This work is supported by a National Scientist Development grant from the American Heart Association: 10SDG3500034 and UTMB-NCB start up funds to SSM. The authors acknowledge the Cryo-EM and Scientific Computing facilities at the Sealy Center for Structural Biology at UTMB (<a href="http://www.scsb.utmb.edu">www.scsb.utmb.edu</a>), as well as Drs. Steve Ludtke and Ed Egelman for help with the 2D and 3D helical reconstruction algorithms.

1. Sample Preparation
<ol>
	<li>Buffer exchange human FVIII-BDD14 and porcine FVIII-BDD15 against HBS-Ca buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH = 7.4) and concentrate to 1.2 mg/ml. Keep the protein solution at -80 &#176;C.</li>
	<li>Prepare lipid nanotubes (LNT) by mixing GalactosylCeramide (GC) and phosphatidylserine (PS) at 1:4 w/w ratio in chloroform. Evaporate the chloroform under argon and solubilize the lipids in HBS buffer to 1 mg/ml. Keep the LNT solution at 4 &#176;C.</li>
</ol>
2. Cryo-electron Microscopy of FVIII-LNT
<ol>
	<li>Cryo-EM Sample Preparation
	<ol>
		<li>Glow discharge the 300 mesh Quantifoil R2/2 copper grids (carbon side up) in a mixture of O2 and H2&#160;gas for 10 sec&#160;at 50 W.</li>
		<li>Mix the FVIII and LNT solutions in HBS-Ca buffer at 1:1 w/w ratio and incubate for 15 min&#160;at room temperature.</li>
		<li>Apply a drop of 2.5 &#181;l of FVIII-LNT sample to the hydrophilic electron microscopy grid in the Vitrobot Mark IV humidified chamber (100% humidity).</li>
		<li>Blot and flash freeze the grid (one blot for 3.5 sec, blot force 1) in liquid C2H6, cooled down by liquid N2 to obtain amorphous ice.</li>
		<li>Store grids in storage boxes under liquid N2 (LN2).</li>
	</ol>
	</li>
	<li>Cryo-EM Data Collection 
	NOTE: The JEM2100-LaB6 (year 2010) is equipped with a TEMCON 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 so-on, and two panels: LEFT and RIGHT, placed on both side of the column. The beam shift X,Y knobs (SHIFT Y, SIFT Y) and the multifunction (DEF/STIG) knobs are on both panels. On the LEFT panel is the illumination (BRIGTHNESS) knob. On the RIGHT panel are: the magnification (MAG/CAM LENGTH) and focus (FOCUS) knobs and the three imaging (MAG1, MAG2, LOWMAG) and a diffraction (DIFF) modes buttons. We acquire our data in MAG1 at alpha 2. The minimum dose illumination conditions (MDS) required for Cryo-EM data acquisition are set with F1 through F6 buttons, top row on RIGHT panel. In this protocol the generic settings are used: F1 - raise/lower screen, F2 - SEARCH mode, F3 &#8211; FOCUS mode, F4 &#8211; PHOTO mode, F5 &#8211; MDS OFF/ON and F6 &#8211; BEAM BLANK, used to shield the specimen from radiation damage by deflecting the beam.
	<ol>
		<li>Place the cryo-holder into the cryo-station and fill the Dewar of the holder and the cryo-station with LN2. When temperature reaches -192 &#176;C, open shutter on the holder tip, place previously frozen Cryo-EM grid into the designated place and secure with a ring clamp.</li>
		<li>Insert the cryo-holder into the electron microscope. Refill the Dewar of the cryo-holder and the anti-contaminator chamber with LN2. Wait for the holder to stabilize for 30-60 min. Press F6 on and turn the filament on. When the filament is saturated, press F6 off and open shutter on holder to view the grid.</li>
		<li>Press Low Mag/alpha 1 (set at 200X&#160;mag) and locate areas with thin ice on the grid.</li>
		<li>Switch to MAG 1/alpha 2 to set minimum dose (MDS) mode and acquire Cryo-EM data at low electron doses, without damaging the specimen.
		<ol>
			<li>Press F2 to set search mode. Set magnification at 40,000x. Enlarge beam with BRIGTNESS knob to minimal electron dose ~ 0.04 e-/&#197;2&#183;s. Press DIFF to switch to diffraction mode. Set camera length to 120 cm with MAG/CAM LENGHT knob. Locate areas on the grid within the pre-selected areas in LOWMAG that have lipid nanotubes in the holes of the carbon film.</li>
			<li>Press F4 to set Photo mode at 40,000X&#160;magnification and set illumination with BRIGHTNESS knob at doses of 16-25 e-/&#197;2&#183;s. Press Standard Focus button to set focus conditions adjusting the Z-height of the specimen with the Z UP/DOWN buttons (RIGHT control panel). Set defocus between -1.5 and -2.5 &#181;m.</li>
			<li>Align SEARCH and PHOTO mode by drawing a square in the LIVE VIEW window on the digital camera monitor in SEARCH mode corresponding to the area to be imaged in PHOTO mode.</li>
			<li>Press F3 to set Focus mode at 100,000X&#160;magnification. Focus illumination to cover the CCD chip (~4 - 5 cm radius) and off axis to not irradiate the area to be imaged in PHOTO mode. Adjust defocus to ~ -1.5 and -2.5 &#181;m with the FOCUS knob and correct for astigmatism of the image with DEF/STIG knobs.</li>
