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Printing protein ribbon-structures on the Form 1+

Before you read, note that I have not yet successfully printed a protein’s ribbon-structure on our lab’s Form 1+ machine. I’m asking for help in getting this working. Thanks in advance for reading my post.

Sincerely,
-Quinton

Edit 2: Significant progress has been made. One user has been able to print the protein on his Form 2. He, along with others, have provided some great tips and feedback for how to get this done. Their responses are also great advice for printing in general on formlabs machines. On my third print with our Form 1, I have had some success but am still looking to improve the results. Thanks again for checking out this thread, and a big thanks to all those that’ve responded.

Sincerely,
-Quinton


Hi!

We are interested in 3D printing the ribbon structure of proteins and our lab has a Form 1+ printer. Our first protein we’d like to print can be found here on the NIH 3D Print Exchange:

http://3dprint.nih.gov/discover/3dpx-003476

As explained there, we downloaded a .pdb of our protein and swapped one molecule inside it, saving this new file as a .pdb as well. We then uploaded the .pdb to the Exchange, which converted the .pdb into .stl and .wrl files. The Exchange uses Netfabb to repair holes and other errors to ensure that we have printable files. Finally, we downloaded from the exchange one of the .stl files and opened it in the PreForm software.

File downloaded: 2hi4zcafdocked1-ribbon.stl

Below is what our protein looks like in the software:

And below is what it looks like all ready to print. We kept the scale at 1.00, and used auto-orient to arrive at the tall and skinny structure. For the supports, a colleague suggested a Density of 1.24mm and Point Size of 0.4 mm. We used the generate supports feature, and added supports for red areas.

Note: There was one small red area that even after adding 4 supports to it did not remove the red shading. We added one extra support and ignored the red shade there.

We printed with the medium print resolution (0.05mm layer thickness) with white resin (FLGPWH01). It was a 5-hour print with 1381 layers and 29.90mL resin to be used. Below is our result.


That was not a good print.

Note: before we printed the protein, we did a test print to be sure our machine was working and it printed as expected.

Brainstorm: What went wrong with our first print?

  • Not a machine issue, we checked that with a test print
  • Not a corrupt file, the Exchange used Netfabb to fix the errors
  • Maybe the print size was too small, and the thickness of the protein ribbon structure too thin.
  • Maybe the active site had too many molecules and was too complex.
  • Maybe we used too many support and it made the print too complex.
  • Maybe the print was oriented in an awkward way, making the print too complex.

Okay, so we had a checklist for what we wanted to change for the second print. The print size, support density, and orientation could all be changed in the Preform software. Simplifying the active site of the protein required another program that could manipulate .stl files.

After some web-surfing, I downloaded Meshmixer and imported the 2hi4zcafdocked1-ribbon.stl file into it. The active site of the protein is like an internal cavity. It has two molecules that I want inside, which are attached to the protein via support rods. It also has molecules (amino acid residues) sticking out of the protein, about a dozen or so. I don’t really want these. With Meshmixer, I was able to literally slice those molecules off the protein, and delete them from the file.

Here’s what it looked like before in Meshmixer:

and here’s what it looks like after slicing:

Note: I also added some rods connecting the heme’s iron to the heme’s porphyrin ring. A little handy-work on my part, I don’t think it foiled the print though.

The slicing was a bit crude, but I figured I could address the stumps post-production. With that done, it was back to the PreForm software.

I scaled the file 1.75 times the original file size and used auto-orient a couple times and decided upon a flat orientation for the protein. This time when we generated supports, we used a density of 0.5 and point size of 0.4mm. There were no red areas this time around, was it too good to be true?

Again, we printed with the medium print resolution (0.05mm layer thickness) and white resin (FLGPWH01). Print time was about 9 hours, with 1686 layers and 82.45mL of resin to be used. Nothing was printed after the first print and before this second print of the protein. When preparing the tank, I did notice a small dot (size of a dull pencil tip) of cloudiness in the tank, but it was faint and I decided to continue on with the print.

Here was the result of our second print:



No good, again!

Upon cleaning the device, I scraped off this piece of resin from the bottom of the tank.

I also noticed a significant cloudy area (a little less than 1/9th the tank) in the center of the tank’s base. I don’t know how old the tank is. We are currently ordering more tanks.

