).</li>
<li>Madine, J., Copland, A., Serpell, L. C., Middleton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross-%CE%B2+spine+architecture+of+fibrils+formed+by+the+amyloidogenic+segment+nfgsvqfv+of+medin+from+solid-state+NMR+and+X-ray+fiber+diffraction+measurements%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Cross-β spine architecture of fibrils formed by the amyloidogenic segment nfgsvqfv of medin from solid-state NMR and X-ray fiber diffraction measurements.</a> Biochemistry. 48 (14), 3089-3099 (2009).</li>
<li>Papapostolou, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+nanoscale+order+into+a+designed+protein+fiber%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Engineering nanoscale order into a designed protein fiber.</a> Proc Natl Acad Sci U S A. 104 (26), 10853-10858 (2007).</li>
<li>Steinmetz, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic+models+of+de+novo+designed+cc%CE%B2-met+amyloid-like+fibrils%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Atomic models of de novo designed ccβ-met amyloid-like fibrils.</a> J Mol Biol. 376 (3), 898-912 (2008).</li>
<li>Blake, C., Serpell, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synchrotron+x-ray+studies+suggest+that+the+core+of+the+transthyretin+amyloid+fibril+is+a+continuous+%CE%B2-helix%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Synchrotron x-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous β-helix.</a> Structure. 4, 989-998 (1996).</li>
</ol>

The authors do not have any conflicts of interest to disclose

X-ray fiber diffraction can be used to evaluate whether a sample is forming an amyloid structure and to gain further insight into the molecular organization of the precursor protein or peptide within the fibers. Here, we have described the methods by which fibrous samples may be prepared for X-ray fiber diffraction, data may be obtained, and then analyzed. Critically, the quality of the data obtained will depend on both the order within the individual fibers. Therefore, initial sample preparation is of major importance, ...

Amyloid fibrils were first identified within a group of diseases known as Amyloidoses, in which proteinaceous fibrils accumulate in the extracellular spaces of tissues1. Each disease is characterized by a particular precursor protein which undergoes a conformational change to form highly ordered, stable protein fibrils2,3. The more recent definition of amyloid fibrils now incorporates fibrils that accumulate within the cellular environment. More recently, a number of functional systems have been identified that utilize the amyloid structure to provide stability, adhesion, or even information storage. Despite the wide range of protein structures that can self-assemble to form amyloid fibrils, they all share a cross-&#946; structure4. This cross-&#946; arrangement was first described by Astbury for hen egg white5 and later explained further by Geddes for cross-&#946; silk6. In this protein architecture, the fibrils are made up of &#946;-strands that run perpendicular to the fibril axis, which are then arranged into &#946;-sheets which associate via the side chains to form what was later referred to as steric zippers7. This creates a highly stable organization held together via hydrogen bonds that run parallel to the fiber, alongside hydrophobic, electrostatic, and polar interactions via side chains8.
Early structural studies used X-ray fiber diffraction to interrogate the structure of amyloid fibrils, whereby the fibrils were aligned and oriented to form a bundle of amyloid fibrils which could be placed in the X-ray beam of a diffractometer9,10. A generic structure of amyloid fibrils was generated to fit the diffraction patterns from several amyloid fibrils extracted from human disease tissue or grown in vitro4, and this has provided a basis for further structural descriptions11. In recent years, advances in solid state nuclear magnetic resonance (ssNMR), microcrystal X-ray crystallography, and cryogenic electron microscopy (CryoEM) have provided atomic detail for the cross-&#946; structure11. However, the X-ray fiber diffraction method remains important and useful, and provides information regarding the repetitive organization of the proteins. Furthermore, in some cases, information relating to a longer-range order may be accessible. Fundamentally, X-ray fiber diffraction is often used to confirm the presence of amyloid fibrils formed from different proteins, and the cross-&#946; pattern is one of the principal requirements for a fibrous assembly to be classified as amyloid12.
Here, we describe the methodology used to prepare amyloid fibrils and align them for X-ray fiber diffraction data collection. Furthermore, we provide details of how the diffraction data may be further analyzed to generate a model structure, its predicted diffraction pattern, and compared to diffraction data from ex-vivo-derived fibrils.

