The PAM sequence is colored in red. Despite low sequence identity 5. Substantial differences in their overall architectures are observed Extended Data Fig.
These structural differences between Cas12i and Cas12b account for their distinct functions to be discussed below. To test the role of these residues, we introduced alanine substitutions at each position. The structural and biochemical analysis suggest that Cas12i processes pre-crRNA by an acid-base catalytic mechanism similar to those of Cas12a 23 and Cas13a 24 , with H and H being the acid-base catalyst pair, and R22 being the stabilizer.
Polar residues potentially involved in pre-crRNA processing tested by mutagenesis are shown in sticks with cryo-EM map shown in mesh. The results shown are representative of three experiments. Activities of wild-type Cas12i and alanine substitutions for residues involved in PAM recognition are tested.
Nicking and double strand breaks in substrate are detected by agarose gel. Uncropped images for panels c,e and g are available as source data online. Alanine substitution of any of these residues resulted in reduced substrate cleavage activity Fig. In addition to the sequence specific recognition, a number of positively charged residues form polar interactions with the phosphate groups of the PAM duplex Extended Data Fig. However, only a few residues of Cas12i contact the bp of the heteroduplex Extended Data Fig.
Indeed, the sequence of the target strand can be modelled into the density, nevertheless unambiguous assignment was not feasible at the present resolution Extended Data Fig. The presence of excess DNA in the catalytic pocket was also previously observed in Cas12b Cas12i binds to substrate DNA mainly through residues surrounding the catalytic site in a sequence-independent manner Extended Data Fig.
However, further studies will be required to investigate substrate preference of Cas12i. Mutagenesis and substrate cleavage assays showed that three acidic residues D, E, and D in the RuvC domain constitute the catalytic center Fig.
A basic residue R from the Nuc domain points towards the catalytic center and is also critical for substrate cleavage Fig. The structure suggests a cleavage site between dG 5 and dA 6 , as the scissile phosphate between the two nucleic acid is well positioned towards the catalytic center Fig. Interestingly, when W from the RuvC domain or T from the Nuc domain are substituted by alanine, cleavage of the non-target strand is sustained but cleavage of the target strand is impaired, indicating that they play a role in target strand cleavage Fig.
By 3D classification, we determined a 3. Furthermore, the 7-nt guide segment located in the central positively charged channel is exposed to solvent Fig. Single-nucleotide mismatch in this segment resulted in significant reduction of substrate cleavage activity Fig.
Together, the 7-nt segment is a plausible seed candidate for Cas12i. Catalytic residues are colored in red. The first lane shows the supercoil plasmid substrate treated by a restriction enzyme to create double-strand breaks DBS , resulting in complete linear product. Uncropped images for panels b and h are available as source data online.
Deletion of this loop resulted in reduced cleavage activity Extended Data Fig. Specifically, a number of polar residues contact the phosphate groups in the heteroduplex, assisting the stabilization of the heteroduplex Extended Data Fig. Rotation of the lid motif opens the substrate binding groove and thus enables substrate binding Fig.
Furthermore, the lid motif also stabilizes the substrate for cleavage such as W described above Fig. Collectively, the lid motif may act as a key regulator for DNA cleavage by Cas12i. To understand the biological significance of the different lengths of crRNA-target DNA heteroduplex observed in our structures, we designed crRNAs with different lengths and measured the cleavage activity.
The results suggested that a nt spacer-derived sequence is sufficient to activate the nickase activity of Cas12i Fig. However, a nt spacer-derived sequence is required for cleavage of the target strand, because deletion or mismatch of positions showed no cleavage of the target strand Fig.
Taken together, Cas12i undergoes a two-step activation, wherein formation of bp crRNA-DNA heteroduplex activates the nickase activity for cleavage of the non-target strand, and further base pairing to 28 bp is required for cleavage of the target strand.
To date, 11 sub-types are reported Cas12a-k 30 - 32 , and structures for three sub-types were determined including Cas12a 23 , 33 - 41 , Cas12b 28 , 29 , 42 and Cas12e Most Cas12 proteins target dsDNA to create double strand breaks; however, Cas12i predominantly cleaves the non-target strand, creating a nick on target dsDNA Cas12i, as well as Cas12a, has several functional features distinct from Cas12b and Cas12e, including utilization of a single guide RNA and autonomous pre-crRNA processing.
