Head density in Nogales Map

[Pellarin]

Yi, Nogales says that the Head density is most probably occupied by the Rrp44 NTD. The head density is 20Ang apart from the Body. How does it reconcile with the crystal structure by E. Conti, where Rrp44 looks like a compact particle?

[wallyshi]

Yes. that looks a bit odd. This extra density was also not observed in other cryoEM studies (or Conti's exosome-11 /+RNA crystal structure) where recombinant proteins were used. I am not sure if it really reflects the difference because of different purification system ( Nogales exosome was purified from endogenously tagged system), or the fac that Rrp44/Dis3 might be quite flexible and seems to have at least two conformations dependent on the RNA, or something is wrong one way or the other.

[Pellarin]

Is it possible that that is the gfp?

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On Apr 14, 2014, at 12:37 PM, wallyshi [email protected] wrote:

Yes. that looks a bit odd. This extra density was also not observed in other cryoEM studies (or Conti's exosome-11 /+RNA crystal structure) where recombinant proteins were used. I am not sure if it really reflects the difference because of different purification system as Rrp44/Dis3 seems to have at least two conformations dependent on the RNA ( Nogales exosome was purified from endogenously tagged system) or something is wrong one way or the other.

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[wallyshi]

nope they used TAP tag... but this TAP is probably larger than GFP.. let me double check the size.

[Pellarin]

What did they tag? Which protein?

On Mon, Apr 14, 2014 at 1:24 PM, wallyshi [email protected] wrote:

nope they used TAP tag... but this TAP is probably larger than GFP.. let
me double check the size.

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[wallyshi]

The tap tag was on Rrp46- the TAP tag should be around 25-30kDa

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On Apr 14, 2014, at 4:55 PM, Riccardo Pellarin [email protected] wrote:

What did they tag? Which protein?

On Mon, Apr 14, 2014 at 1:24 PM, wallyshi [email protected] wrote:

nope they used TAP tag... but this TAP is probably larger than GFP.. let
me double check the size.

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[wallyshi]

Another potential caveat of using Nogales EM was it appears one component (Csl4) was indeed substochiometric in the pullout that it may not be part of the EM density. Alternatively we could think of using cryoEMs genereated by Lorentzen lab ( where Elina Conti got involved in the paper) EMDB (1708 and 1709 w/o RNA) and see how the data are gonna fit. But let's at least see how the final output looks like with the current EM, if possible.

[Pellarin]

It could be that the extra density (the head) that Nogales saw is
structural heterogeneity.
Is that compatible with alternate arrangement of Rrp44 with respect to the
core particle?

On Mon, Apr 14, 2014 at 3:13 PM, wallyshi [email protected] wrote:

Another potential caveat of using Nogales EM was it appears one component
(Csl4) was indeed substochiometric in the pullout that it may not be part
of the EM density. Alternatively we could think of using cryoEMs genereated
by Lorentzen lab ( where Elina Conti got involved in the paper) EMDB (1708
and 1709 w/o RNA) and see how the data are gonna fit. But let's at least
see how the final output looks like, if possible.

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[wallyshi]

If that s structural heterogeneity then the extra density might be at least the major form and such observation should be well documented in the paper ,which I was aware of.

Most likely it is not compatible based on what I remembered the location (as this guy is really off) but I will check it tomorrow.

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On Apr 14, 2014, at 9:27 PM, Riccardo Pellarin [email protected] wrote:

It could be that the extra density (the head) that Nogales saw is
structural heterogeneity.
Is that compatible with alternate arrangement of Rrp44 with respect to the
core particle?

On Mon, Apr 14, 2014 at 3:13 PM, wallyshi [email protected] wrote:

Another potential caveat of using Nogales EM was it appears one component
(Csl4) was indeed substochiometric in the pullout that it may not be part
of the EM density. Alternatively we could think of using cryoEMs genereated
by Lorentzen lab ( where Elina Conti got involved in the paper) EMDB (1708
and 1709 w/o RNA) and see how the data are gonna fit. But let's at least
see how the final output looks like, if possible.

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[wallyshi]

I checked the Nogales paper and I am sure that the extra density does not correlate to the TAP tag (given it s size) because such extra density was very specific to the RE (core components with Rrp44/Dis) but was absent in the CE (core component without Rrp44/Dis3). There was TAP tag in both RE and CE complexes during protein size exclusion chromography .

[Pellarin]

Then it must be some portion of the Rrp44 that localizes in that region (I
guess the N-terminal part). I'm wondering why any of Elena Conti's papers
mention that.

On Tue, Apr 15, 2014 at 2:10 PM, wallyshi [email protected] wrote:

I checked the Nogales paper and I am sure that the extra density does not
correlate to the TAP tag (given it s size) because such extra density was
very specific to the RE (core components with Rrp44/Dis) but was absent in
the CE (core component without Rrp44/Dis3). There was TAP tag in both RE
and CE complexes during protein size exclusion chromography .

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[wallyshi]

Nogales claimed it is the N terminus of Rrp44... it could be real for the endogenously tagged complex which differs from the reconstituted ones by Conti or might be some contaminants in the PO ( when I looked at their gel pic apparently there is a couple of contaminant proteins, albeit substoichiometric).. I am really keen to see what could come out of the initial modeling jobs that you just submitted.. where/ which subunit that extra density can be assigned based on cross-linking (and excluded volume)!

[Pellarin]

From preliminary analysis, the head density is occupied by some other
component.
That's why I'm trying to understand better.

On Tue, Apr 15, 2014 at 3:43 PM, wallyshi [email protected] wrote:

Nogales claimed it is the N terminus of Rrp44... it could be real for the
endogenously tagged complex which differs from the reconstituted ones by
Conti or might be some contaminants in the PO ( when I looked at their gel
pic apparently there is a couple of contaminant proteins, albeit
substoichiometric).. I am really keen to see what could come out of the
initial modeling jobs that you just submitted.. where/ which subunit that
extra density can be assigned based on cross-linking (and excluded volume)!

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[wallyshi]

below is how the other EM paper made the comment. It looks to me that they
were saying Nogales's cryoEM was problematic, although one could always
argue the difference comes from the purification system... It was indeed
quite interesting to see the density was localized by other protein based
on your initial modeling..
[image: Inline image 1]

Yi

On Tue, Apr 15, 2014 at 7:13 PM, Riccardo Pellarin <[email protected]

wrote:

From preliminary analysis, the head density is occupied by some other
component.
That's why I'm trying to understand better.

On Tue, Apr 15, 2014 at 3:43 PM, wallyshi [email protected]
wrote:

Nogales claimed it is the N terminus of Rrp44... it could be real for
the
endogenously tagged complex which differs from the reconstituted ones by
Conti or might be some contaminants in the PO ( when I looked at their
gel
pic apparently there is a couple of contaminant proteins, albeit
substoichiometric).. I am really keen to see what could come out of the
initial modeling jobs that you just submitted.. where/ which subunit
that
extra density can be assigned based on cross-linking (and excluded
volume)!

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https://github.com/salilab/exosome/issues/2#issuecomment-40543585>
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[wallyshi]

in principle, we could always compare using the other cryoEM data from Esben Lorentzen lab. "RNA channelling by the eukaryotic exosome". 2010. In any event, keen to see what it looks like in your first modeling.