<table><tbody><tr><td>0.7 mm diameter X-ray&amp;nbsp; quartzglass capillaries&amp;nbsp;</td><td>GLAS, W. Muller, D-13503, Berlin, Germany</td><td>Q-07-001-80</td><td></td></tr><tr><td>Glass capillaries 1,5 mm borosilicate</td><td>Harvard appatus Ltd, (Edenbridge, Kent, UK)</td><td>BS4 30-0074</td><td>example, available at other manufacturers</td></tr><tr><td>Protein crystallography X-ray diffractometer</td><td>Various</td><td></td><td>Various manufacturers</td></tr></tbody></table>

amyloid and the cross-beta architecture

Amyloid and the Cross-Beta Architecture

Amyloid fibrils are made up of a misfolded, self-assembled protein organised into a well-defined, repetitive structure. They are well known for their important role in protein misfolding diseases, including Alzheimer&#39;s disease and type 2 diabetes, in which the insoluble amyloid fibrils are deposited in the tissues. Furthermore, they have been found to play an important functional role in many organisms, providing strength, scaffolding, and protection. The first structural models of amyloid were informed by X-ray fiber diffraction data collection from bundles of aligned amyloid fibrils. This resulted in early models of the generic cross-&#946; structure which has been superseded by more detailed diffraction analysis from highly oriented and semi-crystalline samples, resulting in details of the organization of peptides into a repetitive architecture. Here, we focused on describing methods of determining the underlying architecture of these fibrils, and we have described the common features of amyloid fibrils from diverse pathogenic, functional, and synthetic sources. We describe the preparation of the samples, data collection, and subsequent data analysis to produce a model structure that can be compared to the experimental data.

CcbMet example24: Figure 1 shows electron micrographs showing a high density of amyloid fibrils on a grid suitable for preparation of an X-ray fiber sample. Figure 2 describes the different textures that can be generated from fibrous samples fiber bundle (Figure 2A), disk (Figure 2B), mat/film (Figure 2C), and shows t...

Amyloid fibrils are formed by numerous proteins, and the resulting fibrils share a &#34;cross &#946;-sheet&#34; structure. Here we describe how amyloid fibril samples may be prepared for X-ray fiber diffraction and how the patterns may be analysed.

Amyloid fibrils are made up of a misfolded, self-assembled protein organised into a well-defined, repetitive structure. They are well known for their ...

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.

Research

JoVE Journal

Neuroscience

Structure-based drug design plays an important role in drug development.  Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

The MultiBac Protein Complex Production Platform at the EMBL

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid β protein on brain tissues of Alzheimer's disease and cerebral amyloid angiopathy samples using MALDI-IMS.

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential β2-microglobulin (β2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous β2m were investigated.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

Here, we describe a yeast system reconstituting human γ-secretase. This system allows for the identification and study of mutations affecting activity and for screening of γ-secretase inhibitors (GSIs). Utilizing the loss of function properties of familial Alzheimer's disease mutants, it is possible to screen for γ-secretase modulators (GSMs).

Saccharomyces cerevisiae Models of Alzheimer's Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by &#947;-Secretase

Protein design involves the construction of amino acid sequences and the incorporation of specific motifs to create functional variants. This approach is critical for the development of antimicrobial peptides (AMPs) to combat antibiotic-resistant pathogens. This paper presents a procedure for protein construction using various bioinformatics tools.

Application of I TASSER, trRosetta, UCSF Chimera, HADDOCK server, and HEX loria for De Novo and In Silico Design of Proteins

This article presents a protocol for data processing of influenza viruses imaged using cryo-electron tomography and subsequent subtomogram averaging of the hemagglutinin glycoprotein. This protocol covers step-by-step data processing, from image preprocessing to final model refinement.

Subnanometer-Resolution Structural Determination of Hemagglutinin from Cryo-Electron Tomography of Influenza Viruses

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Learn X-ray fiber diffraction methods to determine amyloid fibril architecture and cross-beta organization in structural biology. This protocol enables detailed molecular modeling of protein misfolding structures.

Amyloid and the Cross-Beta Architecture

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