		</ol>
		</li>
		<li>Select the FVIII-LNT to be imaged in SEARCH mode by acquiring live images in the LIVE VIEW window on the digital camera monitor. Center the FVIII-LNT in the square drawn on the LIVE VIEW window.</li>
		<li>Record a digital image on the CCD camera at 52,000X&#160;effective magnification and 0.5 sec exposure by switching to Photo mode and clicking the ACQUIRE button on the digital micrograph camera monitor. The image acquisition conditions are set such as the beam blank (SHUTTERS) open only when the image is acquired in PHOTO mode.</li>
		<li>Check the quality and defocus of the acquired image by pressing CTRL-F to obtain a Fast Fourier Transform (FFT).</li>
	</ol>
	</li>
</ol>
3. 3D Reconstruction
NOTE: The image analysis software used for the 2D and 3D analysis: EMAN2 and IHRSR are freely available. EMAN2 can be downloaded from http://blake.bcm.edu/emanwiki/EMAN2/Install. The IHRSR software can be obtained from Professor Egelman: egelman@virginia.edu. The final IHRSR refinements are run on the Texas advanced computing center cluster: http://www.tacc.utexas.edu/ at the University of Texas, Austin. The 3D reconstruction algorithm shown on Figure 1 consists of two main steps: First selecting a homogenous set of helical segments (particles) with the 2D reference free alignment (RFA) algorithms implemented in EMAN 2, second achieving a 3D reconstruction based on the helical parameters and back projection algorithms incorporated in IHRSR. The first step utilizes the programs developed for selecting homogenous particle sets for 3D reconstruction with Single Particle SPA (algorithms) for which EMAN2 has been specifically developed and distributed: http://blake.bcm.edu/emanwiki/EMAN2. This step has been adapted to the Cryo-EM data. The second step is achieved with the IHRSR algorithm, which is specifically designed for the type of helical assemblies obtained with the recombinant Factor VIII forms. This algorithm has been documented extensively through the scientific literature12.
<ol>
	<li>Perform 2D image analysis with the EMAN2 scientific image-processing suite: http://blake.bcm.edu/eman2/doxygen_html16 to select homogenous particle (helical segment) sets for helical reconstruction.
	<ol>
		<li>Select Cryo-EM micrographs with straight and well-organized helical tubes with the digital camera software and visualization tools.</li>
		<li>Invert, normalize and filter for X-ray pixels in the e2workflow.py GUI, single particle reconstruction (spr) option: http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2projectmanager</li>
		<li>Import the inverted, normalized and X-ray pixel filtered images in the e2helixboxer.py GUI. Select FVIII-LNT helical tubes and segment at 256 x 256 pixels (2.9 &#197;/pix) with 90% overlap using: http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2helixboxer</li>
		<li>Evaluate the defocus from the original micrographs and apply contrast transfer function (CTF) correction (only phase correction) to the helical segments from the same micrographs with the e2ctf.py option incorporated in the e2workflow.py: http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2ctf.</li>
		<li>Generate initial particles (helical segments) sets in the e2workflow.py GUI - SPR option: http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2workflow.</li>
		<li>Calculate 2D class averages with the e2refine2d.py algorithm applying reference free k-mean classification to select homogenous data sets with the same diameter LNT and degree of helical order (Figure&#160;2), following the script: &#8216;e2refine2d.py --iter=8 --naliref=5 --nbasisfp=8 --path=r2d_001 --input=INPUT.hdf --ncls=51 --simcmp=dot --simalign=rotate_translate_flip --classaligncmp=dot --classraligncmp=phase --classiter=2 --classkeep=0.8 --classnormproc=normalize.edgemean --classaverager=mean --normproj &#8211;classkeepsig&#8217;, changing the ncls value accordingly.
		<ol>
			<li>Classify the initial data set in 150 classes &#8216;ncls=151&#8217; over 8 iterations with &#8216;classkeep=0.8&#8217;, meaning that particles with less than 80% similarity to the class average are excluded from the given class. http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2refine2d</li>
			<li>Merge particles from class averages with pronounced helical diffraction in e2display.py to create intermediate data set.</li>
			<li>Classify the intermediate data set in 50 classes &#8216;ncls=51&#8217; to differentiate classes with same diameter and degree of order.</li>