At this point, I looked on the forums and surfed the Internet to see if others had some advice or clues to what I could do next. But alas, there is no thread about printing ribbon-structure models of proteins with formlabs 3D printers. I’m interested in getting this working for the Form 1+ if possible.

I don’t know what the problem is. I’m thinking it’s something to do with the supports. My next steps might be to simplify the design in order to see what prints well and what does not.

If you have any comments, questions or suggestions, please respond! I hope we can figure this out.

Sincerely,
-Quinton

Have you tried playing with the internal supports (turn on if off and vice versa)? I’m new to this, too. I just got a Form 2.

What a beautiful protein.
Sounds like we’ve covered some of the same ground this week. I’ve been having support issues because my Form2 just doesn’t work like the FDMs I’m used to:)) I’m wondering if it collapsed from it’s own weight, maybe the point size was too small to support it? I have no idea, it’s very frustrating, you have done so much work…I’m on my 4th try, placing my own…and I know my problem is supports with one of my files, but no where near as complicated as yours. I hope some of our resident experts have an answer.
ann

As someone who has been printing with formlabs equipment since the kickstarter I’ve had a harsh learning curve, and in my opinion you will never successfully print that protein as a single model - it’s very complex with dozens, if not hundreds of overhanging sections. The supports are going to outnumber the model and is going to be impossible/impractical to remove them all neatly. I would take the approach of breaking it up into smaller sections, print those, then glue back together. Good luck, you couldn’t have chosen something more difficult to print in my opinion.

This is the worst thing you could try to print with the printer. Definitely you need to lower the resolution - go with .1mm instead of 0.05mm for starters. Generate more support points.

Great feedback. Good to hear others say how difficult the print is, ha.

Internal supports were turned on for both prints. Not convinced turning them off will help, but it is something I could look into. Changing the point size is an interesting idea. If it did collapse, I think I need to know more of how that happens. Because all I found in both prints were those resin slices. Definitely like the idea of breaking it up into smaller sections, was thinking about doing that next. And thanks for the tip about a lower resolution. Might not of thought to do that.

I’m curious that you say generate more support points. That’s the opposite of what I did going from the first to second print. Some parts of the print are coming out beautifully (that helical structure in the 2nd image of the 2nd print is just fantastic), which had me thinking the number of supports weren’t the issue. Is that a good conclusion to make? I’m totally not the expert here, so please do keep your feedback coming. Thanks a bunch.

-Quinton

That’s kind of what I was thinking about the overhangs collapsing… breaking it up is an interesting idea. I’ve had to use my flex shaft and diamond bits to get to some pesky supports inside small spaces… just had good luck using .55 for the points and my print was heavy 4mm thick …but the supports just literally brushed off and that was what I was looking for. Great discussion…I will learn a lot from how you solve this.
Ann

You’re not going to be able to print that, there’s too many interior parts and too many things that need supports. Even if you got it to print it would be impossible to remove the supports. To get a successful 3D print you would need to use a professional printing service that uses the type of printer that has a wax support structure.

This is true if you want to get your print as a single piece, alternatively, a powder based printer could also print in one piece.

Dow214, from the shape of your supports, it looks like you are using an older version of PreForm. if that is so, then you should try the updated version, as FormLabs have really made an improvement in this area.

I suggest that a Form 2 is not the best printer for this sort of work. Both the 3D Systems Projet and the Stsratasys Objet printers use wax support that is either melted or dissolved away from the finished model. This means that you don’t have to arrange complex supports nor have to remove them from hard-to-get-at places.

No, not all of them.

3D System’s Projet 1000, Projet 1200, and Projet 1500 are DLP resin printers that use the same breakaway supports as the Form 1 and Form 2. Likewise, only high-end Objet models accept dissolvable support material (SUP706) which was just released last year. Older Objet printers and mid-range to low-end machines employ support material that has to be removed mechanically with a water jet – that’s more convenient than break-away material, but it’s still not optimal for delicate or detailed structures.

Neither brand offers meltable/dissolvable supports in a desktop printer AFAIK.

What we really need is a resin developed that would dissolve or melt away depending on the amount of energy delivered by the UV laser. A lite blast of UV makes it soluble and a strong blast makes it non-dissolvable (or vice-versa). That would work with current printers with a firmware upgrade.