Cas12i also displays unique features in type V systems. First, Cas12i pre-orders a 7-nt seed sequence, in comparison to a 5-nt seed sequence in Cas12a 23 , 35 and Cas12b 28 , 29 , Another significant difference between Cas12i and other Cas12 effectors is the length of crRNA-target DNA heteroduplex, wherein Cas12i accommodates 28 bp compared to 20 bp for Cas12a 39 , Cas12b 28 and Cas12e 20 , as well as for Cas9 43 , The presence of more nucleotides for base paring indicates that Cas12i may impose stricter selection for a target sequence in order to create a double strand break Interestingly, formation of a bp heteroduplex is critical for cleavage of the target strand.
The requirement of longer sequence complementation for double strand cleavage implies that Cas12i may exhibit higher fidelity for target selection, therefore reducing off-targeting in genome editing applications.
It has been shown that rationally engineered Cas9 with longer spacer would increase the fidelity 46 , the intrinsic preference for longer spacer base pairing length in Cas12i should motivate the exploration of more Cas12i orthologs with high efficiency and accuracy.
It would therefore be interesting to investigate the genome editing activity of Cas12i in eukaryotic cells. Similar structural patterns stem-loop or pseudoknot are observed in the crRNA repeat region adjacent to the spacer-derived sequence in class 2 CRISPR-Cas systems, which not only stabilize the crRNA and allow for recognition by specific CRISPR effectors, but also likely facilitate the correct guide positioning and faithful target binding.
Interestingly, six nucleotides immediately adjacent to the spacer-derived guide are identical between Cas12i and Cas12b Extended Data Fig. Interestingly, the structural element equivalent to lid in Cas12i is conserved in Cas12a, Cas12b, and Cas12e Fig.
First, the lid engages the heteroduplex in a similar sequence-independent manner at positions Second, conformational changes of the lid open the RuvC catalytic center.
Third, the lid contributes to substrate binding, thereby facilitating DNA cleavage. The lid was demonstrated to be critical for RuvC activation 38 , suggesting a conserved activation mechanism for Cas12 endonucleases Fig. Left panels are the structures without substrate structure for Cas12e is not available , while right panels are structures with substrate. The RuvC catalytic centers are indicated by red circles, with the three acidic amino acids shown in sticks.
In summary, our results shed light on the mechanism of Cas12i system in substrate recognition and activation, and the evolution of the type V endonuclease to acquire distinct functions. Atomic structures would facilitate the manipulation of Cas12i for genome editing with high specificity and diagnostic applications.
The plasmid encoding full-length Cas12i Cas12i1 is obtained from Addgene Catalog Cas12i is fused with an N-terminal 6xHis tag. The mutations were generated by quickchange mutagenesis. The protein expression was induced by adding 0.
The mixture was applied to a Superdex GE column equilibrated with buffer C. Poly-A, -T, -C and -G substrates were generated by substitution at the target-strand of protospacer-derived sequence at positions Cas12i proteins ng were mixed with crRNA at a ratio of The purified plasmids ng were then added into the mixture for another 30 mins.
The cleavage products were resolved on 0. The grids were blotted for 4 s and plunged into liquid ethane using a Gatan Cryoplunge 3 plunger. Cryo-EM data were collected with a Titan Krios microscope FEI operated at kV and images were collected using Leginon 50 at a nominal magnification of 81,x resulting in a calibrated physical pixel size of 1.
For data collection with a K3 detector, a dose rate of 20 electrons per pixel per second and an exposure time of 3. Statistics for cryo-EM data are listed in Table 1. Movie frames were aligned using MotionCor2 52 with a binning factor of 2.
Contrast transfer function CTF parameters were estimated using Gctf A few thousand particles were auto-picked without template to generate 2D averages for subsequent template-based auto-picking. The dataset was split into batches for 2D classifications, which were used to exclude false and bad particles that fall into 2D averages with poor features.