			<li>Merge particles from classes with same diameter and degree of order in e2display.py to create the final data set for the three-dimensional reconstruction: http://blake.bcm.edu/emanwiki/EMAN2/Programs/emselector.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform helical reconstruction with the Iterative Helical Real Space Reconstruction (IHRSR) algorithm, as described in Egelman17,18.
	<ol>
		<li>Estimate the rise, &#916;z&#160;(&#197;) of the FVIII-LNT helix from the combined Fourier transform of the particles in the final data set.</li>
		<li>Define the azimuthal angle &#916;&#934;&#160;(&#186;) by running parallel IHRSR refinements with a constant &#916;z, increasing &#916;&#934;&#160;from 5&#176;&#160;to 60&#176;&#160;in 5&#176;&#160;increments.</li>
		<li>Run 100 consecutive IHRSR cycles for each final data set with a featureless cylinder as an initial volume and the initial helical parameters &#916;z&#160;and &#160;&#916;&#934;&#160;as defined in 3.2.1. and 3.2.2.</li>
		<li>Inspect the final volumes for convergence of the helical parameters and correspondence between class averages from the 2D classification of the final data set and the projections from the final IHRSR reconstruction.</li>
		<li>Impose symmetry to the final 3D reconstructions, corresponding to the asymmetric unit distribution observed in the final asymmetric 3D volumes: 4-fold for the human FVIII-LNT and 5-fold for the porcine FVIII-LNT. Run another 100 refinement cycles to generate a final symmetrized 3D reconstruction.</li>
		<li>Calculate the Fourier Shell Correlation curve for both volumes by first separating the corresponding data set in odd and even: &#8216;e2proc2d.py &#60;infile&#62; &#60;outfile&#62; --split=2&#8217;. Then run 100 IHRSR consecutive refinements as described in 3.2.3. Calculate the FSC of the 3D volumes created from the odd and even datasets: &#8216;e2proc3d.py evenvolume.mrc fsc.txt --calcfsc=oddmap.mrc&#8217;.</li>
	</ol>
	</li>
	<li>Visualize and Segment the FVIII-LNT volumes in UCSF chimera.</li>
	<li>
	<ol>
		<li>Open the final volumes from step 3.2.5. in UCSF Chimera and set the contour level to 0.005 in the TOOLS &#62; VOLUME DATA &#62; VOLUME VIEWER option.</li>
		<li>In TOOLS &#62; VOLUME DATA &#62; SEGMENT MAP, select volume in SEGMENT MAP tab and click on SEGMENT to segment the volume.</li>
		<li>Group segments corresponding to one unit cell by selecting with CTRL+SHIFT and clicking group.</li>
		<li>Color the segments by unit cell and helix with ACTIONS &#62; COLOR option to emphasize the structural features.</li>
	</ol>
	</li>
</ol>
4. Electron Tomography
<ol>
	<li>Negatively Stained FVIII-LNT Sample Preparation
	<ol>
		<li>Prepare FVIII-LNT samples as for the Cryo-EM experiments.</li>
		<li>Glow discharges carbon-coated 300-mesh copper grid (carbon side up) in a mixture of O2 and H2&#160;gas for 10 sec&#160;at 50 W as for Cryo-EM experiments.</li>
		<li>Apply a drop of 2.5 &#956;l FVIII-LNT suspension with 6-nm colloidal gold nanoparticles to the grid, blot the excess liquid and negatively stain by applying 5 &#181;l 1% uranyl acetate solution for 2&#160;min. Blot the excess liquid and air dry the grid.</li>
	</ol>
	</li>
	<li>Electron Tomography Data Collection
	<ol>
		<li>Transfer the grid into the single tilt holder.</li>
		<li>Transfer the holder in the electron microscope.</li>
		<li>Collect tilt series automatically with the SerialEM software19 at 2&#176;&#160;increments over an angular range of -60&#176;&#160;to +60&#176;&#160;and record images with a CCD camera at 52,000X&#160;effective magnification, -6 to -10 &#956;m defocus and electron dose of 150 - 170 electrons/&#197;2&#183;s per tomogram.</li>
	</ol>
	</li>
	<li>Electron Tomography Reconstruction of FVIII-LNT 
	Note: Reconstruct the pFVIII-LNT and hFVIII-LNT tilted series acquired in 4.2. with the ETomo option of the IMOD software following the tutorial: http://bio3d.colorado.edu/imod/doc/etomoTutorial.html
	<ol>
		<li>Using a terminal window, open the tomogram in 3DMOD. http://bio3d.colorado.edu/imod/doc/3dmodguide.html.</li>
		<li>Bin the tomogram by 4 with the IMOD BINVOL command to decrease the size of the tomogram.</li>
		<li>Select the proper angle to rotate the lipid nanotube along the Y-axis in the binned tomogram using the IMOD ROTATEVOL command.</li>
		<li>Rotate the full tomogram with the IMOD ROTATEVOL command.</li>
		<li>Crop the selected lipid nanotube oriented along the Y-axis with the IMOD CLIP RESIZE command.</li>
		<li>Open the cropped sub-tomogram with 3DMOD.</li>
		<li>Click on the tomogram to visualize the arrangement of Factor VIII molecules along the slices in Z-axis and along Y-axis in the sub-tomogram volume.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interest and they can be contacted directly regarding any of the procedures published in this manuscript.