Short of that, what about a resin that is say 90% UV resin, and 10% visible light, or IR curable resin. The UV resin would come out pretty much solid, while the other resin would be weak in comparison and could be even be a soluble formulation. Of course, the machine would need dual lasers then. But for support structures on FDM printers, 10-15% fill is all that is needed…

Anyhow just an idea - not that it will help get this project printed now.

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Have you flipped through all the layers looking for unsupported regions? I have learned that the failed builds I have had almost certainly failed because PreForm didn’t detect some unsupported geometry.

If you haven’t, I would suggest to flip through the layers and look for any geometry that starts floating in space. Also look for any geometry that mostly starts floating in space but may still be slightly connected to the rest of the geometry. These areas would need a manual support added.

I have also learned that one failed area can cascade failures through the rest of the print. So just one unsupported regions could cause catastrophic failure…

Hi Dow214,

I was curious as to why that protein structure wouldn’t print, so I had to try it myself. I downloaded and printed the 2hi4zcafdocked1-ribbon.stl file at 100% on my Form 2.

It worked OK for me, here are the results:


The photos aren’t the best but after comparing it with the stl geometry, it seemed to be all there.

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Good luck knowing what is support and what is the model :slight_smile:

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It wasn’t so hard. Luckily the straight tube bits that are part of the geometry are thicker than the supports, so they are relatively easy to spot.

Other than that, just don’t snip anything that looks like a telephone cord!

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Hahaha

Thanks again for all the feedback. I have not flipped through the layers, but I’m not sure how to do it in the software. I read somewhere that that feature was discontinued too. And thanks for the observation on the cascading misprints; I was wondering if that could happen.

@Macro, that’s fantastic! Literally revitalized me and this project today. Thank you so much for trying the print yourself. I have some questions for you. If you could answer them, I’d greatly appreciate it.

  1. I am running Preform 2.5.0. Is that the updated version you mentioned? What version of Preform are you running?
  2. Can you summarize how you set up (print resolution, scale, orientation, supports, layout)
    the model in Preform? By 100%, do you mean at a scale of 1.000?
  3. Do you think the set up matters for the print’s success?
  4. Do you know if it’s printable on a Form 1+? Or what do you think? And if I have a misprint again, do you think it’s a good conclusion to say it was the fault of my printer and not the file or my support structure?
  5. Could you share the .form file of the print above?

Thank you again so much @Macro, looking forward to your response. And thank you again everyone for your feedback. Looking forward to hearing your responses as well,
Sincerely,
-Quinton


Edit: I hope those questions aren’t too much! If you could answer them all, even if it’s brief, I’d greatly appreciate it. Thank you so much again, I’m really excited to see it worked. -Quinton

Edit #2: See @Hellborn’s post #21 below for how to flip through the layers. -Quinton

You are welcome, Dow214.

Answers to your questions:

  1. It turns out we are running the same version, 2.5.0 of PreForm. I thought yours was older because the supports are thicker at the base than the supports for the Form 2. Compare this screenshot to yours.

  2. These are the settings I used:
    Scale: 1.000 (100% of the imported size)
    Layer Thickness: 0.1
    Material: White FLGWH01
    Density: 0.5
    Point Size: 0.40mm
    Internal Supports: On
    Height Above Base: 10.0mm
    I just used PreForm do the geometry repairs of the original 2hi4zcafdocked1-ribbon.stl file.
    I also let PreForm orient the model for me, and I didn’t need to edit the generated supports.
    I always change the Density and Point Size to the lowest settings, which makes the supports much easier to remove than the default settings. They mostly just snap off.
    I also always change the Height Above Base to the maximum of 10.0mm because of a great tip that Konstantin_Dani gave here. He says that the first hundred layers are always overly compressed so that the model sticks to the build platform, and then the rest are less compressed. There is misalignment at the point at which the compression changes which can cause problems. This could be one of the problems.

  3. The basic setup other than the few points I mentioned probably doesn’t matter. I believe that it should print successfully at different sizes, and at different layer thicknesses (although the 0.025 setting isn’t available for White FLGWH01 resin on the Form 2).

  4. I don’t know if the supports are thicker at the bottom on the Form 1+ prints because Form 2 heats and maintains the resin temperature at 30° Celsius, but if the .form file that I have supplied a link for in 5. doesn’t print on your printer, maybe try and heat the resin yourself.

  5. Yes, here it is.