The dataset was split into six batches for 3D refinement, each converging at resolution of around 3. After Bayesian polishing, each part was classified into six classes in 3D classification.
The classes representing bad particles from Bayesian polishing and the classes with less alignment accuracy were excluded. Remaining , particles were used for 3D refinement, CTF refinement and 3D refinement, which finally converged at 2.
The higher the voltage, the wider the RPM band the motor can produce torque. Our more popular motors that are volt systems only make torque over about a RPM range, making the case for at least a 2-speed transmission to get the torque over rpm range.
So in the end it typically boils down to the type of EV conversion you are building. So, in essence, EVs are not excluded from the realm of the manual transmission.
Instead, the likelihood of an EV having a manual transmission is really dependent on the voltage system. However, the American consumer is enamoured with EVs that have instant power and torque. So, the more voltage power the better. Even if manual transmission EVs were to suddenly become available from every manufacturer it might be too late. Often, in their minds, the standard, or manual transmission, is something relegated to the forgotten past. Now, an electric with no transmission is the natural progression from an automatic.
Manual transmission vehicles garner less than two percent of sales in the United States. So, why would any potential, or active, mass-market EV manufacturer seek to develop a product for such a niche market? However, having said that, there will always be companies that seek to provide an exclusive product. Companies offering exclusivity will always be there with a high-end, pricey, low-volume product regardless of what is going on in the mass-market climate.
Does it make sense? I can't measure winding resistance to prove it is too small. This is why I will gravity test the battery because if I tow the customer's truck to our facilities and have the motor re-winded and the problem still present I will look very bad.
Thanks for the time you take. Dwaive, I want to go back and examine what you are saying about your battery a bit more closely. When you say "44V tilt backward full pressure" it is important to know what the voltage was 'at rest' before you put the load on it, and generally speaking, if this battery charged up to We used to allow a bit more discharge in older machines, but it was not good for the battery's life, we just figured the battery would die from abuse or lack of proper maintenance, anyway.
ALSO you have given us a model number but I did not see your serial number, and if anyone is going to be able to provide you the correct settings, they will need the serial number of your truck and maybe some motor measurements too.
I had a similar overheating problem on the Hyster version of this truck and it was related to the parking brake shoes. The springs broke off and the shoes would lodge into the drum and drag the park brake. Do not know if this truck has a park brake drum on it but as long as the battery is out it is an easy check. Having read most of the posts on this problem I think you do have a failing battery, I dont supose you have another truck you could swap batteries with.
Double post. Already checked the battery, no defect cells, voltage good, 44V tilt backward full pressure. One wheel was dragging a bit, I adjusted the brakes, didn't help. I was suspecting a faulty component ithe EV but after many test, my conclusion was it is overheating and proved it with an infrared thermometer.
In the book it is written that even with amperage reduced at REC1 output, the truck should reach sufficient speed before 1A close. It is clearly not what is happenning on my truck. I will replace the contact at the resolution of this bug. Like I said the truck works in a small area with short distance receiving dept with 4 garage door to cover but it has always been the same applicaton. I dont have the factory specs but i ran through the settings and all seems correct according to logic.
If you havr them it may be a good thing to do too. Many thanks! Is this a short shuttle or a long run operation? If the drive wheels are off the ground, can you turn both drive wheels by hand?
What can be happening is that the contactor pulls in, but does not make good contact, and because the recs keep pulsing, the driver is not aware that they are not really in bypass. I would also go over ever ALL the settings and reset all the settings to the factory specs.
Thanks all, I found it is the thermal cutback that reduce amperage output at rec1 this causing the accelration being insufficient before 1A close. Now I need to know why it is overheating. The operator is doing a lot of stop and go but it was working fine previously and the application still the same.
Thanks, it was what i thought. The pattern when the bug is present is controller hot. With the accelerator fully actuated, the truck will drive but like it was on the brakes so it will accelerate slowly and after the timer set at function 3 timer has reach the end then 1A is closing while the truck has not reached the correct speed. I suspect REC1 becomes resistive while getting hot but the oscillator card doesn't see it so the countdown of function 3 occurs normally.
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