In this work a methodology is presented to differentiate between two membrane-bound organizations of highly homologous proteins: human and porcine FVIII self-assembled on lipid nanotubes in the conditions encountered in the human body.

In the described procedure, human and porcine FVIII are successfully organized helically on lipid nanotubes, which is the most critical step. The next critical step is to preserve the sample in thin amorphous ice by flash freezing&#1...

Blood coagulation Factor VIII (FVIII) is a large glycoprotein of 2,332 amino acids organized in six domains: A1-A2-B-A3-C1-C2&#160;3. Upon Thrombin activation FVIII acts as the cofactor to Factor IXa within the membrane-bound Tenase complex. Binding of activated FVIII (FVIIIa) to FIXa in a membrane-depending manner enhances FIXa proteolytic efficiency more than 105 times, which is critical for efficient blood coagulation4. Despite the important role FVIII plays in coagulation and the Tenase complex formation, the functional membrane-bound FVIII structure is yet to be resolved.
To address this, single lipid bilayer nanotubes (LNT) rich in phosphatidylserine (PS), capable of binding FVIII with high affinity8,9 and resembling the activated platelet surface have been developed10. Consecutive helical organization of FVIII bound to LNT has been proven to be effective for structure determination of FVIII membrane-bound state by Cryo-EM5. Functionalized LNT are an ideal system to study protein-protein and protein-membrane interactions of helically organized membrane-associated proteins by Cryo-EM11,12. Cryo-EM has the advantage over traditional structural methods such&#160;as X-ray crystallography and NMR, as the specimen is preserved at closest to the physiological environment (buffer, membrane, pH), without additives and isotopes. In the case of FVIII, studying the membrane-bound structure with this technique is even more physiologically relevant, as the LNT resemble closely by size, shape and composition the pseudopodia of the activated platelets where the Tenase complexes assemble in vivo.
Defects and deficiency of FVIII cause Hemophilia A, a severe bleeding disorder affecting 1 in 5,000 males of the human population4,6. The most effective therapy for Hemophilia A is life-long administration of recombinant human FVIII (hFVIII). A significant complication of the recombinant FVIII Hemophilia A therapy is the development of inhibitory antibodies to the human form affecting approximately 30% of Hemophilia A patients13. In this case, porcine FVIII (pFVIII) concentrate is used, as porcine FVIII displays low cross-reactivity with inhibitory antibodies against human FVIII and forms functional complexes with human FIXa7. Establishing the membrane-bound organization of both porcine and human FVIII forms is important to understand the structural basis of FVIII cofactor function and implications for blood hemostasis.
In this study, we describe a combination of lipid nanotechnology, Cryo-EM, and structure analysis designed to resolve the membrane-bound organization of two highly homologous FVIII forms. The presented Cryo-EM data and 3D structures for helically organized porcine and human FVIII on negatively charged LNT show the potential of the proposed nanotechnology as basis for structure determination of FVIII and membrane-bound coagulation factors and complexes in a physiological membrane environment.

<table border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">JEM2100 with LaB6&#160;</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;">JEM-2100</td>
			<td style="width:447px;">operated at 200 kV</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">with TEMCON software</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan626 Cryo-holder</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">626.DH</td>
			<td style="width:447px;">cooled to -175 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">with temperature controler unit</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan 4K x 4K CCD camera</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">US4000</td>
			<td style="width:447px;">4096 x 4096 pixel at 15 microns/pixel physical resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Solarus Model 950 plasma cleaner</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Vitrobot Mark IV</td>
			<td style="width:205px;">FEI</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Materials</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Carbon coated 300 mesh 3mm copper grid</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">01821</td>
			<td style="width:447px;">plasma cleaned for 10 s on high power</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">Quantifoil R2/2 300 mesh</td>
			<td style="width:205px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">Q225-CR2</td>
			<td style="width:447px;">Carbon coated 300 mesh Cu grids with 2 mm in diameters holes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Uranyl acetate dihydrate</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">19481</td>
			<td style="width:447px;">1% solution, filtered</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:239px;">Galactosyl ceramide</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">860546</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Dioleoyl-sn-glycero-phospho-L-serine</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">840035</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Software</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:239px;">EM software Digital Micrograph</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.gatan.com/DM/">http://www.gatan.com/DM/</a></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">EM software EMAN</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://blake.bcm.edu/emanwiki/EMAN/">http://blake.bcm.edu/emanwiki/EMAN/&#160;</a></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">EM software Spider</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://spider.wadsworth.org/spider_doc/spider/docs/spider.html">http://spider.wadsworth.org/spider_doc/spider/docs/spider.html</a></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">EM software IHRSR</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;">Programs available from Edward H. Egelman http://people.virginia.edu/~ehe2n/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (IMOD)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://bio3d.colorado.edu/imod/">http://bio3d.colorado.edu/imod/&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (SerialEM)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="ftp://bio3d.colorado.edu/pub/SerialEM/">ftp://bio3d.colorado.edu/pub/SerialEM/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">UCSF-Chimera</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.cgl.ucsf.edu/chimera/download.html">http://www.cgl.ucsf.edu/chimera/download.html</a></td>
		</tr>
	</tbody>
</table>

helical organization of blood coagulation factor viii on lipid nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

Recombinant human and porcine FVIII were successfully organized helically on negatively charged single bilayer LNT, resembling the activated platelet surface. The helical organization of the human and porcine FVIII-LNT was consistent through the collected digital micrographs (Figure&#160;2). &#160;The control LNT and the human and porcine FVIII-LNT helical tubes were selected and segmented with the e2helixboxer.py GUI and initial data sets created with the e2workflow.py GUI, Single parti...

Watch this Scientific Journal Video about Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes at JoVE.com

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

helical-organization-blood-coagulation-factor-viii-on-lipid

Research

JoVE Journal

Bioengineering

12.2K Views.  University of Texas Medical Branch. We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Video: Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in transcription of non-coding RNAs, specifically of microRNAs. Here, we propose a strategy to study the potential role of transcription factor in regulating transcription of microRNAs using publically available data, computational resources and high throughput data. We use the H3K4me3 epigenetic signature to identify microRNA promoters and chromatin immunoprecipitation (ChIP)-sequencing data from the ENCODE project to identify microRNA promoters that are enriched with transcription factor binding sites. By transfecting cells of interest with shRNA targeting a transcription factor of interest and subjecting the cells to microRNA array, we study the effect of this transcription factor on the microRNA transcriptome. As an illustrative example we use our study on the effect of STAT3 on the microRNA transcriptome of chronic lymphocytic leukemia (CLL) cells.

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...